Effect of plant growth-promoting bacteria on the growth and fructan production of Agave americana L. Academic research paper on "Biological sciences"

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BRAZILIAN JOURNAL OF MICROBIOLOGY

SOCIEDADE BRASILEIRA DE MICROBIOLOGIA

http://www.bjmicrobiol.com.br/

Environmental Microbiology

Effect of plant growth-promoting bacteria on the growth and fructan production of Agave americana L.

Neyser De La Torre-Ruiza, Víctor Manuel Ruiz-Valdiviezob, Clara Ivette Rincón-Molinab, Martha Rodríguez-Mendiolaa, Carlos Arias-Castroa, Federico Antonio Gutiérrez-Micelib, Héctor Palomeque-Dominguezb, Reiner Rincón-Rosalesb'*

a Plant Biotechnology, DEPI Instituto Tecnológico de Tlajomulco, Carretera a San Miguel Cuyutlán, Tlajomulco de Zúñiga, Jalisco, Mexico b Laboratory of Biotechnology, Instituto Tecnológico de Tuxtla Gutiérrez, Tuxtla Gutiérrez, Mexico

article info

abstract

Article history:

Received 4 June 2015

Accepted 11 February 2016

Available online xxx

Associate Editor: Ieda de Carvalho

Mendes

Keywords:

Inoculation

Bacteria

Inulin

16S rRNA

The effect of plant growth-promoting bacteria inoculation on plant growth and the sugar content in Agave americana was assessed. The bacterial strains ACO-34A, ACO-40, and ACO-140, isolated from the A. americana rhizosphere, were selected for this study to evaluate their phenotypic and genotypic characteristics. The three bacterial strains were evaluated via plant inoculation assays, and Azospirillum brasilense Cd served as a control strain. Phylo-genetic analysis based on the 16S rRNA gene showed that strains ACO-34A, AC0-40 and AC0-140 were Rhizobium daejeonense, Acinetobacter calcoaceticus and Pseudomonas mosselii, respectively. All of the strains were able to synthesize indole-3-acetic acid (IAA), solubi-lize phosphate, and had nitrogenase activity. Inoculation using the plant growth-promoting bacteria strains had a significant effect (p < 0.05) on plant growth and the sugar content of A. americana, showing that these native plant growth-promoting bacteria are a practical, simple, and efficient alternative to promote the growth of agave plants with proper biological characteristics for agroindustrial and biotechnological use and to increase the sugar content in this agave species.

© 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is

an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

The use of nitrogen-fixing microorganisms and plant growth-promoting bacteria (PGPB) is an important alternative to

replace chemical fertilizers for the cultivation of agricultural 26

plants. 27

The search for PGPB as well as research on their biologi- 28

cal properties are increasing at a rapid pace because efforts 29 are being made to exploit them commercially as inoculants.

* Corresponding author. E-mail: reriro61@hotmail.com (R. Rincón-Rosales). http://dx.doi.Org/10.1016/j.bjm.2016.04.010

1517-8382/© 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Significant improvement on the growth and yield of crops in response to microbial inoculation has been reported by many workers.1-3 Studies confirm that inoculants formulated with PGPB have shown positive effects on the agricultural yield and crop quality.2,4 Regarding the effect of PGPB on plant growth, it has been reported that Glycine max L. Merrill seedlings inoculated with PGPB (Pseudomonas sp. strain AK-1, and Bacillus sp. strain SJ-5) demonstrated enhanced plant biomass and that the plants had a higher proline content than control plants.5 In another study, the efficiency of Mesorhizobium, Azo-tobacter, and Pseudomonas on the growth, yield, and disease suppression in chickpea plants (Cicer arietinum L.) was evaluated. Pseudomonas showed positive IAA production, phosphate solubilization, and antagonistic activities against Fusarium oxysporum and Rhizoctonia solani compared to other strains.6 Martinez-Rodriguez et al.7 also reported that cultivable endophytic bacteria from the leaf base of Agave tequilana Weber var. Blue have the potential to enhance plant growth.

Scientific evidence supports that the agave genus includes several species of economic, social and cultural importance for people around the world.8 Agave plants are greatly relevant to Mexico because this country is considered to be the point of origin of the evolution and diversification of this genus.9 Approximately 163 species grow in Mexico, and 123 species are endemic.10

Agave americana L. has successfully adapted to climatic and edaphic conditions and proliferated in the highlands of Chiapas, Mexico, where it is an important source of natural fibre, medicine, fructans, and traditional alcoholic beverages for the local community. Due to the economic significance of this plant, several commercial plantations have been established in the state of Chiapas to produce sufficient raw materials for agro-industrial use. However, when the plantlets are transplanted to the field, their growth and development is slow, and consequently, 5-7 years are required to obtain mature plants for industrial use.11 An alternative for obtaining mature plants for industrial use is the application of plant growth-promoting bacteria, but it is necessary to assess the possible effects of PGPB on A. americana to increase the survival and growth of plantlets. PGPB are rhizosphere bacteria that enhance plant growth by a wide variety of mechanisms, such as phosphate solubilization, siderophore production, biological nitrogen fixation, phytohormone production, antifungal activity, induction of systemic resistance, promotion of beneficial plant-microbe symbioses, and so on.12,13

Many aspects of the microbial community associated with agaves are still unknown and only a manuscript related to14 suggests that the hypothesis that PGPB inoculation significantly increases the growth and sugar content (mainly inulin) in A. americana is true. Therefore, the objective of this study was to evaluate the effect of PGPB inoculation on plant growth and sugar accumulation in A. americana.

