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African Journal of Biotechnology Vol. 10(47), pp. 9623-9630, 24 August, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB11.641
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Biofertilization of micropropagated Agave tequilana:
Effect on plant growth and production of hydrolytic
enzymes
Sonia Ruiz1*, Lourdes Adriano1, Isidro Ovando1, Cuauhtemoc Navarro2 and Miguel Salvador1
1Centro de Biociencias, Universidad Autonoma de Chiapas, Carretera a Puerto Madero Km 2.0, Tapachula, 30700,
Chiapas, Mexico.
2AGROMOD, S. A. DE C.V. Rancho El Rocio S /N, Canton El Carmen, Frontera Hidalgo, Chiapas, Mexico.
Accepted 27 July, 2011
Three beneficial bacterial strains [Gluconoacetobacter diazotrophicus (Pal5), the diazotrophs (11B) and
Pachaz (008)] and an arbuscular mycorrhizal fungus [Glomus intraradices (AMF)] were evaluated for
their ability to enhance plant growth and the production of hydrolytic enzymes in micropropagated
Agave tequilana Weber var. Blue. Results show that the growth of the agave plants and the production
of hydrolytic enzymes in their roots were influenced by the presence of these microorganisms. AMF +
11B treatment induced the greatest fresh weight, showing significant differences with respect to other
combinations. Microscopic analysis showed dense root colonization in the AMF treated plants. Pal 5
treatment produced taller plants, indicating a better plant nitrogen nutrition and possibly phytohormone
production by Gluconoacetobacter. Treatment Pachaz 008 presented the highest values of the most
important agronomic variables, such as the diameter of the pseudo-stem. On another hand, differential
catalytic activities of the enzymes β
ββ
β-glucosidase, cellobiohydrolase and endo-1,4-β
ββ
β-D-glucanase were
detected in inoculated roots in comparison to the un-inoculated control . We offer explanations about
those results based on nutritional and hormonal relationships between the microorganisms and the
agave plantlets, as well as on the microbial mechanism to colonize the agave roots.
Key words: Bacterial and mycorrhizal inoculants, Agave plantlets, hydrolytic enzymes.
INTRODUCTION
Blue agave (Agave tequilana Weber var. Azul) is a crop
of economical, social and cultural importance in Mexico
because it is the raw material for the “tequila” production;
tequila is a national and centenary alcoholic beverage
(Granados, 1993). In the last decade, the volume of
exportation of that drink showed a sustained increment
(7.49% in average per year) (INEGI, 1997; Valenzuela,
2003; Macías and Valenzuela, 2009). For that reason,
the tequila corporations have identified the availability of
agave plants among their priorities. Several millions of
agave are planted per year in the states possessing the
denomination of origin of tequila. This necessity along
*Corresponding author. E-mail: sonia.ruizgonzalez@gmail.com.
with the restrictions in the use of pesticides in this crop
have pushed the search for pest and disease-free
propagules.Some corporations are using the
micropropagation in order to achieve their goals of mass
propagation of good quality agaves. Between 2000 and
2010, about ten millions of plantlets of A. tequilana were
produced by means of plant tissue culture only for the
new plantations of the firm SAUZA, S.A. (AGROMOD, S.
A., Pers. Comm.).
Even though micropropagated plants have many
advantageous characteristics, they have some
limitations, such as lack of capacity for an adequate
acclimatization to the field conditions (Hartmann et al.,
1997), due to physiologic changes during the in vitro
phase (Ovando et al., 2005; Ovando-Medina et al.,
2007), and for the fact that their roots do not have
9624 Afr. J. Biotechnol.
symbiotic microorganisms. In agave ex vitro plants, poor
performance have been observed during the first months
in the field in comparison to the conventional plants pro-
duced by farmers (A. tequilana is a monocot propagated,
principally through vegetative methods).
Biofertilization of micropropagated plants, using plant
growth promoting microorganisms (PGPM’s), such as
diazotrophic bacteria and mycorrhizal fungi, produces an
improved growth, development and increases the rate of
ex vitro survival (Jaizme-Vega et al., 2004; Ovando-
Medina et al., 2007).
