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Page 1 of 34
1
IAA-producing rhizobacteria from chickpea (Cicer arietinum L.) induce changes in root
2
architecture and increase root biomass†
3 4
Fierro-Coronado, Rosario Aliciaa,1; Quiroz-Figueroa, Francisco Robertoa,1; García-Pérez,
5
Luz Maríaa; Ramírez-Chávez, Enriqueb; Molina-Torres, Jorgeb and Maldonado-Mendoza,
6
Ignacio Eduardoa,*
7 8
a
9
el Desarrollo Integral Regional Unidad Sinaloa, Instituto Politécnico Nacional (CIIDIR-
Departamento de Biotecnología Agrícola, Centro Interdisciplinario de Investigación para
10
IPN Unidad Sinaloa ). Blvd. Juan de Dios Bátiz Paredes No. 250, Col. San Joachin,
11
Guasave Sinaloa, México C.P. 81100
12
b
13
Politécnico Nacional. Irapuato, Guanajuato, México
14
1
15
E-mails: RAFC ([email protected]), FRQF ([email protected]), LMGP
16
([email protected]), ERC ([email protected]), JMT
17
([email protected]) and IEMM ([email protected])
18
*Corresponding author.
19
Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional Unidad
20
Sinaloa, Instituto Politécnico Nacional (CIIDIR IPN - Unidad Sinaloa). Blvd. Juan de Dios
21
Bátiz Paredes No. 250, Col. San Joachin, Guasave Sinaloa, México C.P. 81100. Phone (52)
22
687 872 9626 ext. 87652, Fax (52) 6878729626. [email protected]
23
† We dedicate this paper in memoriam to Juan Cristóbal Pérez-Carranza, head of PURP and
24
the person who promoted the present work.
Departamento de Biotecnología y Bioquímica, CINVESTAV-Irapuato, Instituto
These authors contributed equally to this work.
1
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Page 2 of 34
25 26
ABSTRACT
27
Rhizobacteria promote and have beneficial effects on plant growth, making them useful to
28
agriculture. Nevertheless, the rhizosphere of the chickpea plant has not been extensively
29
examined. The aim of the present study was to select indole-3-acetic acid (IAA) producing
30
rhizobacteria from the rhizosphere of chickpea plants for their potential use as
31
biofertilizers. After obtaining a collection of 864 bacterial isolates, we performed an screen
32
using the Salkowski reaction for the presence of auxin compounds (such as IAA) in
33
bacterial LB supernatant (BLBS). Our results demonstrate that the Salkowski reaction has a
34
greater specificity for IAA detection than other tested auxins. Ten bacterial isolates
35
displaying a wide range of auxin accumulation were selected, which produced IAA levels
36
from 5 to 90 µM (according to the Salkowski reaction). Bacterial isolates were identified on
37
the basis of 16S rDNA partial sequences: nine isolates belonged to Enterobacter and one
38
was classified as Serratia. The effect of BLBS on root morphology was evaluated in
39
Arabidopsis thaliana. IAA production by rhizobacteria was confirmed by means of a
40
DR5::GFP construct that is responsive to IAA, and also by HPLC-GC/MS. Finally, we
41
observed that IAA secreted by rhizobacteria: 1) modified the root architecture of
42
Arabidopsis thaliana; 2) caused an increase in chickpea root biomass; and 3) activated the
43
GFP reporter gene driven by the DR5 promoter. These findings provide evidence that these
44
novel bacterial isolates may be considered as putative PGPRs modifying root architecture
45
and increasing root biomass.
46 47
Keywords
48
Rhizobacteria; Cicer arietinum; chickpea; root morphology; indole-3-acetic acid (IAA). 2
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Page 3 of 34
49 50 51
INTRODUCTION Chickpea (Cicer arietinum L.) was one of the first cultivated legumes, and is now
52
the third most important legume in the world. India, Australia, Pakistan, Turkey and
53
Myanmar account for 88% of its worldwide production (Food and Agriculture
54
Organization 2013). Chickpea is regarded as a good source of plant proteins, carbohydrates
55
and vitamins for humans (Jukanti et al. 2012), and it is also used as feed for livestock
56
(Soltero-Díaz et al. 2008). It is widely grown in soils with poor moisture status, due to its
57
minimum management requirements (Upadhyaya et al. 2011).
58
Organic matter is required for a good chickpea crop production, as it prevents loss
59
of nutrients by retaining and making them available for plant use (Reeves 1997; FAO
60
2005). However, the majority of agricultural soils are characterized by low organic matter
61
content. Soil erosion is also exacerbated by traditional management of agricultural fields,
62
which involves chemical fertilization and the use of machinery for soil conditioning and
63
establishment of monocultures (Toro et al. 2008). As an alternative, the nutritional status of
64
infertile soils can be improved through biological fertilization. Research in the field of
65
biofertilizers includes soil nutrient management, biological control and the use of plant
66
growth-promoting rhizobacteria, all of which are economic and renewable sources of
67
nutrition suited for increasing agricultural production and improving soil fertility (Dodd et
68
al. 1990; Vessey 2003). This use of bacteria to stimulate plant growth is an
69
environmentally friendly approach, since the molecules of bacterial origin are
70
biodegradable, as opposed to agrochemicals, which are more persistent (Lugtenberg and
71
Kamilova 2009).
