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Potential of Rhizobium strains isolated from Agave americana L. as Plant Growth Promoting Rhizobacteria.

Plants establish a relationship with a variety of soil microorganisms in order to obtain essential nutrients (Hayat et al., 2010). Bacteria of the genera Rhizobium, Bradyrhizobium, Sinorhizobium and Azorhizobium are known for their capacity to fix atmospheric nitrogen in a symbiotic connection with the roots of leguminous plants. However, Rhizobia also have the ability to form non-specific associative interactions with roots of other plants without forming nodules. It has been suggested that rhizobial strains may be able to produce plant growth regulators, and some of them have already been considered as plant growth promoting rhizobacteria (PGPR) (Galleguillos et al., 2000). Numerous rhizobial species have been isolated from various plants and environmental samples. For example, Rhizobium cellulosilyticum was isolated from the sawdust of Populus alba L. (Garcia-Fraile et al., 2007). Rhizobium oryzae was obtained from surface-sterilized roots of wild rice Oryza alta L. (Peng et al., 2008) and R. daejeonense was isolated from a cyanide treatment bioreactor (Zhe-Xue et al., 2005). These bacteria can establish colonies in plant roots and cause beneficial effects on plant development, and are referred to as plant-growth-promoting rhizobacterias (PGPR). Some of these PGPR are diazotrophic bacteria and have the ability to develop root associations with different plants (such as grasses), an ability which may therefore be significant for their use as biofertilizers (Santi et al., 2013).

The agave genus includes several species of economical, social, and cultural importance for people around the world. Various Agave species have even been introduced to different international regions such as Australia, Brazil, Tanzania, Kenya, Madagascar, Mexico, China, and across the Caribbean and Mediterranean regions, where it is grown to obtain various products of great importance in the food and pharmaceutical industries (Garcia-Mendoza, 2007; Ahumada-Santos et al., 2013, Yang et al., 2015).

For instance, Agave salmiana is grown in the Eastern Cape province of South Africa for the production of fibers and fructans (Smith and Figueiredo, 2012). Agave plants are of course of great relevance to Mexico, since this country is considered to be the point of origin for the evolution and diversification of the genus (Garcia-Mendoza, 2002). Approximately 163 species occur in Mexico, 123 of these being endemic to this country (Delgado-Lemus et al., 2014). One of these, Agave americana L. (Agavaceae), is an agave species found in the highlands of Chiapas, Mexico, where it is an important source of natural fiber, medicine, and fructans, as well as being used for the production of alcoholic beverages (Garcia-Mendoza, 2002). Local farmers have established plantations of A. americana to obtain sufficient raw materials for agro-industrial use. However, low water availability and nutrient content (especially phosphorus) in the soil limits growth, and consequently, plants only become mature after 5 to 7 years. An alternative already being used in other crops is to apply plant growth-promoting rhizobacteria (PGPR); however, it is necessary to assess the possible effects in A. americana specifically, in order to improve chances for the survival and growth of plantlets. PGPR are the rhizosphere bacteria which have the capacity to enhance plant growth by means of 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 more (Saharan and Nehra, 2011; Bhattacharyya and Jha, 2012).

As part of a project to promote sustainable agriculture and organic farming in Chiapas, the objective of this study was to determine the phenotypic and genotypic characteristics of four Rhizobium strains isolated from A. americana, and to evaluate their symbiotic potential as plant-growth-promoting rhizobacterias (PGPR)


Bacterial Strains

The bacterial strains ACO-R05, ACO-R19, ACO-R35, and ACO-R62 were selected from 235 strains, which were isolated previously from the rhizosphere of Agave americana. They were preselected for their potential as biofertilizers and were provided by the 'Instituto Tecnologico de Tuxtla Gutierrez'. All strains were grown in yeast extract-mannitol (YEM) medium at 28[degrees]C and then conserved at 4[degrees]C until used (Vincent, 1970). Phenotypic Characteristics of Strains

The cell morphology of the strains was analyzed using an optic microscope (Zeiss[R] PS7, Germany). The Gram reaction was done using a stain kit (Merck[R], Germany) according to the manufacturer's procedure and colony morphology was determined using cells grown on YEM agar plates at 28oC for 5 days (Toledo et al., 2003).