Materials and methods

82 Bacterial strains

83 The bacterial strains ACO-34A, ACO-40, and ACO-140 were

84 chosen subsequent to a study of approximately 235 strains

previously isolated from the rhizosphere of A. americana. These three strains were selected based on their capacity for nitrogen fixation, auxin production, P-solubilization and biosynthesis of IAA (Table S1) and were provided by the Instituto Tecnológico de Tuxtla Gutiérrez, while the reference strain Azospirillum brasilense Cd was provided by the Centro de Ciencias Genómicas, Cuernavaca, México. All strains were grown in yeast extract-mannitol (YMA) medium15 at 28 ° C and preserved at 4 °C until use.

Phenotypic and genotypic analysis of strains

The cell morphologies of the strains isolated from A. americana were examined by light microscopy (Zeiss® PS7, Germany). The Gram reaction was determined using a kit (Merck®, Germany), according to the manufacturer's procedure, and colony morphology was determined with cells grown on YMA plates at 28 °C for 5 days.16

Bacteriological and physiological characterization of strains ACO-34A, ACO-40, and ACO-140 were performed with isolates from YMA medium. Salt tolerance was evaluated at 28°C with 0.5,1.0, 2.0, 3.0 and 5.0% (w/v) NaCl and pH levels of 4.0, 5.0, 9.0 and 11.0. Acid or alkali production was determined on the same medium supplemented with 25mgmL-1 bro-mothymol blue as a pH indicator.16 Antibiotic resistance was tested on YMA plates following the process recommended by Martinez-Romero et al.17. In addition, the Al and Cu tolerance of the strains were determined on solid YMA medium.18

16S rRNA gene sequencing and phylogenetic analysis

The strains were grown in 2.0 mL of YMA medium overnight. Total genomic DNA was extracted using a DNA Isolation Kit for Cells and Tissues (Roche®, Switzerland), according to the manufacturer's specifications. PCR was performed with the bacterial universal 16S rRNA primers fD1 (5'-AGAGTTTGATCCTGGCTCAG-3') and rD1 (5'-AAGGAGGTGATCCAGCC-3'), which amplified products of approximately 1500 bases, and procedures were performed as described by Weisburget al.19. The PCR products were purified using the PCR Product Purification System Kit from Roche® and sequenced (Macrogen®, Korea). All sequences were compared with the reference sequences obtained by a BLAST search.20 The sequences were aligned using the CLUSTAL X (2.0) software with the default settings.21 Minor modifications in the alignment were made using the BIOEDIT sequence editor. Phylogenetic and molecular evolutionary analyses were performed with MEGA v5.2.22 The phylogenetic tree of the 16S rRNA gene sequences from type strains was constructed by Neighbour-Joining23 and a Bootstrap analysis with 1000 pseu-doreplicates using the Tamura-Nei model.24 The 16S rRNA gene sequence of strains ACO-34A, ACO-40, and ACO-140 were deposited in the GenBank database under the accession numbers KM349967, KM349968, and KM349969, respectively. Additionally, strains ACO-34A, ACO-40, and ACO-140 were deposited in DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen), Germany as an open collection under the deposit numbers DSM 101606, DSM 01771 and DSM 01784, respectively.

100 101 102

120 121 122

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Measurement ofPGPB efficiency

Quantification ofIAA production

Bacteria were grown in conical flasks containing 50 mL of YMA medium composed of mannitol (0.25 gL-1), K2HPO4 10% (5 mLL-1), MgSO4 10% (2 mLL-1), NaCl 10% (1mLL-1), CaCO3 (1gL-1) and yeast extract (3gL-1) at a pH of 6.8, supplemented with 100 mgL-1 of L-tryptophan. After incubation at 30 ±2 °C on a rotary shaker for 48 h, the culture medium was centrifuged at 5000 x g for 10 min, and the supernatant was filtered through a 0.22-^m membrane filter. The IAA levels in the filtered supernatants of each strain were measured by high-performance liquid chromatography (HPLC) using a PerkinElmer model series 200A equipped with a Supelco LiChrosorb RP Ci8 column (5 ^m; 4.6 by 150 mm). The mobile phase was acetonitrile-50 mM KH2PO4 (pH 3) (30/70) at a flow rate of 1mLmin-1. Eluates were analyzed with a diode array detector at 220 nm, and IAA was quantified by integrating the area under the peak; authentic IAA (Sigma) was used as a standard. The IAA produced by each strain was measured in triplicate.25