In nature, interactions between PGPM’s and plant
growth promoting microorganism (PGPM’s) and plant
roots play an essential role in the plant health, through
different mechanisms: 1) solubilization of nutrients and
breakdown of the organic matter; 2) nitrogen fixation; 3)
root zone extension by fungi hyphae; 4) production of
phyto-hormones and 5) suppression of soil-borne
pathogens (Klibansky and González, 1996; Azcón, 2000;
Salvador et al., 2001).
In this work, we studied the effect of inoculation of
PGPM's on the growth of agave plantlets as well as on
the production of hydrolytic enzymes in their roots.With
the aim of increasing the probabilities of infection,
PGPM’s must be inoculated in the starting of the ex vitro
phase, but is not possible to assure that the roots will be
colonized; a thumb rule is that a second inoculation must
be done just before the transplant to the field. There are
several hypotheses to explain the mechanisms of
microbial colonization of root tissues, including the
production of hydrolytic enzymes by the PGPM’s (García-
Garrido et al., 2000) to degrade cell walls of epidermal
and cortex cells of the root, in a process similar to the
pathogenic infections. Therefore, in the programs of
biofertilization of micropropagated plants, an increment in
the activity of hydrolytic enzymes could be taken as an
indicator of effective PGPM colonization.
MATERIALS AND METHODS
This study was carried out during 2004 in the Soconusco region
(the most Southern site of Mexico), which has a typical tropical
climate with an intense six-month rainy period.
Plant
4800 plantlets of A. tequilana W eber var. Azul micropropagated by
the biotechnology firm AGROMOD, S. A. (Frontera Hidalgo,
Chiapas, MEXICO) were used. Mother plants for the tissue culture
procedure were sampled from fields of the company SAUZA, S. A.
(Tequila, Jalisco, MEXICO). A pre-acclimatization stage was
required, in which plantlets from laboratory were transferred to
nursery trays containing a steam-sterilized substrate (1:1 w/w mix of
peat moss and coconut fiber). Substrates were sterilized separately
injecting steam (100°C) to piles of 1 m3 during 40 min. Plants were
placed during one month in a glass greenhouse with controlled
humidity (90%) and temperature (25°C); photoperiod was provided
by the daylight. The experiment of biofertilization was carried out in
the hardening-off phase, using pre-acclimatized plants of 6 to 7 cm
of height.
Microbial inoculants
PGPM’s studied consisted of three bacterial and one fungal strains:
Gluconoacetobacter diazotrophicus (PAL 5, a c ollection strain), the
strain11B (a diazotrophic bacterium isolated from the rhizosphere of
a banana cr op) (Martínez, 2004), the strain Pachaz 008 (a
diazotrophic bacterium isolated of the rhizosphere of a papaya
crop) (Becerra, 2001), the arbuscular mycorrhizal fungus Glomus
intraradices Schenk and Smith (AMF, a collection strain). Bacterial
inocula were prepared in 1 L Erlenmeyer flasks with nutritive broth,
incubating them during 12 h on a rotatory shaker (28°C, 200 rpm)
adjusted to 1 × 108 cells/ml by dilution with sterilized distillated
water. The AMF was produced in a system of co-cultivation f ungal
spores/transgenic roots of carrot (Daucus carota) in Petri dishes
with minimal medium (Becard and Fortín, 1988); cultures were
maintained during two months in darkness to 28°C.
Biofertilization trial
The experiment was done in a plant nursery during 10 months;
plants were sowed in celled trays with a mixture of perlite,
pulverized coconut fiber and coffee husks (1:1:1 weight based) as
substrate, which was previously pasteurized. Roots of each plant
was inoculated, at the start of the experiment period (day 0), with 3
ml of the bacterial suspension and/or one squared centimeter of
AMF culture medium containing 50 spores (in average), carrot root
fragments and AMF mycelia. A factorial experiment (24) was
designed combining the presence/absence of the four inoculants,
totalizing 16 treatments with 300 randomly distributed plants for
each one (Table 1). Treatment 1 was the abs olute control and
treatments 2 to 16 contained the four inoculants.