3
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Page 4 of 34
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Plant growth-promoting rhizobacteria can colonize roots to increase growth and
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crop yields; they are also able to control plant-pathogenic microorganisms and enhance
74
resistance to abiotic stresses (van Peer et al. 1991; Van Loon 2007; Lugtenberg and
75
Kamilova 2009; Kavamura et al. 2013). Numerous rhizobacteria are reported to be plant
76
growth promoters, including Azotobacter, Azospirillum, Burkholderia, Enterobacter,
77
Pseudomonas, Bacillus, Methanobacterium, Clostridium, and Rhizobia (Reis et al. 2000;
78
Ahmad et al. 2008; Hayat et al. 2010; Gamalero and Glick 2011; Bhattacharyya and Jha
79
2012). The beneficial effects of rhizobacteria on plant growth are manifested in three ways:
80
there is the synthesis of compounds that act on the plants; there is the increase in nutrient
81
uptake by plants; and there is a reduction in or prevention of plant diseases caused by
82
pathogens (Hayat et al. 2010).
83
Plant growth and plant protection are promoted either directly or indirectly. Direct
84
promotion works through production of plant regulators such as auxins, cytokinins,
85
gibberellins and abscisic acid. Alternatively, direct promotion can function through
86
degradation of ethylene, solubilization of minerals such as phosphate (to increase their
87
uptake), symbiotic nitrogen fixation, or synthesis of vitamins (Hayat et al. 2010). As for
88
indirect promotion, plant growth-promoting rhizobacteria synthesize siderophores (high-
89
affinity iron chelating compounds), antibiotics such as agrocin substances, herbicolin, 2,4-
90
diacetylphloroglucinol, oomycin, pyoluteorin, phenazines, cyclic lipopeptides, pyrrolnitrin,
91
hydrogen cyanide and lytic enzymes or fungal cell wall degrading enzymes (e.g. chitinase
92
and ß-1,3-glucanase) (Vessey 2003; Fernando et al. 2006; Lugtenberg and Kamilova 2009;
93
Hayat et al. 2010; Gamalero and Glick 2011).
94 95
Indole acetic acid (IAA) is one of the most physiologically active auxins in plants (Zhao 2010; Taiz and Zeiger 2010), although it is also biosynthesized by fungi and bacteria 4
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Page 5 of 34
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(Frankenberger and Poth 1987; Arshad and Frankenberger, Jr. 1991). It is a common
97
product of L-tryptophan metabolism in several microorganisms, including plant growth-
98
promoting rhizobacteria (Frankenberger and Poth 1987; Arshad and Frankenberger, Jr.
99
1991). IAA regulates many aspects of plant development, including stem elongation, apical
100
dominance, tropism, and lateral root initiation, as well as fruit, meristem and root hair
101
development by cell division and elongation (Taiz and Zeiger 2010). IAA synthesized by
102
bacteria can enhance the production of root hairs and the lateral and adventitious roots,
103
leading to improved mineral and nutrient uptake and root exudation, which stimulates
104
bacterial proliferation in the rhizosphere (Gamalero and Glick 2011).
105
To date, only a limited number of reports have been published about growth-
106
promoting rhizobacteria in the chickpea plant (Sarma et al. 2002; Singh et al. 2003; Joseph
107
et al. 2007; Rokhzadi et al. 2013). To improve their application to agricultural practices
108
will require a greater exploration of rhizobacteria diversity, and an evaluation of their traits,
109
such as IAA production. In the present study we have screened chickpea rhizobacteria for
110
their capacity to produce IAA as a plant growth regulator, and we have analyzed their root
111
promotion ability with the main goal of selecting those with potential to be used as
112
biofertilizers.
113 114
MATERIALS AND METHODS
115
Bacterial collection
116
A bacterial collection of 864 isolates (Scientific Collection CIIDIR-006) from the
117
chickpea rhizosphere (C. arietinum L.) was created following the methodology of Cordero-
118
Ramírez et al. (2012). A total of thirty chickpea rhizosphere samples were collected from
119
six chickpea fields in Guasave, Sinaloa, Mexico. Five plants were sampled from each field 5
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Page 6 of 34
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and were homogenized together and stored at room temperature. Only the soil particles
121
strongly added to the roots (rhizospheric soil) were taken to isolate the microorganisms
122
using serial dilutions by plating 100 µL per dilution. Luria Bertani (LB) medium was used
123
to generate the chickpea rhizospheric bacterial collection. Single colonies were taken from
124
LB after 24 hours growth at 25º C. After two rounds of purification in LB medium and
125
microscopic confirmation of a uniform bacterial morphology for each isolates they were
126
cryopreserved in 96-well plates at -70°C, using 200 µL of LB containing 15% glycerol by
127
triplicate (Pasarell and McGinnis, 1992). Frozen stocks were made from each isolate and
128
were grown at 25°C and 200 rpm in 2 mL 96-well plates containing 1 mL liquid LB
129
medium for 24 hours. The isolate was considered nonviable if no visible growth was
130
observed after thawing. Only viable isolates were included in the collection.
131 132 133
Screening of bacterial isolates by Salkowski reaction The bacterial collection was screened for IAA-like compound production using
134
Salkowski colorimetric assays (Glickmann and Dessaux 1995). Isolates were cultured in 2
135
mL 96-well plates containing 1 mL of LB (without any tryptophan supplement) at 28 ± 2°C
136
for 48 h and 200 rpm, followed by centrifugation at 3,020 g for 25 min (Beckman Coulter
137
Avanti J-30I, JS-5.9 rotor). Pellets were discarded and the supernatants were filtered
138
through 0.22 µm pore size filters (Cat. SLGV 013 SL Millex® Millipore). A 100 µl volume
139
of the bacterial LB supernatant (BLBS) was mixed with 100 µl of Salkowski reagent (20 g
140
per liter of FeCl3•6H2O in 7.9 M H2SO4). The mix was incubated for 30 min in the dark at
141
25 ± 2°C, the absorbance was recorded at 530 nm with a spectrophotometer (Spectro
142
Multiskan GO, Thermo Scientific), and the concentration of IAA produced was estimated
6
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Page 7 of 34
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by using a standard IAA (Cat. 12886, Sigma) curve as a reference. Three replicates were
144
evaluated for each BLBS sample or control.