Estimation of phosphate solubilization in broth assay by PGPB

The isolates were individually grown in YM broth medium overnight, and the OD600 nm was adjusted to 1.0. The cells were washed twice in 0.85% sterile Ringers solution before inoculating in National Botanical Research Institute Phosphate (NBRIP) growth medium containing insoluble tricalcium phosphate (Ca3(PO4)2).26 The pH of the NBRIP medium was adjusted to 7.0 before autoclaving. The strains were inoculated in 20-mL vials containing NBRIP medium and incubated at 30 °C on a shaker (150 rpm) for 5 days. After incubation, 5.0 mL of each strain was taken and centrifuged, and the pH of the supernatant was recorded. The available phosphorus content in the culture supernatant as well as the control (supernatant obtained from medium not inoculated with bacteria) was estimated using the vanado-molybdate colorimetric method27 by measuring the absorbance at a wavelength of 420 nm. Each treatment was replicated three times.

Assay for acetylene reduction activity (ARA) The nitrogen fixation ability of the PGPB strains was determined by the acetylene reduction activity.28 The tubes were sealed with rubber stoppers after inoculation and then incubated for 24h in NFB medium.29 Ten percent of the air was removed from each tube, and an equal volume of acetylene was injected using a syringe, before incubating the tubes at 30 °C for 24 h. A parallel uninoculated control tube was prepared. A 100-^L air sample was taken, and the amount of ethylene produced from acetylene was determined by gas chromatography (Perkin Elmer, USA). The ARA value was reported to as [ARA nmol C2H4 per culture h-1].

Plant inoculation assay

The PGPB strains ACO-34A, ACO-40, and ACO-140 isolated from the A. americana rhizosphere and the reference strain A. brasilense Cd were evaluated in a biofertilization test. A. americana plantlets obtained by micropropagation were planted in polystyrene trays that contained peat as the substrate (the pH

of the peat was adjusted to 6.7 using 35 g Na2CO3 for each 100 g of material) and covered with a polyethylene sheet to maintain the humidity and prevent dehydration. Plants were maintained in a growth chamber at 28 °C and 38% relative humidity (RH) with a 14 h light/10 h dark photoperiod. Lighting was provided by fluorescent lamps with 50 ^Em-2 s-1.30

After two months (60 days after transplantation), the plants were transferred to pots containing peat moistened with free N Fahraeus medium [CaCl2,0.1 g; MgSO4-7H2O,0.12 g; KH2PO4, 0.1 g; Na2HPO4-2H2O, 0.15 g; Fe citrate, 0.005 g; Mn, Cu, Zn, B, Mo traces; dist. water, 1000 mL; pH 6.5] as a nutrient source31 and placed in a greenhouse at 25 ±2 °C (natural illumination). The plants were inoculated with 2 mL of a suspension of each PGPB strain at a concentration of 1 x 106UFCmL-1.30'32 Uninoculated plants and others treated with 30 mg of KNO3-N per plant served as controls. Four replicates were used per treatment, and the plants were arranged in a completely randomized design. The plants were grown under greenhouse conditions for 90 days. Measurements of the plants, the dry weight, the diameter of the stem, the number of leaves, and the length of the roots were made on the plants during the transplantation phase (mi) and after 90 days (m2) in the greenhouse. The data used for statistical analysis were the difference between the m2-m1 measurements.

Extraction and quantification of carbohydrates The carbohydrates were extracted and quantified from the dried leaves and roots, which were washed with ultrapure water. The samples were crushed and placed in a water bath at 80 ° C for 40 min, the supernatant was removed by centrifu-gation at 16,000 x g for 15 min at room temperature, and the precipitate was removed. Samples were adjusted to pH 7.0, filtered through 20- and 45-^m nylon membranes, and stored at 8 °C until use. Carbohydrates were identified with a previously described modified method.33 Briefly, thin layer chromatography was performed using propanol-butanol-water (12:3:4) for the mobile phase and Merck® F254 silica gel plates for the stationary phase. Spots were developed with a solution containing 45% (v/v) aniline (4% v/v in acetone), 45% (v/v) diphenylamine (4% (v/v) in acetone) and 9.1% (v/v) of 85% phosphoric acid. The developing solution was applied to the plates and allowed to dry. The plates were heated to 80 °C for 5, 10, and 15 min until a clearly defined colour appeared. The aldoses had a blue-grey colour, and the ketoses and sucrose were red or a mixture of both colours.