Bacterial growth was determined in a YEM medium with 0.5, 1.0, 2.0, 3.0 or 5.0% (w/v) NaCl and at pH 4.0, 5.0, 9.0 and 11.0 at 28[degrees]C. Tolerance to high temperatures was also tested at 37[degrees]C and 44[degrees]C. The acid/alkaline reaction was verified by spreading the inoculum on YEM plates (pH 7.0) containing 25 mg [mL.sup.-1] bromothymol blue (Vincent, 1970). The resistance to antibiotics was tested on YEM agar plates as recommended by Martinez-Romero et al. (1991). Following the same procedures, tolerance to [Al.sup.3+] and [Cu.sup.2+] was tested on a solid YEM medium. Al[Cl.sub.3]. 6[H.sub.2]O, 500 [micro]g [mL.sup.-1] and Cu[Cl.sub.2].2[H.sub.2]O, 100 [micro]g [mL.sup.-1] solutions were filtered, sterilized, and added to the sterilized YEM medium. The ACO-R05, ACO-R19, ACO-R35, and ACO-R62 strains were then added to the medium and incubated at 28[degrees]C for 5 days.

16S rDNA Gene Sequencing and Phylogenetic Analysis

The strains were grown in 2.0 ml YEM medium overnight. Total genomic DNA was extracted using the DNA isolation kit for cells and tissues (Roche[R], Basel, Switzerland), according to the manufacturer specifications. PCR was done with the universal bacterial 16S rRNA primers fD1 (5'-AGAGTTTGATCCTGGCTCAG-3') and rD1 (5'-AAGGAGGTGATCCAGCC-3'), which amplify products of approximately 1500 bases. Procedures were done as described by Weisburg etal. (1991). The PCR products were purified using the PCR product purification system kit of Roche[R] (Basel, Switzerland) and sequenced at Macrogen[R] (Seoul, Korea). Partial sequences obtained were matched against nucleotide sequences present in GenBank using the BLASTn program. The phylogenetic tree with the 16S rDNA gene sequences from the type strains was constructed by neighbour-joining (Saitou and Nei, 1987) and through a bootstrap analysis with 1000 replicates with the Tamura-Nei model (Tamura and Nei, 1993).

Measurement of Rhizobium Strains Efficiency Phosphate solubilizing capacity

The P solubilizing capacity of the strains isolated from the Agave americana and of the reference strains was tested by plate assay using Pikovskaya medium (PVK) supplemented with 1.5% agar (Nautiyal, 1999). The strains were stabbed in triplicate using sterile toothpicks. The halo and colony diameters were measured after incubating the plates at 28[degrees]C for 5 days.

Indole-3-acetic acid (IAA) production by the Rhizobium strains

The colorimetric Salkowski's method was used to determine the amounts of IAA produced by each Rhizobium strain (Ehmann, 1977). Each strain (3'109 CFU [mL.sup.-1]) was grown in triplicate in 100 mL conical flasks containing 10 mL YEM broth, supplemented with L-tryptophan (100 mg [mL.sup.-1]) at 28 [+ or -] 2[degrees]C in the dark, and shaken for 24 h. The culture medium after growth was centrifuged and the cell-free supernatant was used for IAA extractions. The extracted IAA was estimated colorimetrically at 530 nm using a standard curve with IAA obtained from Sigma Chemical (St. Louis, MO, USA). Determination of gibberellins produced by the Rhizobium Strains

The method described by Holbrook et al. (1961) was used with minor modifications in order to determine gibberellins. All strains (3'109 CFU [mL.sup.-1]) were grown in 25 mL of nutrient broth in triplicate at 28[degrees]C in the dark, and shaken for 24 h. Culture supernatants were recovered after centrifugation. 2 mL of zinc-acetate reagent was added to 15 mL supernatant. After 2 min, 2 mL 10.6% potassium ferrocyanide was added and the solution was centrifuged at 2000 rpm for 15 min. A 5 mL aliquot of the supernatant was added to 30% HCl, mixed, and then incubated in a dark room at 20[degrees]C for 75 min. 5 ml 5% HCl served as control and absorbance was read at 254 nm. Concentration of GA was calculated by preparing a standard curve using Gibberellic acid ([GA.sub.3], Hi-Media) as standard (100-1000 [micro]g [mL.sup.-1]). The experiment was done in triplicates.