145 146 147
Spectrophotometric scanning measurement A spectrophotometric scan was performed at a wavelength range from 400 to 700
148
nm and a 2 nm resolution. A 1:1 v/v mix was prepared with a combination of Salkowski
149
reagent and either the bacterial supernatant or auxin-activity compounds in LB. Absorption
150
peaks (λmax) of the mixes containing IAA, indole butyric acid (IBA), naphthalene acetic
151
acid (NAA), LB, tryptophan, sodium hydroxide (NaOH) and BLBS were compared.
152 153 154
Effect of BLBS and IAA on root architecture in Arabidopsis thaliana Three-day old Arabidopsis thaliana ecotype Columbia-0 seedlings (n=3) were
155
planted on Petri dishes with 0.5X MS medium (Murashige and Skoog 1962). Seedlings
156
were supplemented with either LB, 50 nM IAA, or BLBS (equivalent to 50 nM of IAA as
157
measured by the Salkowski reaction). Each experiment, comprising three Petri dishes with
158
three plants each (n=9), was evaluated after five days of treatment, and the experiment was
159
repeated three times independently.
160
Petri dishes were maintained upright for five days in growth chambers (Binder Mod.
161
KBW 400) at 25 ± 2ºC (16 h:8 h light:dark photoperiod). Primary root growth was recorded
162
every 24 h, and the number of lateral roots was quantified on the fifth day. Root
163
architecture was evaluated by staining roots with the general dye propidium iodide (Cat.
164
P4170, Sigma), followed by observation with a confocal laser scanning microscope (Leica
165
TCS SP5X). Samples were excited at 488 nm and visualized under emission ranges from
166
502 to 548 nm. All micrographs were taken under the same settings. 7
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167 168
Evaluation of the biological functionality of IAA-like compounds secreted by
169
rhizobacteria in pDR5::GFP transgenic Arabidopsis thaliana plants
170
Fourteen-day old seedlings of A. thaliana pDR5::GFP (Friml et al. 2003) were
171
immersed in either 500 µL of BLBS (equivalent to 100 µM IAA), LB, or sterile distilled
172
water at 25 ± 2°C for 12 h. The concentration of IAA-like compounds in the BLBS was
173
estimated by the Salkowski reaction as described above. All samples were analyzed in
174
triplicate at pH 7 after the BLBS was filtered through a 0.22 µm Millipore filter (Cat.
175
SLGV 013 SL Millex®, Millipore). A. thaliana leaves were observed with a confocal laser
176
scanning microscope (Leica TCS SP5X) using a 488 nm excitation laser, and visualized
177
with both beam lines for chlorophyll fluorescence emission (red, 640-700 nm) and GFP
178
(green, 496-540 nm). All micrographs were taken under the same settings.
179 180 181
Evaluation of IAA-producing rhizobacteria in chickpea plants Rhizobacteria were evaluated under greenhouse conditions in substrate composed of
182
sand and vermiculite (1:1 v/v) with pH 6.8 and electrical conductivity of 0.14 mS/cm. The
183
substrate was sterilized twice at 121°C (15 PSI) for one hour followed by a 24 h waiting
184
period before use. Chickpea (var. Costa 2004) seeds (obtained from Productores Unidos del
185
Río Petatlán/INIFAP JJR, Sinaloa, Mexico) were surface-sterilized with sodium
186
hypochlorite (0.3%) for 10 min and rinsed with sterile distilled water five times and then
187
planted in 1 L pots. Seven-day old plant root systems were inoculated with bacterial
188
suspensions prepared in water containing 3 x 109 CFU per plant. Negative controls only
189
received water. Plants were supplemented with Long-Ashton nutritive solution (Hewitt
8
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Page 9 of 34
190
1966) once per week. Plant irrigation was performed every three days with sterile water.
191
Thirty days after inoculation, plants were evaluated by recording their biomass (e.g. dry
192
weight of roots and shoots). This experiment was repeated three times independently with
193
similar results.
194 195
Statistical analyses Results for IAA production by Salkowski reaction, primary root growth, lateral root
196 197
(LR) density, LR length and root growth (measured as dry weight) were subjected to
198
variance analysis. All experiments were performed at least three times independently,
199
producing similar results. The means were compared by the least significant difference
200
(LSD) test at P = 0.05 with the Statistical Analysis System 9.0 (SAS Institute, Inc., Cary,
201
NC).
202 203
Mass spectrometric identification of IAA in rhizobacterial culture supernatants
204
Samples for mass spectrometry analysis were prepared from 100 ml bacterial
205
cultures. Bacteria were removed by centrifugation at 10,000 g for 25 min, and the
206
supernatants were filtered through 0.22 µm pore size filters (Cat. SLGV 013 SL Millex®,
207
Millipore). The supernatants were extracted twice with equal volumes of ethyl acetate, and
208
both extractions were combined and freeze-dried in a liophilizer (Freezone, Labconco).
209
Samples were then dissolved in 1 mL of HPLC-grade ethanol. 100 µl of sample were dried
210
with nitrogen gas and silylated using 20 µl of pyridine and 80 µl of 1% (v/v) N,N-bis(tri-
211
methyl-silyl) tri-fluoro-acetamide in tri-methyl chloro-silane (TMS) at 80°C for 30 min.
212
Samples (1 µl) were injected and analyzed in an Agilent Technologies 7890A GC System
213
equipped with a capillary column (HP5-MS, 30 m × 0.25 mm i.d. x 250 µm film thickness) 9
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coupled to a mass selective detector (Agilent Technologies 5975C). The following
215
operating conditions were used: injector temperature 250ºC, with oven temperature
216
programmed as initial temperature 150ºC for 3 min, then increasing at a rate of 4ºC/min to
217
a final temperature of 280ºC, which was then maintained for 25 min. Helium was used as
218
the carrier gas, with a constant flow rate of 1 ml/min. The EI source had a mass
219
spectrometer temperature of 230ºC, and the mass spectrometer Quadrupole temperature
220
was 150ºC. The spectra of indolic compounds were then identified using the Deconvolution
221
AMDIS software from the National Institute of Standards and Technology (NIST), as well
222
as the spectra library from the NIST MS Data Base Search version 2 software.