For the quantification of carbohydrates, samples were adjusted to pH 7.0 and filtered through 20- and 45-^m membranes before being placed in 2-mL vials. A 10-^L sample of plant extract was injected into a HPLC-IR (Thermo Finnigan®, Ontario, Canada) equipped with a Rezex RCM-monosaccharide Ca2+ column, with water as the mobile phase at a temperature of 85 °C, a flow rate of 0.3mLmin-1, and a pressure of 300 psi. Inulin from chicory root, fructose (Sigma®, USA), sucrose, and glucose (Baker®, USA) were used as standards.33

Statistical analysis

All of the data obtained in the tests for inoculation, P-solubilization, IAA production, acetylene reduction activity

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253 (ARA), and carbohydrate quantification using different PGPB

254 strains were statistically analyzed by analysis of variance

255 (ANOVA), and the means were compared by TUkey's test

256 (p <0.05).34

Results and discussion

257 Investigation of PGPB strains from different plants with the

258 potential to be used as inoculants has increased in recent

259 years, and many are commercially available.12,13,35 Taxonomic

260 polyphasic studies are commonly used to analyze bacterial

261 diversity and include morphological, physiological, biochem-

262 ical, metabolic, and principally, phylogenetic studies.36-38

263 The morphological and physiological characteristics of the

264 bacterial strains isolated from the A. americana rhizosphere

265 that have potential as inoculants are shown in Table S2. The

266 strains evaluated were aerobic, Gram-negative, rod-shaped,

267 and grew rapidly in YMA medium. The cells grew at 37 °C and

268 had an acidic reaction. The ACO-40 strain formed circular,

269 lightly opaque yellow colonies from 0.5 to 1.5 mm in diameter

270 with a mucoid appearance. On the other hand, the ACO-140

271 strain formed circular, creamy beige colonies with regular

272 borders, measured approximately 3-4 mm in diameter, and

273 had no observable pigmentation. Finally, strain ACO-34A was

274 characterized by the formation of circular, semitranslucent

275 creamy colonies that were 1.5-3.0 mm in diameter with a

276 mucoid appearance. These three strains could grow in a pH

277 range from 4.0 to 9.0. As for NaCl tolerance, the AC0-140

278 strain grew in 5.0% NaCl, but the AC0-34A and AC0-40

279 strains tolerated no more than 2.0% NaCl. The AC0-34A

280 strain was resistant to ampicillin (100 ^gmL-1), carbenicillin

281 (20 ^gmL-1), chloramphenicol (100 ^gmL-1), and kanamycin

282 (100 ^gmL-1). The AC0-40 strain showed a greater sensitivity

283 to the antibiotics assessed. The strains grew in the presence of

284 Al3+ (500 ^gmL-1) and Cu2+ (100 ^gmL-1). These results indi-

285 cated that some of the biological qualities that distinguished

286 these strains included their capacity to grow in a wide pH

287 range, as well as their ability to tolerate different NaCl con-

288 centrations and heavy metals, such as Al and Cu. For example,

289 strain AC0-140 managed to grow in 5.0% NaCl. This outcome

290 is of great significance because salinity is one of the harshest

291 environmental factors that prevents a high crop yield, and

292 most agricultural plants are sensitive to a high salt concen-

293 tration in the soil. Therefore, plant growth promoting bacteria

294 (PGPB) act as one of the most effective tools for the alleviation

of salt stress. Azospirillum lipoferum JA4, a genetically tagged strain, was capable of colonizing the roots of wheat seedlings in the presence of a high NaCl concentration.39 Puente et al.40 reported that strains of Azospirillum halopraeferens and A. brasilense survived in seawater and were capable of colonizing the root surfaces of black mangrove seedlings. In light of this, the AC0-140 strain could be an alternative to promote A. americana growth under salt stress conditions, as has been previously demonstrated with barley and oats.41,42 Additionally, this study demonstrated that these three strains could grow at low pH and tolerate high concentrations of aluminium and copper. PGPB, such as Pseudomona fluorescens, can produce oxalic acid, which also might be a possible mechanism for reducing Al toxicity.43 Rhizobium sp. strain BICC 651 produced a threefold higher level of siderophore in the presence of 100 ^M Al3+, which could be a mechanisms to relieve Al stress,44 suggesting that the application of such bacteria may be an efficient approach to alleviate aluminium toxicity and eventually improve soil fertility. Strain AC0-34A was resistant to various antibiotics, which could be important because it could protect the plant against phytopathogens and establish an area in the rhizosphere with a greater capacity to assimilate soil nutrients.45

We used a polyphasic approach in this study that combined the phenotypic characteristics of the bacterial strains with a phylogenetic analysis of their 16S rRNA gene sequences to determine the taxonomic status of strains isolated from the A. americana rhizosphere (Fig. 1). Strain AC0-40 had a sequence size of 1381 bp, was affiliated with members of the genus Acinetobacter, and showed a 99.1% genetic identity with the type strain Acinetobacter calcoaceticus NCCB22016T (Table 1). The 16S rRNA gene sequence of strain AC0-140 was 1387 bp and was classified in the genus Pseudomonas, with a 100.0% genetic similarity to Pseudomonas mosselii CIP 105259T.46 Finally, the strain AC0-34A sequence was 1333bp and clustered with members of the genus Rhizobium; its 16S rRNA gene sequences had a 96.1% similarity with Rhizobium daejeonense L61T.36