After six months (185 days after the transplant, DAT), plants
were transferred to 500 cm3 pots containing the same substrate
than that in the previous phase; at the same time, a s econd
inoculation was realized with the same mix of microorganisms,
doubling t he inocula ( 6 ml of bacterial s uspension and/or 100 AMF
spores). All the treatments were irrigated by automated aspersion
twice a day and fertilized each month with the Steiner’s nutritive
solution (Steiner, 1984). Variables registered monthly included:
height (expressed as the length of the longest leaf), leaf number,
leaf width, fresh and dry weights. The variable stem diameter was
only measured 285 DAT. The presence of bacteria was determined
in the roots of ten plants per treatment by the method of most
probable number (MPN) and to verify the mycorrhizal colonization,
roots were stained by the technique of Phillips and Hayman (1970)
and observed under the light microscope.
Preparation of enzymatic extracts
The roots s ampled at random monthly from each of the treatments
were k ept cold during transport t o the laboratory, and then were
pulverized in a mortar with liquid nitrogen. The extraction was made
by mixing 1 g of fresh powdered root, 15% (w/w)
Ruiz et al. 9625
Table 1. Treatment matrix resulting from the combination of four microbial strains inoculated to ex vitro plants of A.
tequilana Weber var. Azul.
Treatment Microbial strain
Diazotroph Pachaz 008 Diazotroph 11B G. diazotrophicus PAL 5 G. intraradices AMF
1 - - - -
2 + - - -
3 - + - -
4 + + - -
5 - - + -
6 + - + -
7 - + + -
8 + + + -
9 - - - +
10 + - - +
11 - + - +
12 + + - +
13 - - + +
14 + - + +
15 - + + +
16 + + + +
Presence (+); absence (-) of the inoculants.
polyvinylpyrrolidone ( Sigma-Aldrich™) with 3 ml of buffer B (Tris
12.11 g/L, MgCl2 2.03 g/L, NaHCO3 0.84 g/L, β - mercaptoethanol
700 µl/L, phenylmethylsulfonylfluoride (PMSF) 0.026 g/L, Triton X-
100 3 ml/L, pH 7.0). The resulting suspension was filtered and
centrifuged at 10,000 rpm for 5 min. The supernatant was frozen
until use.
Enzyme assays
The extracts were used to determine cellulase activity comprising
the following enzymes: endo 1,4-β-D-glucanase, c ellobiohydrolase
and β-glucosidase, using the methods of Burke et al. (1998) and
Coughlan (1985), modified for each enzyme. The activity of endo
1,4-β-D-glucanase was measured using carboxylmethylcellulose as
a substrate; to determine the activity of cellobiohydrolase, Avicel PH
101 was used as substrate ,and for the activity of β-glucosidase the
substrate was ρ-nitrophenol-β-D-glucopyranoside (all reagents were
from Sigma-Aldrich™).
Determination of protein
Total protein was determined in the extracts by the Bradford
method (1976; Sigma-Aldrich reagent). For the calibration curve, a
standard protein (bovine serum albumin from Sigma-Aldrich™ to 6
g/dL) was used.
Statistical analysis
The experiment was organized in a completely randomized design
totaling 16 treatments; the final data of the morphological and
biomass variables were processed by ANOVA and the averages
were compared by the method of least significant difference
(α=0.05).
RESULTS AND DISCUSSION
The results showed that the growth of agave plants and
the production of hydrolytic enzymes in their roots were
influenced by the presence of PGPMs, since all variables
analyzed in the control treatment presented a different
behavior.
Effect of biofertilization on plant growth
The fresh and dry weight had a tendency to rise through-
out the study period; until the third month, the treatments
had very similar values, with a gradual differentiation from
the fourth month and became v ery different at sixth
months. They showed a significant increase after the
second inoculation (185 DAT), particularly in treatments
11 (11B and AMF), 9 (AMF), 8 (Pachaz 008, 11B, Pal 5),
13 (Pal 5, AMF) and 4 (Pachaz 008, 11B), although the
un-inoculated treatment (1) had a moderate increase.