223 224 225
Molecular identification of bacteria Bacterial isolates were grown on LB plates for 48 h at 28°C. Bacterial pellets were
226
resuspended in ultrapure distilled water (Cat. 10977, Gibco) and boiled to obtain DNA
227
template, and debris was removed by centrifugation at 10,000 g for 15 min at 4°C.
228
The universal primers F2C (AGAGTTTGATCATGGCTC) and C
229
(ACGGGCGGTGTGTAC) were used for 16S rDNA partial amplification (Shi et al. 1997).
230
The PCR mixture contained 1 – 2.5 µL of bacterial lysates, 1X reaction buffer, 1 mM
231
MgCl2, 0.5 mM of each primer, 500 µM of each deoxy-nucleoside-tri-phosphate (dNTP),
232
and 0.5 U of Taq DNA polymerase (Cat. 10966-030, Invitrogen) in a total volume of 25
233
µL. PCR amplification was performed with a C-1000 thermocycler (Bio-Rad), using the
234
following program: an initial denaturation step at 95°C for 4 min; 32 cycles consisting of a
235
1 min denaturation step at 95°C, 1 min annealing step at 60°C, and 2 min extension step at
236
72°C; and a final step at 72°C for 5 min. PCR products were separated by 1.2% agarose gel
237
electrophoresis in 0.5X Tris-boric acid-EDTA (TBE) buffer and stained with Gel-RedTM 10
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Page 11 of 34
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(Cat. 41003, Biotium). A Chemidoc photodocumentation system (Bio-Rad) was used to
239
visualize the amplicons. Only one band was observed per rhizobacteria PCR amplification
240
(~1.4 kb). Amplicons were purified using the QIAquick PCR Purification Kit (Cat. 28106,
241
Qiagen) according to the manufacturer’s indications. The amplicons were sequenced bi-
242
directionally with an ABI Prism 3100 Automated Sequencer at the National Laboratory of
243
Genomics (Langebio, CINVESTAV Irapuato, Mexico) using the primers
244
U1(CCAGCAGCCGCGGTAATACG) and C (ACGGGCGGTGTGTAC) (Shi et al. 1997;
245
Lu et al. 2000).
246 247
Sequence analysis
248
Homologous 16SrDNA sequences were searched against bacterial databases from
249
the Ribosomal Database project (http://rdp.cme.msu.edu), using the BLAST-N nucleotide
250
software with the Megablast algorithm (Cole et al. 2009). Our identification criterion was
251
based on a > 98% sequence identity. The sequences were aligned using the multiple
252
sequence alignment software Clustal W (Yadav et al. 2010), and a phylogenetic tree was
253
then generated with the MEGA 5 software (Tamura et al. 2011). Phylogenetic tree
254
construction also utilized the neighbor joining method (Saitou and Nei 1987) with a Kimura
255
2 substitution model. Variation rate was modeled with a gamma distribution, using five
256
categories. The neighbor joining topology strength was evaluated by bootstrap test with
257
1000 replicates. Ten chickpea rhizobacteria sequences (GenBank KF303795-KF303804)
258
were included in our phylogenetic tree, along with reference strains of closely related
259
genera (Enterobacter spp.). Pectobacterium carotovorum (NR044980) and Proteus
260
vulgaris (NR025336) were used as outgroups.
261 11
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Page 12 of 34
262
RESULTS
263
Selection of auxin-like secreting bacteria by Salkowski reaction screening
264
Two molecules with auxin activity were used to evaluate the specificity of the
265
Salkowski reaction for IAA-like compounds: IBA (a synthetic auxin with an indole ring
266
whose radical group differs from IAA) and NAA (which has the same radical group as IAA
267
but lacks the indole ring). Auxins and BLBS as well as a number of controls were all
268
spectrophotometrically scanned and plotted from 440 to 600 nm. Only the Salkowski
269
reaction with IAA exhibited a maximum absorbance at 530 nm. Neither the auxin activity
270
compounds (NAA and IBA) nor the controls (LB and Trp) had a maximum at 530 nm (Fig.
271
1A). Following the Salkowski reaction, the BLBS of isolate 476 showed a maximum
272
absorbance at the same wavelength as IAA. Scans from all bacterial isolates displayed an
273
absorbance pattern that was identical to isolate 476, and the same absorbance peak as IAA
274
(data not shown). In the absence of the Salkowski reagent, no compounds were able to
275
exhibit similar absorbance patterns or a maximum absorbance at 530 nm, as compared to
276
IAA (Fig. 1A). These results suggest that the Salkowski reaction is specific for IAA or
277
IAA-like compounds with an acyl radical group in the C-3 position of the indole ring
278
including indoleacetamide and indolepyruvic (Glickmann and Dessaux 1995) .