The highest amount of IAA was produced by the P. mos-selii strain AC0-140 (15.7 mgL-1), followed by the R. daejeonense strain AC0-34A and A. calcoaceticus strain AC0-40 (Table 2). The reference strain A. brasilense Cd also produced a significant amount of IAA (10.4 mgL-1), according to Tukey's test (p < 0.05). The capacity of the rhizobial strains to solubilize phosphate was evaluated with a colorimetric method using NBRIP growth medium that contained insoluble tricalcium phosphate. All

Table 1 - Molecular identification of the PGPB strains isolated from Agave americana L.

Strain Closest partial 16S rRNA gene sequence Accession No. 16S rRNA seq. (bP) Closest NCBI match/species identity Reference

ACO-34A Rhizobium daejeonense KM349967 1333 R. daejeonense L61T (AY341343) 96.1% Zhe-Xue et al.36

ACO-40 Acinetobacter calcoaceticus KM349968 1381 A. calcoaceticus NCCB22016T (AJ888983) 99.1% Unpublished

ACO-140 Pseudomonas mosselii KM349969 1387 P. mosselii CIP 105259T (AF072688) 100% Dabboussi et al.46

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76 ACO-40 (KM349968)

100p-Acinetobactercalcoaceticus D10 (JQ031270)

'-Acinetobacter calcoaceticus B9 (JQ579640)

—Acinetobactercalcoaceticus NCCB 22016T (AJ888983)

i-Acinetobacter baylyi B2T (AF509820)

—AcinetobactersoliB1T (EU290155)

......,df

-Acinetobacterradioresistens DSM6976T (X81666)

—Acinetobacterbaumannii DSM30007 (X81660) —Acinetobacterjunii DSM69641" (X816649) -Acinetobactergerneri 9A01T (AF509829)

-Acinetobacterlwoffii DSM2403T (X81665)

-Acinetobacterparvus LUH4616T (AJ293691)

—Acinetobacterbeijerinckii LUH 4759T(AJ626712)

-Acinetobacterhaemolyticus DSM6962T (X81662)

-Acinetobacterjohnsonii ATCC 17909T (Z93440)

i-Acinetobacterschindleri LUH5832T (AJ278311)

—Acinetobacterbouvetii 4B02T (AF509827)

97|ACO-140 (KM349969)

Pseudomonasmosselii CIP 105259T(AF072688) Pseudomonas taiwanensis BCRC 17751T (EU103629) Pseudomonas monteilii CIP104883T(AF064458) —Pseudomonas plecoglossicida FPC951T(AB009457)

Pseudomonas japonica IAM 15071T(AB126621) Pseudomonas putida IAM 1236T (D84020) Pseudomonas oryzihabitans IAM 1568T (D84004) Pseudomonas asplenii ATCC 23835T(AB021397)

Pseudomonas fluorescens CCM 2115T(DQ207731) Pseudomonas mohnii IpA-2T (AM293567) Pseudomonas jessenii CIP105274T(AF068259) 96| i—Pseudomonas koreensis Ps9-14T(AF468452) ■Pseudomonas reinekei MT1T (AM293565)

Pseudomonas aeruginosa ATCC 10145T(X06684)

54— Rhizobium loessense CCBAU 7190B (AF364069)

971-Rhizobium gallicum R602spT (U86343)

-Rhizobium mongolense USDA 1844T (U89817) -Rhizobium suiiae IS123T (Y10170) -Rhizobium etli CFN 4? (U28916)

-Rhizobium indigoferae CCBAU 7104? (AY034027) 92 I-Rhizobium tropici CIAT 899T (U89832)

-Rhizobium hainanense I66T (U71078) Rhizobium huautlense SO2T (AF025852)

■Rhizobium galegae ATCC 4367^ (D11343)

95 |-Rhizobium undicola LMG11875T (Y17047)

'-Rhizobium larrymoorei AF3-10T (Z30542)

Rhizobium giardinii H152T (U86344)

76 |-ACO-34A (KM349967 )

-Rhizobium daejeonense R2-60 (JQ659582)

Rhizobium daejeonense NBRC 102494 (AB681831) Rhizobium daejeonense L61T (AY341343)

Fig. 1 - Neighbour-joining phylogenetic trees based on 16S rRNA gene sequences. (A) Acinetobacter calcoaceticus strain ACO-40 (1381 bp), (B) Pseudomonas mosselii strain ACO-140 (1387 bp) and (C) Rhizobium daejeonense strain ACO-34A (1333 bp). Only bootstrap values > 50% are shown. Type strains are indicated by the superscript T. The accession numbers for the sequences are indicated within the parentheses. Those generated in this work are shown in bold.