The behavior described may have a double cause: the
microbial re-inoculation and the change of plants into
pots; the latter allowed more space, reducing the
mechanical root stress and increasing the penetration of
water. This explains the increase in fresh and dry weight
in non-inoculated plants. However, the fact that in
treatments 16, 14, 7, 10 and 15 no major changes were
manifested between the sixth and eighth month, indicates
that the change to the pots does not fully explain the
weight gain.
The variables of height and width of the blade showed
a clear distinction between treatments until the sixth month
9626 Afr. J. Biotechnol.
Table 2. Growth data of A. tequilana vitro plants treated with biofertilizers 236 days after transplant.
Treatment Fresh weight
(g)
Dry weight
(g) Height (cm) Width of leaf
(cm) Number of leaf Diameter of stem*
(mm)
1 107.72ab 9.69abc 26.28ab 2.97cd 8.55a 32.50bc
2 112.73ab 8.89abc 27.55ab 3.02cd 7.25abc 39.85a
3 102.09ab 7.83abc 26.08ab 3.26bcd 6.75bc 35.25abc
4 126.94ab 10.41abc 29.08ab 3.08bcd 8.00abc 36.10abc
5 107.96ab 8.12abc 26.41ab 3.44abc 7.25abc 35.70abc
6 112.10ab 9.28abc 30.90a 3.31bcd 7.50abc 31.75bc
7 83.98b 7.93abc 24.54b 2.94cd 6.875abc 35.55abc
8 136.84ab 11.50abc 28.12ab 3.09bcd 8.00abc 33.40bc
9 129.93ab 12.43a 29.06ab 2.72cd 7.75abc 32.74bc
10 91.08b 7.70abc 28.34ab 4.60a 6.75bc 37.60ab
11 157.13a 12.85a 31.10a 3.35bc 8.12abc 31.35c
12 112.59ab 9.35abc 27.52ab 3.24bcd 7.37abc 34.80abc
13 124.20ab 11.13abc 26.31ab 3.27bcd 8.25ab 33.00bc
14 85.48b 6.44c 24.22b 3.81b 6.37c 36.80abc
15 108.93ab 9.18abc 27.04ab 3.40bc 7.25abc 36.37abc
16 76.20b 6.90bc 27.06ab 2.60cd 7.37abc 34.78abc
* This variable was measured 285 days after transplant. The data are averages of 50 randomly s elected repetitions. Different letters mean statistical
difference (DMS, α = 0.05). Treatments are combinations of f our microbial strains: Diazotroph Pachaz 008, Diazotroph 11B, G. diazotrophicus and G.
intraradices.
month, with a substantial increase at the end, although
this was not immediately after the second inoculation.
The number of leaves and appearance had irregular
kinetics throughout the study, and therefore, were not
considered reliable variables for evaluating the effect of
biofertilization on the growth of micropropagated agave.
Final data of the growth variables, including the
diameter of the stem are shown in Table 2.
Treatment 11 had the highest fresh weight at the end of
the experiment (a 35-fold increase), been statistically
different from all the other combinations of strains. Plants
inoculated with G. intraradices (treatment 9) had a 28.9-
fold fresh weight increase, whilst those treated with
individual 11B (treatment 3) had a 22.7-fold fresh weight
increase. These data suggest that, in the interaction, the
main effect was caused by the AMF. The AMF-induced
increase is explained by an enhanced effective root zone
and root mass of the plant, facilitating the entry of water;
similar findings have been reported previously for
different mycorrhizal systems (Bago et al., 2000).
Treatments 11 (G. intraradices + diazotroph 11B) and 9
had the greatest dry weight data, so again the AMF can
be a promoter of increased biomass of agave plant
micropropagated in the phase of acclimatization. Some
authors report that arbuscular mycorrhizal fungi, as well
as transporting phosphorus and other minerals to the
roots, act as stimulants for greater efficiency in photo-
synthesis, so that relative fresh weight to dry weight is
usually increased in mycorrhizal plants (Gianinazzi-
Pearson et al., 1991; Bago et al., 2000). Microscopic
analysis revealed that the roots of the plants of the treat-
ments 11 and 9 were densely colonized by mycelium,
vesicles and spores at the end of the experiment.