279
The complete chickpea rhizosphere bacterial collection was screened using the
280
Salkowski reaction. The initial selection criterion for IAA-producing bacteria was the
281
development of pink color upon contact with this reagent. Seventy-four out of the 864
282
rhizobacteria exhibited this pink color, from which we repeated the Salkowski reaction. Ten
283
isolates showing the most intense pink color were further selected after quantification of
284
IAA-like compounds (Fig. 1B). We observed that rhizobacteria secreted IAA-like
12
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Page 13 of 34
285
compounds in the concentration range of 5 to 80 µM. Four of these rhizobacteria (476, 715,
286
727 and 758) secreted between 65 – 80 µM. The bacteria could thus be separated into two
287
subgroups: those with high (> 60 µM) or low (
Page 1 of 34
1
IAA-producing rhizobacteria from chickpea (Cicer arietinum L.) induce changes in root
2
architecture and increase root biomass†
3 4
Fierro-Coronado, Rosario Aliciaa,1; Quiroz-Figueroa, Francisco Robertoa,1; García-Pérez,
5
Luz Maríaa; Ramírez-Chávez, Enriqueb; Molina-Torres, Jorgeb and Maldonado-Mendoza,
6
Ignacio Eduardoa,*
7 8
a
9
el Desarrollo Integral Regional Unidad Sinaloa, Instituto Politécnico Nacional (CIIDIR-
Departamento de Biotecnología Agrícola, Centro Interdisciplinario de Investigación para
10
IPN Unidad Sinaloa ). Blvd. Juan de Dios Bátiz Paredes No. 250, Col. San Joachin,
11
Guasave Sinaloa, México C.P. 81100
12
b
13
Politécnico Nacional. Irapuato, Guanajuato, México
14
1
15
E-mails: RAFC ([email protected]), FRQF ([email protected]), LMGP
16
([email protected]), ERC ([email protected]), JMT
17
([email protected]) and IEMM ([email protected])
18
*Corresponding author.
19
Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional Unidad
20
Sinaloa, Instituto Politécnico Nacional (CIIDIR IPN - Unidad Sinaloa). Blvd. Juan de Dios
21
Bátiz Paredes No. 250, Col. San Joachin, Guasave Sinaloa, México C.P. 81100. Phone (52)
22
687 872 9626 ext. 87652, Fax (52) 6878729626. [email protected]
23
† We dedicate this paper in memoriam to Juan Cristóbal Pérez-Carranza, head of PURP and
24
the person who promoted the present work.
Departamento de Biotecnología y Bioquímica, CINVESTAV-Irapuato, Instituto
These authors contributed equally to this work.
1
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Page 2 of 34
25 26
ABSTRACT
27
Rhizobacteria promote and have beneficial effects on plant growth, making them useful to
28
agriculture. Nevertheless, the rhizosphere of the chickpea plant has not been extensively
29
examined. The aim of the present study was to select indole-3-acetic acid (IAA) producing
30
rhizobacteria from the rhizosphere of chickpea plants for their potential use as
31
biofertilizers. After obtaining a collection of 864 bacterial isolates, we performed an screen
32
using the Salkowski reaction for the presence of auxin compounds (such as IAA) in
33
bacterial LB supernatant (BLBS). Our results demonstrate that the Salkowski reaction has a
34
greater specificity for IAA detection than other tested auxins. Ten bacterial isolates
35
displaying a wide range of auxin accumulation were selected, which produced IAA levels
36
from 5 to 90 µM (according to the Salkowski reaction). Bacterial isolates were identified on
37
the basis of 16S rDNA partial sequences: nine isolates belonged to Enterobacter and one
38
was classified as Serratia. The effect of BLBS on root morphology was evaluated in
39
Arabidopsis thaliana. IAA production by rhizobacteria was confirmed by means of a
40
DR5::GFP construct that is responsive to IAA, and also by HPLC-GC/MS. Finally, we
41
observed that IAA secreted by rhizobacteria: 1) modified the root architecture of
42
Arabidopsis thaliana; 2) caused an increase in chickpea root biomass; and 3) activated the
43
GFP reporter gene driven by the DR5 promoter. These findings provide evidence that these
44
novel bacterial isolates may be considered as putative PGPRs modifying root architecture
45
and increasing root biomass.
46 47
Keywords
48
Rhizobacteria; Cicer arietinum; chickpea; root morphology; indole-3-acetic acid (IAA). 2
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49 50 51
INTRODUCTION Chickpea (Cicer arietinum L.) was one of the first cultivated legumes, and is now
52
the third most important legume in the world. India, Australia, Pakistan, Turkey and
53
Myanmar account for 88% of its worldwide production (Food and Agriculture
54
Organization 2013). Chickpea is regarded as a good source of plant proteins, carbohydrates
55
and vitamins for humans (Jukanti et al. 2012), and it is also used as feed for livestock
56
(Soltero-Díaz et al. 2008). It is widely grown in soils with poor moisture status, due to its
57
minimum management requirements (Upadhyaya et al. 2011).
58
Organic matter is required for a good chickpea crop production, as it prevents loss
59
of nutrients by retaining and making them available for plant use (Reeves 1997; FAO
60
2005). However, the majority of agricultural soils are characterized by low organic matter
61
content. Soil erosion is also exacerbated by traditional management of agricultural fields,
62
which involves chemical fertilization and the use of machinery for soil conditioning and
63
establishment of monocultures (Toro et al. 2008). As an alternative, the nutritional status of
64
infertile soils can be improved through biological fertilization. Research in the field of
65
biofertilizers includes soil nutrient management, biological control and the use of plant
66
growth-promoting rhizobacteria, all of which are economic and renewable sources of
67
nutrition suited for increasing agricultural production and improving soil fertility (Dodd et
68
al. 1990; Vessey 2003). This use of bacteria to stimulate plant growth is an
69
environmentally friendly approach, since the molecules of bacterial origin are
70
biodegradable, as opposed to agrochemicals, which are more persistent (Lugtenberg and
71
Kamilova 2009).