0.—b

341 four strains had the capacity to solubilize phosphate after

342 2-4 days of incubation. Strain AC0-140 exhibited the high-

343 est phosphate solubilizing activity (37.8mgL-1), followed by

344 strain AC0-40 (29.8mgL-1) and strain AC0-34A (24.7mgL-1)

345 (Table 2). The pH value of the culture medium decreased from

an initial pH of 6.8 to 4.5 as bacterial growth progressed, 346

suggesting that the bacteria might secrete organic acids to 347

solubilize the insoluble phosphorus. 348

Recently, endophytic bacteria belonging to the genera 349 Acinetobacter, Bacillus and Pseudomonas with a capacity for 350

Table 2 - Phosphate solubilization, IAA production, and acetylene reduction activity (ARA) in the PGPB strains isolated from Agave americana L.

Treatment IAA production (mgL-1) P-solubilization (mgL-1) ARAb nmol C2H4 per culture h-1

Pseudomonas mosselii 15.7 Aa 37.8 A 167.1 B

ACO-140

Acinetobacter calcoaceticus 8.5 C 29.8 B 106.1 B

AC0-40

Rhizobium daejeonense 10.3 B 24.7 B 627.4 A

AC0-34A

Azospirillum brasilense 10.4 B 24.2 B 821.3 A

MSDc (p <0.05) 1.65 7.63 224.39

a Mean values of four replicates. Means followed by the same letter do not show any significant differences (p < 0.05). b ARA, acetylene reduction assay (^mol C2H4 per culture fresh weighh-1). c MSD, minimum significant difference.

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Table 3 - Growth parameters for Agave americana plants inoculated with plant growth-promoting bacteria.

Treatment Plant dry weight (g) Stem diameter (cm) Number of leaves Root length (cm)

Pseudomonas mosselii 2.44 B* 0.48 A 3.0 A 11.3 CD

ACO-140

Acinetobacter calcoaceticus 1.23 D 0.27 CD 2.7 AB 11.3 CD

ACO-40

Rhizobium daejeonense 3.41 A 0.37 B 4.0 A 26.6 A

ACO-34A

Azospirillum brasilense Cd 1.65 C 0.34 BC 1.5 BC 16.2 B

KNO3-N 1.07 D 0.23 D 1.5 BC 14.3 BC

Uninoculated 0.76 E 0.18 D 1.2 C 9.8 D

MSD? (p <0.05) 0.2975 0.0994 1.4510 3.1339

* Mean values of four replicates. Means followed by the same letter do not show a sig ® MSD, minimum significant difference. nificant difference (p < 0.05).

351 nitrogen fixation, auxin production and phosphate solubi-

352 lization were isolated from blue agave plants (A. tequilana)

353 from Nayarit, Mexico.7 Pseudomonas putida M5TSA, isolated

354 from an endemic cactus Mammillaria fraileana that grows

355 in the Sonoran Desert, solubilized inorganic phosphate and

356 demonstrated rock-weathering capacity.47 These pseudomon-

357 ads are characterizedbybiochemicalmechanisms and specific

358 enzymes that facilitate rock degradation, as well as the biosyn-

359 thesis of important secondary metabolites, under varied biotic

360 and abiotic stress conditions.48

361 Inoculation of plants with PGPB enhances the assimilation

362 of essential nutrients and plant-associated biological nitrogen

363 fixation.13,49 In this study, nitrogenase activity was assessed

364 with the acetylene reduction assay (ARA) to determine the

365 ability of PGPB to fix nitrogen. A. brasilense Cd and R. daejeonense

366 AC0-34A strains showed maximum nitrogenase activity (821.3

367 and 627.4nmol C2H per cultureh-1, respectively) compared

368 to the other strains evaluated (Table 2). Thus, the three strains

369 showed the potential to produce IAA and solubilize phosphate,

370 as well as nitrogen-fixation capacity. These results are impor-

371 tant because nitrogen and phosphorus are key elements for

372 the growth and metabolism of agave plants.

373 Furthermore, inoculation with PGPB had a positive effect

374 on the growth of A. americana plants (Table 3). A. calcoaceticus

375 strain AC0-40, P. mosselii strain AC0-140, and R. daejeonense

376 strain AC0-34A all had positive effects on the plant dry weight,

377 stem diameter, number of leaves, and root length compared

378 to uninoculated control plants and to those with added KN03.