The presence of low mycorrhizal colonization in the
control plants at the end of the experiment can be
explained by 'contamination' with atmospheric dust; as
from 185 DAT, the plants were potted in nursery condi-
tions. Another possible explanation is that the substrate,
based on coffee husks, may contain mycorrhizal fungi
spores that survived the pasteurization process and that
functioned as a natural inoculum.
Treatment 6 (diazotrophs Pachaz 008 + Pal 5) and 11
had the highest height and showed significant differences
with the other treatments. Since the length of the third
leaf represented the height of the plant, biofertilizers can
be said to induce the elongation of the leaves, which may
be due to better plant nutrition and production of active
metabolites of phytohormones by microorganisms.
In this regard, several studies have shown that
biofertilizers, either bacterial or fungal, improve the plant
nutrition by phosphate, nitrogen and trace elements. For
example, Johansen et al. (1992, 1993) showed, using
radioactive labeled phosphorus and/or nitrogen (15N and
32P), that those elements can be mobilized by AMF
hyphae into roots of Trifolium subterraneum and other
plants. It was noticed that in treatment 10 (diazotroph
Pachaz 008 + G. intraradices) plants had wider leaves,
having statistically significant differences with the other
Ruiz et al. 9627
0
2
4
6
8
10
12
14
16
18
20
0 28 56 84 118 185 236
B-glucosidase activit y (nKatal/mg protein)
Days after transplant
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
Figure 1. Biofertilization effect on the activity of the enzyme β-glucosidase in vitroplants of A. tequilana W eber var. Blue.
Treatments are c ombinations of four microbial strains: Diazotroph Pachaz 008, Diazotroph 11B, G. diazotrophicus, G.
intraradices.
treatments. The possible explanation is that such micro-
bial strains could be producing plant growth regulators
(phytohormones) of cytokinin type, since these promote
leaf expansion (Salisbury and Ross, 1995).
Several authors stated that Gluconoacetobacter
produce phytohormones; Fuentes-Ramirez et al. (1993)
stated that Acetobacter diazotrophicus (later renamed G.
diazotrophicus) is a species with high production of
auxins; Albores (2003) reports that the beneficial effect of
several Azospirillum strains on banana plants was due in
part to the production of indole-3-acetic acid auxin.
Several other microorganisms associated with plants are
capable of producing auxins, cytokinins, gibberellins and
abscisic acid (Costacurta and Vanderleyden, 1995);
however, it has not yet been verified that the strains used
in this study produce cytokinins.
In the variable number of leaves, only the whole leaves
were taken into account while throughout the experi-
mental period (285 days), the plants renovated their
leaves; in this case, the non-inoculated treatment had the
highest number of leaves at the end. Since the increase
of leaves in plants of the class Liliopsida (monocots) is
the result of apical growth of the stem, it follows that the
bacterial strain Pachaz 008 (T2) induced the growth of
agave stalk through the production of metabolites of the
auxin family. The total production of leaves (few at the
end and lost throughout the period) was correlated with
the diameter of the stem. With respect to the variable
diameter of the stem, treatment 2 (diazotroph Pachaz
008) presented the highest values. The main variable in
the selection of agave plants for planting in the field is the
diameter of the stem, due to the fact that tequila
beverage is prepared from sugars extracted from the
stem. For the later reason, it is possible that the best
inoculant is that based on diazotroph Pachaz 008. Again
the most likely explanation lies in the production of
phytohormones by the microbial strain and improved
nitrogen nutrition of the agave plant.
Effect of biofertilization on the production of
enzymes
Figure 1 shows the pattern of activity of β-glucosidase
enzyme. Treatment-dependent differential activity is
shown.