3
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Plant growth-promoting rhizobacteria can colonize roots to increase growth and
73
crop yields; they are also able to control plant-pathogenic microorganisms and enhance
74
resistance to abiotic stresses (van Peer et al. 1991; Van Loon 2007; Lugtenberg and
75
Kamilova 2009; Kavamura et al. 2013). Numerous rhizobacteria are reported to be plant
76
growth promoters, including Azotobacter, Azospirillum, Burkholderia, Enterobacter,
77
Pseudomonas, Bacillus, Methanobacterium, Clostridium, and Rhizobia (Reis et al. 2000;
78
Ahmad et al. 2008; Hayat et al. 2010; Gamalero and Glick 2011; Bhattacharyya and Jha
79
2012). The beneficial effects of rhizobacteria on plant growth are manifested in three ways:
80
there is the synthesis of compounds that act on the plants; there is the increase in nutrient
81
uptake by plants; and there is a reduction in or prevention of plant diseases caused by
82
pathogens (Hayat et al. 2010).
83
Plant growth and plant protection are promoted either directly or indirectly. Direct
84
promotion works through production of plant regulators such as auxins, cytokinins,
85
gibberellins and abscisic acid. Alternatively, direct promotion can function through
86
degradation of ethylene, solubilization of minerals such as phosphate (to increase their
87
uptake), symbiotic nitrogen fixation, or synthesis of vitamins (Hayat et al. 2010). As for
88
indirect promotion, plant growth-promoting rhizobacteria synthesize siderophores (high-
89
affinity iron chelating compounds), antibiotics such as agrocin substances, herbicolin, 2,4-
90
diacetylphloroglucinol, oomycin, pyoluteorin, phenazines, cyclic lipopeptides, pyrrolnitrin,
91
hydrogen cyanide and lytic enzymes or fungal cell wall degrading enzymes (e.g. chitinase
92
and ß-1,3-glucanase) (Vessey 2003; Fernando et al. 2006; Lugtenberg and Kamilova 2009;
93
Hayat et al. 2010; Gamalero and Glick 2011).
94 95
Indole acetic acid (IAA) is one of the most physiologically active auxins in plants (Zhao 2010; Taiz and Zeiger 2010), although it is also biosynthesized by fungi and bacteria 4
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(Frankenberger and Poth 1987; Arshad and Frankenberger, Jr. 1991). It is a common
97
product of L-tryptophan metabolism in several microorganisms, including plant growth-
98
promoting rhizobacteria (Frankenberger and Poth 1987; Arshad and Frankenberger, Jr.
99
1991). IAA regulates many aspects of plant development, including stem elongation, apical
100
dominance, tropism, and lateral root initiation, as well as fruit, meristem and root hair
101
development by cell division and elongation (Taiz and Zeiger 2010). IAA synthesized by
102
bacteria can enhance the production of root hairs and the lateral and adventitious roots,
103
leading to improved mineral and nutrient uptake and root exudation, which stimulates
104
bacterial proliferation in the rhizosphere (Gamalero and Glick 2011).
105
To date, only a limited number of reports have been published about growth-
106
promoting rhizobacteria in the chickpea plant (Sarma et al. 2002; Singh et al. 2003; Joseph
107
et al. 2007; Rokhzadi et al. 2013). To improve their application to agricultural practices
108
will require a greater exploration of rhizobacteria diversity, and an evaluation of their traits,
109
such as IAA production. In the present study we have screened chickpea rhizobacteria for
110
their capacity to produce IAA as a plant growth regulator, and we have analyzed their root
111
promotion ability with the main goal of selecting those with potential to be used as
112
biofertilizers.
113 114
MATERIALS AND METHODS
115
Bacterial collection
116
A bacterial collection of 864 isolates (Scientific Collection CIIDIR-006) from the
117
chickpea rhizosphere (C. arietinum L.) was created following the methodology of Cordero-
118
Ramírez et al. (2012). A total of thirty chickpea rhizosphere samples were collected from
119
six chickpea fields in Guasave, Sinaloa, Mexico. Five plants were sampled from each field 5
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and were homogenized together and stored at room temperature. Only the soil particles
121
strongly added to the roots (rhizospheric soil) were taken to isolate the microorganisms
122
using serial dilutions by plating 100 µL per dilution. Luria Bertani (LB) medium was used
123
to generate the chickpea rhizospheric bacterial collection. Single colonies were taken from
124
LB after 24 hours growth at 25º C. After two rounds of purification in LB medium and
125
microscopic confirmation of a uniform bacterial morphology for each isolates they were
126
cryopreserved in 96-well plates at -70°C, using 200 µL of LB containing 15% glycerol by
127
triplicate (Pasarell and McGinnis, 1992). Frozen stocks were made from each isolate and
128
were grown at 25°C and 200 rpm in 2 mL 96-well plates containing 1 mL liquid LB
129
medium for 24 hours. The isolate was considered nonviable if no visible growth was
130
observed after thawing. Only viable isolates were included in the collection.
131 132 133
Screening of bacterial isolates by Salkowski reaction The bacterial collection was screened for IAA-like compound production using
134
Salkowski colorimetric assays (Glickmann and Dessaux 1995). Isolates were cultured in 2
135
mL 96-well plates containing 1 mL of LB (without any tryptophan supplement) at 28 ± 2°C
136
for 48 h and 200 rpm, followed by centrifugation at 3,020 g for 25 min (Beckman Coulter
137
Avanti J-30I, JS-5.9 rotor). Pellets were discarded and the supernatants were filtered
138
through 0.22 µm pore size filters (Cat. SLGV 013 SL Millex® Millipore). A 100 µl volume
139
of the bacterial LB supernatant (BLBS) was mixed with 100 µl of Salkowski reagent (20 g
140
per liter of FeCl3•6H2O in 7.9 M H2SO4). The mix was incubated for 30 min in the dark at
141
25 ± 2°C, the absorbance was recorded at 530 nm with a spectrophotometer (Spectro
142
Multiskan GO, Thermo Scientific), and the concentration of IAA produced was estimated
6
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by using a standard IAA (Cat. 12886, Sigma) curve as a reference. Three replicates were
144
evaluated for each BLBS sample or control.