Biofertilizer Test

The efficiency of PGPR strains ACO-R05, ACO-R19, ACO-R35, and ACO-R62 isolated from A. americana rhizosphere was evaluated in a biofertilization test. A. americana plantlets, previously obtained through micropropagation, were planted in polystyrene trays which contained peat-moss as substrate, and covered with a polyethylene sheet to maintain humidity and prevent dehydration. The plantlets were placed in the growth chamber at 25[degrees]C to achieve acclimatization. After two months (60 days after transplantation, DAT), the plants were transferred to pots containing peat-moss with the Fahraeus medium as nutrient (Fahraeus, 1957) and placed in the greenhouse. The plants were inoculated with each of the PGPR strains, with 2 mL of the bacterial suspension at a concentration of 1 x [10.sup.6] cel [mL.sup.-1]. Plants without inoculum, served as control. Four replicas were used per treatment, and these were arranged in a completely randomized design. The plants were grown under greenhouse conditions for 90 days. Measurements of the plants' fresh weight, the diameter of the stem, the number of leaves, and the length of the roots were carried out on the plants during the transplantation phase ([m.sub.1]) and after 90 days ([m.sup.2]) at the greenhouse. The data used for statistical analysis were the difference between [m.sub.2]-[m.sub.1] measurements.

Statistical Analysis

The effect of the four Rhizobium strains on inoculation, P-solubilizing, GA, and IAA production was determined by analysis of variance (ANOVA) and comparison of means with the Tukey test (P < 0.05) (SAS Institute, 1989).


Characteristics of the Strains

The morphological and physiological characteristics of the bacterial strains isolated from the Agave americana rhizosphere with the potential to be use as biofertilizers are shown in Table 1. The strains ACO-R05, ACO-R19, ACOR35, and ACO-R62 present the typical phenotypic characteristics of the Rhizobium genus. Cells are motile and rod-shaped, gram-negative, strictly aerobic, and of rapid growth (24-48 h). The optimum growth temperature was of 28 [+ or -] 2[degrees]C, and they presented an acidic reaction. In the YEM medium, the strains ACO-R05 and ACO-R3 5 formed circular colonies, a cream-white in colour, between 1.5 and 3.0 mm in diameter and of a glossy appearance. The strains ACO-R19 and ACO-R62 formed circular colonies of a cream-white colour, with regular borders and between approximately 1.0 and 1.5 mm in diameter and of a mucoid appearance.

The four strains can grow at 37[degrees]C but not at 44[degrees]C. The strains ACO-R19 and ACO-R62 can grow within a wide pH range between 4.0 and 11.0, in contrast to the strains ACO-R05 and ACO-R35, which only grow between a pH of 5.0 and 9.0. As for the capacity to tolerate NaCl, the strains ACO-R05 and ACO-R35 have the ability to grow within a NaCl range of 0.5 to 3.0, with the exception of the strain ACO-R19 and ACO-R62, which tolerates up to 2.0% salt. The same behavior is observed in the reference strains Rhizobium radiobacter NCPPB 2437 (Sawada et al., 1993) and R. radiobacter NF20A (Verastegui-Valdes et al., 2014). However, the strain ACO-R05 and ACO-R35 were most resistant to the tested antibiotics in comparison to the other evaluated strains, and at the same time proved to be sensitive to the Amikacin (10 [micro]g [ml.sup.-1]) and Netilmicin (20 [micro]g [ml.sup.-1]). It was also determined that all strains have the capacity to grow in the presence of [Al.sup.+3] (500 [micro]g [mL.sup.-1]) and of [Cu.sup.+2] (100 [micro]g [mL.sup.-1]).