Un-inoculated control plants (treatment 1) showed no
significant variation in enzyme activity during the eight
months of monitoring. Microbial inoculated treatments 4,
6, 7, 8, 9, 10, 11, 13, 14 and 15 showed increased
activity during the first month after inoculation and subse-
quently, the activity decreased, and became, in some
cases, similar to the control plants. The activity of β-
glucosidase in treatments 2, 3 (11B), 5 (G.
diazotrophicus) and 16 (all microorganisms) increased
more slowly, because its maximum was observed two
months after inoculation and, as in the other treatments,
then declined.
The results indicate that endophytic beneficial micro-
organisms penetrate the root cortical cells probably
through a generic mechanism and that the speed
depends on the type and composition of population, since
9628 Afr. J. Biotechnol.
-200
0
200
400
600
800
1000
1200
0 28 56 84 118 185 236
Activity of cellobiohydrolase (nKatal/mg protein)
Days after transplant
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
Figure 2. Biofertilization effect on cellobiohydrolase enzyme activity in vitroplants of A. tequilana Weber var. Blue.
Treatments are combinations of four microbial strains: Diazotroph Pachaz 008, Diazotroph 11B, G. diazotrophicus, G.
intraradices.
0
100
200
300
400
500
600
700
800
900
0 28 56 84 118 185 236
Activity of glucanase (nKatal/mg protein)
Days after transplant
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16
Figure 3. Effect of biofertilization on glucanase enzyme activity in vitroplants of A. tequilana Weber var. Blue.
Treatments are combinations of four microbial strains: Diazotroph Pachaz 008, Diazotroph 11B, G.
diazotrophicus, G. intraradices.
it was observed that treatments with individual bacteria or
with all the microorganisms express the maximum
hydrolytic activity.
Figure 2 shows the pattern of activity of the enzyme
cellobiohydrolase during the experimental period. Un-
inoculated control plants (treatment 1) had an increase at
56 DAT, stabilized during the experiment and declined to
almost basal levels at 236 DAT. Treatments 8 and 13
(also had a peak of cellobiohydrolase activity up to 56
DAT. For its part, treatment 15 (11B, G. diazotrophicus
and G. intraradices) had a significantly higher value up to
84 DAT, with its peak intensity at 118 DAT. The rest of
the treatments significantly increased the production of
the enzyme in the first month of culture.
In general, cellobiohydrolase activity increased in
biofertilized plants after inoculation (28 DAT) and later
showed a second increase (around 118 DAT), which is
not associated with inoculation. This may be due to
endogenous production of the enzyme by the plant for
the generation of new roots.
Figure 3 shows the pattern of activity of the enzyme
cellobiohydrolase during the experimental period. Un-
inoculated control plants (treatment 1) showed variation
in enzyme activity during the eight months of monitoring,
although with low values, when compared with bio-
fertilized treatments. The plants of treatments 2, 3, 6, 7,
10, 11, 13 and 16 had a significant increase in the first
month, while other treatments had their maximum around
118 DAT.
The microorganism-plant interaction mediated by the
hydrolytic enzyme production of cell wall polymers
depends on the type of microorganism and/or composi-
tion of the population of inocula. However, no correlation
was found between the morphological variables and the
production of hydrolytic enzymes.
It can be seen that at the end of the experimental
period, the increased activity of the enzymes β-
glucosidase and cellobiohydrolase occurred in the roots
inoculated with Gluconoacetobacter, 11B and PACHAZ
008, either alone or combined, however, activity of both
enzymes was minimal when the two bacteria were
inoculated together so that there was perhaps an
antagonism that does not allow the development of both
microorganisms and decreased the production of
enzymes. Adriano-Anaya et al. (2006) found that G.
intraradices and G. diazotrophicus population decreased
when inoculated on roots of sorghum.
As for the glucanase enzyme activity, higher values
were obtained in treatments where the diazotrophic
bacterium 11B was present, while in treatment 9, which
contained only G. intraradices, there was no activity of
this enzyme, indicating that the fungus penetrates the
roots of the agave using hydrolytic enzymes in the cell
wall other than the glucanase, as throughout the study it
had very low activity values (Figure 3). According to
Garcia-Garrido et al. (1999, 2000) and Adriano-Anaya et
al. (2005, 2006) hydrolytic activity produced and/or
Ruiz et al. 9629
induced by G. intraradices differs according to plant
species.