145 146 147
Spectrophotometric scanning measurement A spectrophotometric scan was performed at a wavelength range from 400 to 700
148
nm and a 2 nm resolution. A 1:1 v/v mix was prepared with a combination of Salkowski
149
reagent and either the bacterial supernatant or auxin-activity compounds in LB. Absorption
150
peaks (λmax) of the mixes containing IAA, indole butyric acid (IBA), naphthalene acetic
151
acid (NAA), LB, tryptophan, sodium hydroxide (NaOH) and BLBS were compared.
152 153 154
Effect of BLBS and IAA on root architecture in Arabidopsis thaliana Three-day old Arabidopsis thaliana ecotype Columbia-0 seedlings (n=3) were
155
planted on Petri dishes with 0.5X MS medium (Murashige and Skoog 1962). Seedlings
156
were supplemented with either LB, 50 nM IAA, or BLBS (equivalent to 50 nM of IAA as
157
measured by the Salkowski reaction). Each experiment, comprising three Petri dishes with
158
three plants each (n=9), was evaluated after five days of treatment, and the experiment was
159
repeated three times independently.
160
Petri dishes were maintained upright for five days in growth chambers (Binder Mod.
161
KBW 400) at 25 ± 2ºC (16 h:8 h light:dark photoperiod). Primary root growth was recorded
162
every 24 h, and the number of lateral roots was quantified on the fifth day. Root
163
architecture was evaluated by staining roots with the general dye propidium iodide (Cat.
164
P4170, Sigma), followed by observation with a confocal laser scanning microscope (Leica
165
TCS SP5X). Samples were excited at 488 nm and visualized under emission ranges from
166
502 to 548 nm. All micrographs were taken under the same settings. 7
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167 168
Evaluation of the biological functionality of IAA-like compounds secreted by
169
rhizobacteria in pDR5::GFP transgenic Arabidopsis thaliana plants
170
Fourteen-day old seedlings of A. thaliana pDR5::GFP (Friml et al. 2003) were
171
immersed in either 500 µL of BLBS (equivalent to 100 µM IAA), LB, or sterile distilled
172
water at 25 ± 2°C for 12 h. The concentration of IAA-like compounds in the BLBS was
173
estimated by the Salkowski reaction as described above. All samples were analyzed in
174
triplicate at pH 7 after the BLBS was filtered through a 0.22 µm Millipore filter (Cat.
175
SLGV 013 SL Millex®, Millipore). A. thaliana leaves were observed with a confocal laser
176
scanning microscope (Leica TCS SP5X) using a 488 nm excitation laser, and visualized
177
with both beam lines for chlorophyll fluorescence emission (red, 640-700 nm) and GFP
178
(green, 496-540 nm). All micrographs were taken under the same settings.
179 180 181
Evaluation of IAA-producing rhizobacteria in chickpea plants Rhizobacteria were evaluated under greenhouse conditions in substrate composed of
182
sand and vermiculite (1:1 v/v) with pH 6.8 and electrical conductivity of 0.14 mS/cm. The
183
substrate was sterilized twice at 121°C (15 PSI) for one hour followed by a 24 h waiting
184
period before use. Chickpea (var. Costa 2004) seeds (obtained from Productores Unidos del
185
Río Petatlán/INIFAP JJR, Sinaloa, Mexico) were surface-sterilized with sodium
186
hypochlorite (0.3%) for 10 min and rinsed with sterile distilled water five times and then
187
planted in 1 L pots. Seven-day old plant root systems were inoculated with bacterial
188
suspensions prepared in water containing 3 x 109 CFU per plant. Negative controls only
189
received water. Plants were supplemented with Long-Ashton nutritive solution (Hewitt
8
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1966) once per week. Plant irrigation was performed every three days with sterile water.
191
Thirty days after inoculation, plants were evaluated by recording their biomass (e.g. dry
192
weight of roots and shoots). This experiment was repeated three times independently with
193
similar results.
194 195
Statistical analyses Results for IAA production by Salkowski reaction, primary root growth, lateral root
196 197
(LR) density, LR length and root growth (measured as dry weight) were subjected to
198
variance analysis. All experiments were performed at least three times independently,
199
producing similar results. The means were compared by the least significant difference
200
(LSD) test at P = 0.05 with the Statistical Analysis System 9.0 (SAS Institute, Inc., Cary,
201
NC).
202 203
Mass spectrometric identification of IAA in rhizobacterial culture supernatants
204
Samples for mass spectrometry analysis were prepared from 100 ml bacterial
205
cultures. Bacteria were removed by centrifugation at 10,000 g for 25 min, and the
206
supernatants were filtered through 0.22 µm pore size filters (Cat. SLGV 013 SL Millex®,
207
Millipore). The supernatants were extracted twice with equal volumes of ethyl acetate, and
208
both extractions were combined and freeze-dried in a liophilizer (Freezone, Labconco).
209
Samples were then dissolved in 1 mL of HPLC-grade ethanol. 100 µl of sample were dried
210
with nitrogen gas and silylated using 20 µl of pyridine and 80 µl of 1% (v/v) N,N-bis(tri-
211
methyl-silyl) tri-fluoro-acetamide in tri-methyl chloro-silane (TMS) at 80°C for 30 min.
212
Samples (1 µl) were injected and analyzed in an Agilent Technologies 7890A GC System
213
equipped with a capillary column (HP5-MS, 30 m × 0.25 mm i.d. x 250 µm film thickness) 9
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coupled to a mass selective detector (Agilent Technologies 5975C). The following
215
operating conditions were used: injector temperature 250ºC, with oven temperature
216
programmed as initial temperature 150ºC for 3 min, then increasing at a rate of 4ºC/min to
217
a final temperature of 280ºC, which was then maintained for 25 min. Helium was used as
218
the carrier gas, with a constant flow rate of 1 ml/min. The EI source had a mass
219
spectrometer temperature of 230ºC, and the mass spectrometer Quadrupole temperature
220
was 150ºC. The spectra of indolic compounds were then identified using the Deconvolution
221
AMDIS software from the National Institute of Standards and Technology (NIST), as well
222
as the spectra library from the NIST MS Data Base Search version 2 software.