The taxonomic position of the bacterial strains was determined according to the phylogenetic analysis performed with partial sequences of the chromosomal gene 16S rDNA (Figure 1). The 16S rDNA gene sequences of strains ACO-R05, ACO-R19, ACO-R35, and ACO-R62 have been deposited in the GenBank database under accession numbers KR232945, KR232948, KR232946, and KR232947 respectively. The four strains were affiliated with the genus Rhizobium. The ACO-R05, ACO-R19, ACO-R35, and ACO-R62 strains showed a 98.6% genetic similarity with Rhizobium radiobacter (Sawada et al., 1993). Considering the relationship with R. radiobacter (previously considered Agrobacterium radiobacter), an additional experiment was performed to determine the pathogenicity of the Rhizobium strains on sunflower plants (Helianthus annus) and found that these strains are not tumorogenic. Also, the nifH gene was amplified for all isolates with primer pair nifHF/nifH1 and the protocol described by Laguerre et al., 2001. Only, the ACO-R05 and ACO-R35 strain could amplify the gene nifH (Table 1). Similar results were reported by Verastegui-Valdes et al. (2014),

Efficiency of the Rhizobium Strain

The abilities for phosphate solubilization by plant-associated bacterial species have been the object of various research projects (Alikhani et al., 2006; Chen et al., 2006). Nevertheless, a very limited number of studies have reported strains of

Rhizobium as P-solubilizing plant-associated bacteria (Sridevi and Mallaiah, 2009). In the current study, results showed that the solubilization of inorganic phosphate by the four Rhizobium strains was accompanied by a significant drop in pH after 72 h (Table 2). The maximum P-solubilization was recorded by the strain ACO-R35 (9.2 mm) with a maximum drop in the pH to 4.3, followed by the ACO-R62 (8.0 mm), ACO-R05 (6.3 mm), and ACOR19 (5.7 mm) with a maximum drop in the pH to 5.3, 5.5, and 5.8, respectively. The P-solubilizing activity is determined by the microbial biochemical ability to produce and release organic acids, which through their carboxylic groups chelate the cations (mainly Ca) bound to phosphate converting them into the soluble forms (Chen et al., 2006).

On the other hand, the biosynthesis of phytohormones by the Rhizobium strains used in the current study showed that all differed in their capacity to produce IAA and GA (Table 2). After 72 h of growth, the IAA production was significantly higher (P < 0.05) in ACO-R35 (17.6 mg [mL.sup.-1]) as compared with the others strains, which did not show significant changes in IAA concentrations according to the Tukey test (P < 0.05). The production of gibberelins by Rhizobium strains showed that ACO-R35 produced a significantly higher amount of GA (4.5 mg [L.sup.-1]), followed by ACO-R05 (3.3 mg [L.sup.-1]), ACO-19 (2.9 mg [L.sup.-1]), and ACO-R62 (2.7 mg [L.sup.-1]). The production of IAA and gibberellins has been confirmed in numerous rhizobia species (Bottini et al., 2004; Boiero et al., 2007). Therefore, these rhizobial strains can be used as biofertilizer and improve the growth of A. americana.