This study demonstrates for the first time that the
PGPMs use enzymes that degrade the primary wall to
colonize the roots of agave, since most of the treatments
induced an activity of cellulases above that of the control
treatment, which represents the hydrolytic activity
produced by the plant cells per se.
REFERENCES
Adriano-Anaya ML, Salvador-Figueroa M, Ocampo JA, García-Romera
I (2005). Plant cell-wall degrading hydrolytic enzymes of
Gluconacetobacter diazotrophicus. Symbiosis, 40: 151-156.
Adriano-Anaya ML, Salvador-Figueroa M, Ocampo JA, García-Romera
I (2006). H ydrolytic enzyme activities in maize (Zea mays) and
sorghum (Sorghum bicolor) roots inoculated with Gluconoacetobacter
diazotrophicus and G lomus intraradices. Soil Biol. Biochem., 38: 879-
886.
Albores VJ (2003). Biofertilización del Banano Clon “Gran Enano”.
Master Thesis. University of Chiapas, Mexico, pp. 15-34.
Azcón R (2000). Papel de la simbiosis micorrízica y su interacción con
otros microorganismos rizosféricos en el crecimiento vegetal y
sostenibilidad agrícola. In: Alarcón A, Ferrera-Cerrato R (eds).
Ecología, fisiología y biotecnología de la micorriza arbuscular.
Colegio dePostgraduados / MundiPrensa. Montecillo, Mexico, pp. 1-
15.
Bago B, Azcón-Aguilar C, Shachar-Hill Y, Pfeffer PE (2000). El micelio
externo de la micorriza arbuscular como puente simbiótico entre la
raíz y su entorno. In: Alarcón A, Ferrera-Cerrato R (eds). Ecología,
fisiología y biotecnología de la micorriza arbuscular. Colegio de
Postgraduados / MundiPrensa. Montecillo, Mexico, pp. 78-92.
Bécard G, Fortín JA (1987). Early events vesicular-arbuscular
mycorrhiza formation on Ri T-DNA transformed roots. Soil Biol.
Biochem., 21: 639-644.
Becerra O (2001). Selección de cepas de Azotobacter sp. Promotoras
de crecimiento de Carica papaya variedad Maradol. Thesis.
University of Chiapas. Mexico.
Bradford MM (1976). A rapid and sensitive method f or quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal. Biochem., 72: 248-254.
Burke RM, Cairney JWG (1998). Measuring production and activity of
plant cell wall-degrading enzymes in ectomycorrhizal fungi. In: Varma
A (ed). Mycorrhiza Manual. Springer. India, pp. 173-186.
Costacurta A, Vanderleyden J (1995). Synthesis of phytohormones by
plant-associated bacteria. Crit. Rev. Microbiol., 21: 1-18.
Coughlan MP (1985). Cellulases, with comment on their production and
application. Biotechnol. Genet. Eng., 3: 39-109.
Fuentes-Ramirez LE, Jimenez-Salgado T, Abarca-Ocampo IR,
Caballero-Mellado J (1993). Acetobacter diazotrophicus,
anindoleacetic acid producing bacterium is olated from sugarcane
cultivars of México. Plant Soil, 154(2): 145-150.
García-Garrido JM, Cabello MN, García-Romera I, Ocampo JA (1992).
Endoglucanase activity in lettuce plants colonized with the vesicular-
arbuscular mycorrhizal fungus Glomus fasc iculatum. Soil Biol.
Biochem., 24: 955-959.
García-Garrido JM, Tribak M, Rejón-Palomares A, Ocampo JA, García-
Romera I (2000). Hydrolytic enzymes and ability of arbuscular
mycorrhizal fungi to colonize roots. J. Exp. Bot., 51: 1443-1448.