223 224 225
Molecular identification of bacteria Bacterial isolates were grown on LB plates for 48 h at 28°C. Bacterial pellets were
226
resuspended in ultrapure distilled water (Cat. 10977, Gibco) and boiled to obtain DNA
227
template, and debris was removed by centrifugation at 10,000 g for 15 min at 4°C.
228
The universal primers F2C (AGAGTTTGATCATGGCTC) and C
229
(ACGGGCGGTGTGTAC) were used for 16S rDNA partial amplification (Shi et al. 1997).
230
The PCR mixture contained 1 – 2.5 µL of bacterial lysates, 1X reaction buffer, 1 mM
231
MgCl2, 0.5 mM of each primer, 500 µM of each deoxy-nucleoside-tri-phosphate (dNTP),
232
and 0.5 U of Taq DNA polymerase (Cat. 10966-030, Invitrogen) in a total volume of 25
233
µL. PCR amplification was performed with a C-1000 thermocycler (Bio-Rad), using the
234
following program: an initial denaturation step at 95°C for 4 min; 32 cycles consisting of a
235
1 min denaturation step at 95°C, 1 min annealing step at 60°C, and 2 min extension step at
236
72°C; and a final step at 72°C for 5 min. PCR products were separated by 1.2% agarose gel
237
electrophoresis in 0.5X Tris-boric acid-EDTA (TBE) buffer and stained with Gel-RedTM 10
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(Cat. 41003, Biotium). A Chemidoc photodocumentation system (Bio-Rad) was used to
239
visualize the amplicons. Only one band was observed per rhizobacteria PCR amplification
240
(~1.4 kb). Amplicons were purified using the QIAquick PCR Purification Kit (Cat. 28106,
241
Qiagen) according to the manufacturer’s indications. The amplicons were sequenced bi-
242
directionally with an ABI Prism 3100 Automated Sequencer at the National Laboratory of
243
Genomics (Langebio, CINVESTAV Irapuato, Mexico) using the primers
244
U1(CCAGCAGCCGCGGTAATACG) and C (ACGGGCGGTGTGTAC) (Shi et al. 1997;
245
Lu et al. 2000).
246 247
Sequence analysis
248
Homologous 16SrDNA sequences were searched against bacterial databases from
249
the Ribosomal Database project (http://rdp.cme.msu.edu), using the BLAST-N nucleotide
250
software with the Megablast algorithm (Cole et al. 2009). Our identification criterion was
251
based on a > 98% sequence identity. The sequences were aligned using the multiple
252
sequence alignment software Clustal W (Yadav et al. 2010), and a phylogenetic tree was
253
then generated with the MEGA 5 software (Tamura et al. 2011). Phylogenetic tree
254
construction also utilized the neighbor joining method (Saitou and Nei 1987) with a Kimura
255
2 substitution model. Variation rate was modeled with a gamma distribution, using five
256
categories. The neighbor joining topology strength was evaluated by bootstrap test with
257
1000 replicates. Ten chickpea rhizobacteria sequences (GenBank KF303795-KF303804)
258
were included in our phylogenetic tree, along with reference strains of closely related
259
genera (Enterobacter spp.). Pectobacterium carotovorum (NR044980) and Proteus
260
vulgaris (NR025336) were used as outgroups.
261 11
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262
RESULTS
263
Selection of auxin-like secreting bacteria by Salkowski reaction screening
264
Two molecules with auxin activity were used to evaluate the specificity of the
265
Salkowski reaction for IAA-like compounds: IBA (a synthetic auxin with an indole ring
266
whose radical group differs from IAA) and NAA (which has the same radical group as IAA
267
but lacks the indole ring). Auxins and BLBS as well as a number of controls were all
268
spectrophotometrically scanned and plotted from 440 to 600 nm. Only the Salkowski
269
reaction with IAA exhibited a maximum absorbance at 530 nm. Neither the auxin activity
270
compounds (NAA and IBA) nor the controls (LB and Trp) had a maximum at 530 nm (Fig.
271
1A). Following the Salkowski reaction, the BLBS of isolate 476 showed a maximum
272
absorbance at the same wavelength as IAA. Scans from all bacterial isolates displayed an
273
absorbance pattern that was identical to isolate 476, and the same absorbance peak as IAA
274
(data not shown). In the absence of the Salkowski reagent, no compounds were able to
275
exhibit similar absorbance patterns or a maximum absorbance at 530 nm, as compared to
276
IAA (Fig. 1A). These results suggest that the Salkowski reaction is specific for IAA or
277
IAA-like compounds with an acyl radical group in the C-3 position of the indole ring
278
including indoleacetamide and indolepyruvic (Glickmann and Dessaux 1995) .
279
The complete chickpea rhizosphere bacterial collection was screened using the
280
Salkowski reaction. The initial selection criterion for IAA-producing bacteria was the
281
development of pink color upon contact with this reagent. Seventy-four out of the 864
282
rhizobacteria exhibited this pink color, from which we repeated the Salkowski reaction. Ten
283
isolates showing the most intense pink color were further selected after quantification of
284
IAA-like compounds (Fig. 1B). We observed that rhizobacteria secreted IAA-like
12
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Page 13 of 34
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compounds in the concentration range of 5 to 80 µM. Four of these rhizobacteria (476, 715,
286
727 and 758) secreted between 65 – 80 µM. The bacteria could thus be separated into two
287
subgroups: those with high (> 60 µM) or low (