379 0n average, plants inoculated with R. daejeonense strain AC0-

380 34A weighed 2.65 g more than uninoculated plants at 90 days

381 post inoculation. The stem diameter of plants inoculated with

382 P. mosselii strain AC0-140 differed significantly (p <0.05) from

383 that obtained in the other treatments. Plants treated with the

384 PGPB strains AC0-40, AC0-140, and AC0-34Aexperienced sim-

385 ilar effects for the number of leaves compared to uninoculated

386 plants according to analysis by Tukey's test (p <0.05). Plants

387 inoculated with strain AC0-34A showed a significantly higher

388 root length compared to other treatments (p <0.05). Bashan

389 et al.,50 reported that the inoculation of pachycereid cacti

390 species (Pachycereus pringlei, Stenocereus thurberi and Lopho-

391 cereus schottii) enhanced the establishment and development

392 of the cacti that were inoculated with A. brasilense and trans-

393 planted into a disturbed urban desert soil. Puente et al.,51

demonstrated that an association between the giant cardon 394

cactus P. pringlei and endophytic bacteria helped the seedlings 395

become established and grow in a soilless environment. It has 396

also been reported that Enterobacter sakazakii M2PFe, Azoto- 397

bacter vinelandii M2Per and P. putida M5TSA, isolated from the 398

rock-dwelling cactus M. fraileana, affected plant growth and 399

the mobilization of elements from rocks.52 In this study, simi- 400

lar results were found for R. daejeonense strain AC0-34A, which 401

significantly influenced A. americana growth, demonstrating 402

its potential as a PGPB due to its capacity for nitrogen fixation, 403

phosphate solubilization, and IAA biosynthesis. PGBP strains 404

such as A. brasilense and R. daejeonense are biological models 405

that may potentially contribute to the revegetation of eroded 406

soil. Similar results concerning the occurrence and diversity 407

of diazotrophic bacteria in rhizosphere soil and in root and 408

leaf tissues of Agave sisalana plants have been reported by 409

Santos et al.,53 as well as a test of their potential for plant 410

growth promotion. Therefore, PGPB strains investigated in this 411

study could be alternative A. americana inoculants that would 412

improve its growth and development. 413

The sugar content in the leaves, stems, and plant roots was 414

measured with a qualitative and quantitative analysis. Thin 415

layer chromatography (Fig. 2) showed that fructan synthesis 416

was different in different plant tissues and that bacterial inoc- 417

ulation influenced the degree of polymerization (PD) of the 418

fructans. Fructose and sucrose were detected in the leaves, 419

stems, and roots of A. americana plantlets inoculated with 420

the four bacteria. However, kestose was detected in A. amer- 421

icana plantlets inoculated with all of the bacterial strains, 422

except in plantlets inoculated with A. calcoaceticus strain AC0- 423

40 (Fig. 2B). Spots corresponding to sucrose were detected with 424

greater intensity in the leaves and the stems compared to roots 425

(Fig. 2B and C). Nystose and other spots corresponding to fruc- 426

tans with PD>4 were detected in the stems of A. americana 427

plantlets inoculated with P. mosselii strain AC0-140 (Fig. 2A). 428

Kestose was only detected in plants inoculated with P. mos- 429

selii. This result may indicate that the induction of kestose 430

and nystose biosynthesis is specific and depends on the inocu- 431

lated microbial species. Therefore, this phenomenon requires 432

further investigation. 433

In addition, the inulin concentration varied from 0.23 434

to 1.09 mgg-1 in the leaves and was higher in plantlets 435

inoculated with R. daejeonense strain AC0-34A (Table 4). 436

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mM ♦

mM ♦

Fructose-Sucrose-

Kestosi Nystosi

Leaves

—f—

seudomonas mosselii ACO-140^-

Leaves

—f— Stem

Fructose Sucrose

Kestose Nystose

mM ♦

Fructose Sucrose

Kestos Nystos

mM ♦

'flcinetobacter calcoaceticus ACO-4^-

mM ♦

f ♦ »

Leaves

» « 4»

'Rhizobium daejeonense ACO-34A •

—*—

Leaves

Azospirillum brasilense C

Fructose Sucrose

Kestose Nystose

Fig. 2 - Thin layer chromatography (TLC) analysis of fructan exohydrolase and fructan:fructan 1-fructosyltransferases (1-FF1) enzymatic activity in extracts from leaves, stems, and root tissue of in vitro-cultured Agave americana plantlets inoculated with (A) Pseudomonas mosselli strain ACO-140, (B) Acinetobacter calcoaceticus strain ACO-40, (C) Rhizobium daejeonense strain ACO-34A, and (D) Azospirillum brasilense Cd. The markers represent the mobility of fructose, sucrose, kestose, and nystose.

437 Nevertheless, Tukey's test showed that the inulin concentra-

438 tion was not significantly different between the uninoculated

439 plants and KNO3 fertilized plants. This demonstrates the bio-

440 logical potential of the agave species to biosynthesize various

441 types of metabolites such as sugars despite unfavourable

442 growth conditions.54 In the roots, the uninoculated plants had

443 a greater inulin concentration than the plants inoculated with

444 the various PGPB.

445 Significant differences (p <0.05) in the sucrose content of

446 A. americana leaves were observed among treatments. Plants

447 inoculated with the PGPB strains had a greater sucrose con-

448 centration than the uninoculated plants and those treated

449 with KNO3. In the plant roots, no significant difference was

450 observed among treatments for sucrose concentration.