Gianinazzi-Pearson V, Azcón-Aguilar C (1991). Fisiología de las
micorrizas vesículo-arbusculares. In: Olivares J, Barea JM (eds).
Fijación y Movilización Biológica de Nutrientes. Vol. II. Nuevas
tendencias, CSIC. Madrid, SPAIN, pp. 175-202.
Granados SD (1993). Los Agaves en México. Universidad Autónoma
Chapingo. Mexico, pp. 1-35.
Hartmann TH, Kester DE, Davies FT, Geneve RL (1997). Plant
Propagation. Principles and Practices. 6th edition. Prentice Hall. N ew
Jersey, USA, p. 425.
INEGI (1997). El Agave Azul en el Estado de Jalisco. Instituto Nacional
9630 Afr. J. Biotechnol.
de Estadística, Geografía e Informática. Mexico, p. 69.
Jaizme-Vega MC, Rodríguez-Romero AS, Piñero Guerra MS (2004).
Potencial use of rhizobacteria from the Bacillus genus to stimulate
the plant growth of micr opropagated banana. Fruits, 59: 83-90.
Johansen A, Jakobsen I, Jensen SE (1992). Hyphal transport of 15N-
labelled nitrogen by a vesicular-arbuscular mycorrhizal fungus and its
effect on depletion of inorganic soil N. New Phytol., 122: 281-288.
Johansen A, J akobsen I, Jensen SE (1993). External hyphae of
vesicular-arbuscular mycorrhizal fungi associated with Trifolium
subterraneum L. 3. Hyphal transport of 32P and 15N. New Phytol., 124:
61-68.
Klibansky M, González L (1996). Algunas experiencias en la aplicación
de la biotecnología en la producción de biofertilizantes en Cuba. In:
Fronteras en Biotecnología y Bioingeniería. Galindo E (ed). Soc.
Mex. Biotecnol. Bioing. Mexico, pp. 143-150.
Macías A, Valenzuela AG (2009). El tequila en tiempos de la
mundialización. Comerc. Ext., 59 (6): 459-472.
Martínez ND (2004). Caracterización bioquímica e isoenzimática de
cepas de bacterias diazotróficas. Thesis. University, Chiapas,
Mexico, pp. 1-30.
Ovando I, Damon A, Ambrosio D, Albores V, Bello R, Adriano L,
Salvador M (2005). Isolation of endophytic fungi and their mycorrhizal
potential for the tropical epiphytic orchids Cattleya sk inneri, C.
aurantiaca and Brassavola nodosa. Asian J. Plant Sci., 4: 309-315.
Ovando-Medina I, Adriano-Anaya L, Chavez-Aguilar A, Oliva-Llaven A,
Ayora-Talavera T, Dendooven L, Gutierrez-Miceli F, Salvador-
Figueroa M (2007). Ex vitro Survival and Early Growth of Alpinia
purpurata Plantlets Inoculated with Azotobacter and Azospirillum.
Pakistan J. Biol. Sci., 10: 3454-3457.
Phillips JM, H ayman DS (1970). Improved procedures for clearing roots
and staining parasitic and vesicular-arbuscular mycorrhizal fungi for
rapid assesment of inf ection. Trans. Br. Mycol. Soc., 55: 158 – 161.
Salisbury FB, Ross CW (1995). Fisiología Vegetal. Grupo editorial
Iberoamérica. Mexico, 759 pp.
Salvador-Figueroa M, Adriano-Anaya ML, Herrera-López D, Albores-
Flores VJ (2001). Biofertilización microbiana de banano. In: Pohlan J
(Ed). La fruticultura orgánica en el Cauca, Colombia-un manual para
el campesinado. Shaker-Verlag. Germany, pp. 147-150.
Steiner AA (1984). The universal nutrient solution. In: Proceeding of the
Sixth International Congress on Soilless Culture. International Society
for Soilless Culture. Lunteren, The Netherlands, pp. 633-649.
Valenzuela-Zapata AG (2003). El Agave Azul, cultivo e industria de
México. 3rd Edition. MundiPrensa. Mexico, p. 215.