Plant growth of A. americana inoculated with Rhizobium strains

The inoculation with Rhizobium strains had a positive effect on the growth parameters of A. americana plants (Table 3). The ACO-R05, ACO R19, ACO-R35, and ACO-R62 strains had a positive effect on plant fresh weight, stem diameter, number of plant, as compared to the uninoculated control plants. Plants inoculated with ACO-R35 were on average weighted at 2.34 g more than uninoculated plants at 90 days post inoculation. The stem diameter obtained with ACO-R35 differed significantly (P < 0.05) from that obtained with the rest of the treatments. Plants treated with Rhizobium strains ACO-R35, ACO-R19, and ACO-R62 experiences similar effects as to number of leaves, compared to uninoculated plants according to the Tukey test (P < 0.05). The plants inoculated with ACO-R35 showed significantly higher root length as compared to other treatments (P < 0.05). As for Rhizobium's potential as a PGPR, an important quantity of rhizobial species isolated from various types of plants and environmental samples have been reported to date. For example, Rhizobium cellulosilyticum was isolated from the sawdust of Populus alba L. (Garcia-Fraile et al, 2007). R. oryzae was obtained from surface-sterilized roots of wild rice Oryza alta L. (Peng et al., 2008), andR. daejeonense was isolated from a cyanide treatment bioreactor (Zhe-Xue et al., 2005). These Rhizobium species were isolated from nonleguminous plants or other natural sources, and have shown high potential for nitrogen fixation, as well as presenting other desirable biological features for their use as biofertilizers. Similar results have been reported by Santos et al. (2014), which concern the occurrence and diversity of diazotrophic bacteria in rhizosphere soil and root and leaf tissues of Agave sisalana plants, as well as to test for their potential for plant growth promotion. Therefore, the PGPR strains from this study could constitute an alternative as an A. americana biofertilizer, in order to improve its growth and development.


Finally, the four strains evaluated in the present study, which were isolated from the rhizosphere of the Agave americana, proved to possess the ability to solubilize phosphate and biosynthesize IAA and GA, which suggests that these strains have a high potential to be used as growth promoters in this agave species


We thank the 'Coordinacion de Posgrado en Ciencias Biologicas-UNAM'. The project was funded by 'Tecnologico Nacional de Mexico' (TNM, Mexico) and the 'Infraestructura 251805' project from the 'Consejo Nacional de Ciencia y Tecnologia' (CONACyT, Mexico). We also thank Elisabeth Tremblay for reading this manuscript.


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Luis Galdino Garcia-Perez [1], Clara Ivette Rincon-Molina [2], Victor Manuel Ruiz-Valdiviezo [2], Federico Antonio Gutierrez-Miceli [2], Nestor Hugo Cruz-Perez [2], Juan Jose Villalobos Maldonado [2] and Reiner Rincon-Rosales [2] *

[1] Centro de Ciencias Genomicas, Universidad Nacional Autonoma de Mexico, Cuernavaca, Morelos, Mexico.

[2] Laboratorio de Biologia Molecular. Instituto Tecnologico de Tuxtla Gutierrez, Carretera Panamericana km. 1080, C.P. 29050, Tuxtla Gutierrez, Mexico.

(Received: 05 May 2015; accepted: 06 July 2015)

* To whom all correspondence should be addressed. Tel: +52 (961) 6150461; E-mail:

Table 1. Characteristics that distinguish Rhizobium strain
Isolated from Agave americana L from Phylogenetically
Closely Related Taxa

Characteristics             Rhizobium isolated from A. americana

                              1        2        3        4

Gram stain                    -        -        -        -
Colony aspect on            Cream-   Cream-   Cream-   Cream-
                            white    white    white    white
YEM medium:                 Glossy   Mucoid   Glossy   Mucoid

Growth on YEM agar at:

37[degrees]C                  +        +        +        +
44[degrees]C                  -        -        -        -

Tolerance to NaCl:

  0.5 %                       +        +        +        +
  1.0 %                       +        +        +        +
  2.0 %                       +        +        +        +
  3.0 %                       +        -        +        -
  5.0 %                       -        -        -        -

Growth in YEM medium to
different pH at 28[degrees]C:

  4.0                         -        +        -        +
  5.0                         +        +        +        +
  7.0                         +        +        +        +
  9.0                         +        +        +        +
  11.0                        -        +        -        +
Acid production in YEM        +        +        +        +
  (25 [micro]g [L.sup.-1]
  bromothymol blue)

Tolerance to antibiotics (ug [mL.sup.-1]):