451 Similarly, the concentration of glucose and fructose in the

452 leaves was higher in plants treated with the R. daejeonense

strain AC0-34A and P. mosselii strain AC0-140, indicated by 453

Tukey's test (p <0.05). The glucose concentration in the roots 454

was significantly higher in plants inoculated with the R. dae- 455

jeonense strain AC0-34A, and the fructose concentration was 456

higher in plants inoculated with the A. calcoaceticus strain 457

AC0-40 compared to the rest of the treatments. These results 458

indicate that fructan synthesis is different in different tissues, 459

and with respect to the inoculated bacteria (Fig. 2). The sugar 460

content in agave plants was different amounts for plants of 461

different physiological ages.55 Cedeno11 reported that young 462

A. tequilana plants had higher levels of free monosaccharides 463

(glucose, fructose), than adult plants that accumulated fructan 464

from 8 to 12 years. The evaluation of sucrose, fructose and glu- 465

cose is important because Trevisan et al.56 reported that these 466

sugars act on the metabolism of fructans, such as kestose 467

and nystose. The concentration of fructose and glucose in the 468

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Table 4 - Effect of inoculation with plant growth-promoting bacteria on sugar accumulation in leaves and roots of Agave americana L.

Treatment Inulin Sucrose Glucose Fructose Inulin Sucrose Glucose Fructose

(mgg~ _1) Leaves (mg ;g-1) Root

Pseudomonas mosselii 0.99 A' 1.04 AB 1.97 A 0.561 A 0.35 D 0.17 A 0.02 D 0.031 B

ACO-140

Acinetobacter calcoaceticus 0.23 C 1.50 A 0.78 BC 0.054 C 0.84 B 0.16 A 0.19 B 0.040 A

ACO-40

Rhizobium daejeonense 1.09 A 1.59 A 1.43 A 0.856 A 0.76 BC 0.23 A 0.35 A 0.033 B

ACO-34A

Azospirillum brasilense Cd KNO3-N 0.28 B 0.77 BC 0.91 BC 0.217 BC 0.43 E 0.15 A 0.08 C 0.032 B

0.44 AB 0.68 BC 0.63 C 0.131 BC 0.28 E 0.14 A 0.03 D 0.033 B

Uninoculated 0.63 AB 0.75 BC 0.84 BC 0.140 BC 0.93 A 0.27 A 0.08 C 0.012 C

MSD£ (p <0.05) 0.740 0.726 0.628 0.318 0.022 0.146 0.108 0.045

* Mean values of four replicates. Means followed by the £ MSD, minimum significant difference. same letter do not show significant difference (p <0.05).

stems results from the active hydrolysis of fructans by fruc-tanexohydrolase. Sucrose is biosynthesized via crassulacean acid metabolism in A. americana plants. This disaccharide is a precursor to the synthesis of fructans and is hydrolysed by the action of vacuolar invertase, generating glucose and fructose.57,58 The 1-FFT enzyme subsequently converts the fructose moiety to 1-kestose, which is synthesized by the 1-SST enzyme, causing glucose to be released.59 1-kestose is the precursor of all fructans.33 Detection of neo-kestose indicated 6G-FFT enzyme activity.60,61 Fructans with degrees of polymerization DP >4 that were found in stem samples from plantlets inoculated with the P. mosselii strain ACO-140 could be the result of microbial fructosyltransferase activity involved in microbial fructan levan, inulin, or fructo-oligosaccharide biosynthesis.

It is also worth noting that the concentration of inulin and other sugars was increased in plantlets inoculated with the R. daejeonense strain ACO-34A and P. mosselii strain ACO-140 primarily in the leaves but not in the roots (Table 4). In another study, bacterial endophytes isolated from A. tequilana leaves showed the capacity for nitrogen fixation, auxin production, and phosphate solubilization and also increased inulin production.7

Conclusions

The R. daejeonense strain ACO-34A, A. calcoaceticus strain ACO-40, and P. mosselii strain ACO-140 showed potential as PGPB due to their phenotypic characteristics as well as their capacity for nitrogen fixation, phosphate solubilization, and IAA biosynthesis. Additional studies are necessary to evaluate the use of these bacteria for commercial application in A. americana culture to improve its growth and development and, most importantly, to increase its fructan and inulin contents.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

We thank the Laboratory of Ecology Genomics (CCG-UNAM) 501

for their technical assistance. The project was funded by 502 Tecnológico Nacional de México (TNM, Mexico) (5665.15-P and Q3 503

5663.15-P) from the project 'Infraestructura 251805' from the 504

Consejo Nacional de Ciencia y Tecnología (CONACyT, Mexico). 505

Appendix A. Supplementary data

Supplementary data associated with this article can be found, 506

in the online version, at doi:10.1016/j.bjm.2016.04.010. 507

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