  Ampicillin (100)            +        +        +        +
  Amikacin (10)               -        -        -        -
  Carbencillin (20)           +        +        +        -
  Chloramphenicol (100)       +        +        +        +
  Gentamicin (10)             +        -        +        -
  Kanamycin (100)             +        +        +        +
  Netilmicin (20)             -        -        -        -

Tolerance to heavy metals (ug [mL.sup.-1]):

  [Al+.sup.3] (500)           +        +        +        +
  [Cu.sup.+2] (100)           +        +        +        +
Pathogenicity test on:        -        -        -        -
  sunflower plants
  (Helianthus annus)
nifH (b)                      +        -        +        -

Characteristics             Reference Strains

                              5        6

Gram stain                    -        -
Colony aspect on            Cream-   Cream-
                            white    white
YEM medium:                 Mucoid   Mucoid

Growth on YEM agar at:

37[degrees]C                  +        +
44[degrees]C                  -        -

Tolerance to NaCl:

  0.5 %                       +        +
  1.0 %                       +        +
  2.0 %                       +        +
  3.0 %                       -        -
  5.0 %                       -        -

Growth in YEM medium to
different pH at 28[degrees]C:

  4.0                         +        +
  5.0                         +        +
  7.0                         +        +
  9.0                         +        +
  11.0                        +        +
Acid production in YEM        +        +
  (25 [micro]g [L.sup.-1]
  bromothymol blue)

Tolerance to antibiotics (ug [mL.sup.-1]):

  Ampicillin (100)            +        +
  Amikacin (10)               -        -
  Carbencillin (20)           -        -
  Chloramphenicol (100)       +        +
  Gentamicin (10)             -        -
  Kanamycin (100)             +        +
  Netilmicin (20)             -        -

Tolerance to heavy metals (ug [mL.sup.-1]):

  [Al+.sup.3] (500)           +        +
  [Cu.sup.+2] (100)           +        +
Pathogenicity test on:        +        -
  sunflower plants
  (Helianthus annus)
nifH (b)                      -        +

(b) nifH gene was detected by PCR after isolation.

Taxon 1, ACO-R05; 2, ACO-R19; 3, ACO-R35; 4, ACO-R62;
5, Rhizobium radiobacter NCPPB 2437 (Sawada et al.,
1993) and 6, Rhizobium radiobacter NF20A (Verastegui-Valdes
et al., 2014). +, Positive; -, Negative'.

Table 2. Phosphate Solubilizing, Production IAA and GA
(Gibberellic acid) by PGPR- Rhizobium Strains Isolated
from Agave americana L.

Strain             P-solubilizingAgar     IAA (mg       GA (mg
                    (halo size (mm))    [L.sup.-1])   [L.sup.-1])

ACO-R05                6.3 C (a)           9.1 B         3.3 B
ACO-R19                  5.7 C             9.0 B         2.9 B
ACO-R35                  9.2 A            17.6 A         4.5 A
ACO-R62                  8.0 B            12.5 B         2.7 B
MSD (b) (P<0.05)         1.1810           4.6659        0.8435

(a) Mean values of three replicates. The means followed by
the same letter are not significantly different (P<0.05).

(b) MSD: Minimum Significant Difference.

Table 3. Growth Parameters for Agave americana
Plants Inoculated with Rhizobium Strains

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

ACO-R05          1.15 C         0.30 B       1.25 B       10.18 B
ACO-R19          1.38 B         0.27 B       1.75 AB      13.0 AB
ACO-R35          2.85 A         0.77 A       2.25 A       15.62 A
ACO-R62          0.75 D         0.24 B       1.75 AB     10.60 AB
Uninoculated     0.51 E         0.21 B       1.00 B       10.05 B
MSD ([yen])      0.1434         0.2027       0.9799       5.1173

* Mean values of four replicates. Means followed by the same
letter do not show a significant difference (P<0.05).

?MSD: Minimum significant difference.
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Author:Garcia-Perez, Luis Galdino; Rincon-Molina, Clara Ivette; Ruiz-Valdiviezo, Victor Manuel; Gutierrez-M
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Sep 1, 2015
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