Applied Soil Ecology 129 (2018) 84–93

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Applied Soil Ecology

journal homepage: www.elsevier.com/locate/apsoil

Dry micro-polymeric inoculant of Azospirillum brasilense is useful for T

producing mesquite transplants for reforestation of degraded arid zones
⁎
E. Jairo Gonzaleza,b,1, Juan-Pablo Hernandeza,b,c, Luz E. de-Bashana,c,d, Yoav Bashana,c,d,
a
Environmental Microbiology Group, Northwestern Center for Biological Research (CIBNOR), Av. IPN 195, La Paz B.C.S. 23096, Mexico
b
Environmental Microbiology, Biology Program, El Bosque University, No. 131 A, Cra. 9 #131a2, Bogota, Colombia
c
The Bashan Institute of Science, 1730 Post Oak Court, Auburn, AL 36830, USA
d
Dept. of Entomology and Plant Pathology, 301 Funchess Hall, Auburn University, Auburn, AL 36849, USA

A R T I C LE I N FO A B S T R A C T

This study is dedicated for the memory of the Massive clear-cutting of wild stands of mesquite trees in the Mexican part of the Sonoran Desert result from high
Mexican agricultural molecular biologist demand for this wood by the charcoal industry. Consequently, there is a need to develop techniques for refor-
Alberto Mendoza Herrera (1957–2017) of The estation of this tree in the desert and maintain its natural diversity at the same time. An outdoor nursery pro-
Center of Biotechnology on Genomics of the cedure to produce mesquite transplants from diversely originated seeds for reforestation of arid zones was de-
National Polytechnical Institute of Mexico in
veloped. This procedure involved: 1) inoculation of the seedlings in the nursery with the plant growth-promoting
Reynosa, Mexico.
bacteria (PGPB) Azospirillum brasilense immobilized in dry microbeads of alginate, and 2) developing a reliable
Keywords: way to monitor plant development and aerial volume in the nursery for the entire growth period of seven months
Alginate
before transplantation. Dry microbeads containing the PGPB and maintained at room temperature were tested
Azospirillum
for survival of bacteria for up to seven months. These dry microbeads maintained sufficient population levels of
Desert restoration
Inoculants A. brasilense to inoculate the plant for the entire period. Inoculation with the PGPB enhanced all growth para-
Mesquite meters of the plants, including biomass, aerial volume, root system, and chlorophyll pigments, but not the
Plant growth-promoting bacteria auxiliary photosynthetic pigments. The PGPB was specifically identified colonizing the roots of the transplants
by fluorescent in situ hybridization for the entire growth period. Measuring a few simple parameters allowed
development of a workable model for plant growth. This model was confirmed by data obtained from sacrificed
plants whose parameters were measured directly. This study shows that outdoor nursery cultivation of in-
oculated mesquite transplants is feasible.

1. Introduction colorant (Goycoolea et al., 1995), ornamental value, recreation sites

Mesquite legume trees are globally one of the most useful trees of
arid zones. About 45 species of the genus Prosopis have significant
ecological, economic, and social roles for all human communities living
in deserts (Felker, 2009). Mesquite shrubs or trees serve as anchor tree
in the desert phenomenon of “resource islands”, improve soil fertility,
allow other species to grow under its canopy, serve as a refuge for
wildlife, and is a source of nectar for foraging insects (Bashan and de-
Bashan, 2010; Carrillo-Garcia et al., 1999; Garcia et al., 2018). Eco-
nomically, some species are used as hard wood for furniture (Felker and
Guevara, 2003), intensive charcoal production (Galindo Almanza and
Garcia Moya, 1986; Taylor, 2006), livestock feed (Felker and
Bandursky, 1979) honey production, a source of food and energy for
indigenous and poor communities (Wojtusik et al., 1993), a gum and
and shade in deserts where shade is in short supply. The latter are
difficult to quantify economically. In areas such as southwestern USA
and parts of northern Mexico where the wild trees are not commercially
harvested for wood, they are ubiquitous (Johnson and Mayeux, 1990).
The mesquite species used in this study, Prosopis articulata S. Watson
(Johnston, 1962; León de la Luz et al., 2000), is an endemic, common
species of the southern Sonoran Desert and in other parts of Mexico. It is
a thorny shrub or small tree (2–15 m tall) and serves as a climax ve-
getation of this desert. When mature, its main role is as the dominant
tree in resource island formation and, therefore, directly contributes to
the natural vegetation of these deserts and well-being of the desert. Its
role as a common source of charcoal makes it a candidate for desert
restoration (Moreno et al., 2017), a trend that gained momentum
during the last decade, using many species of desert plants (Armada

⁎
Corresponding author.
E-mail addresses: bashan@bashanfoundation.org, ybashan@cibnor.mx (Y. Bashan).
1
Current address: Laboratorio de Microbiología de Suelos, Centro de Biotecnología y Bioindustria, Corporación Colombia de Investigación Agropecuaria (Corpoica), Mosquera 250014,
Colombia.

https://doi.org/10.1016/j.apsoil.2018.04.011
Received 13 February 2018; Received in revised form 17 April 2018; Accepted 27 April 2018
0929-1393/ © 2018 Elsevier B.V. All rights reserved.
E.J. Gonzalez et al.
et al., 2014; Bacilio et al., 2006; Bashan et al., 1999, 2009a,b, 2012; de-
Bashan et al., 2010; Felker et al., 2005; Founoune et al., 2002; Gao
et al., 2002; Grandlic et al., 2008; Medina et al., 2004; Mengual et al.,
2014a,b; Puente et al., 1999; Requena et al., 2001; Toledo et al., 1995;
Valdenegro et al., 2001; Vovides et al., 2011).
Although mesquite is considered an invasive plant of grasslands in
Arizona (Gibbens et al., 1992), in Mexico it is a highly exploitable re-
source for charcoal production, mainly for export to the US market.
Consequently, large stands of wild mesquite have been removed and
have become scarce, with no re-planting efforts (Taylor, 2006). Re-
vegetation to conserve the remaining natural stands is necessary. Pro-
pagation of mesquite seedlings from seeds is a relatively slow process
and faster procedures for mesquite production were proposed, such as
rooting of mesquite cuttings (Felker and Clark, 1981; Klass et al., 1984;
Leakey et al., 1990), mini-grafting (Ewens and Felker, 2003), and tissue
cultures and clonal propagation (Batchelor et al., 1989; Buendía-
González et al., 2007; Ramawat and Nandwani, 1991). While these
methods allow rapid development of seedlings for re-forestry or in-
dustrial plantation, for ecological restoration, seed propagation that
maintains the natural diversity of the species is preferable, since natural
dissemination and colonization of pastoral land by mesquite is always
done by seeds. Any means of shortening the growth period in nurseries
and better plant development is economically preferable.
Plant growth-promoting bacteria (PGPB) of the genus Azospirillum
are motile (Bashan and Levanony, 1987), non-pathogenic bacteria
(Bashan, 1998), known to promote growth and performance of over
130 species of plants (Pereg et al., 2016), affecting the entire plant
growth, but especially enhances root growth (Bashan and Dubrovsky,
1996; Levanony and Bashan, 1989). This includes cultivating mesquite
in hydroponic growth solution (Leyva and Bashan, 2008), in small
growth cell masses (Dominguez-Nuñez et al., 2012), or root cuttings
dipped in bacteria culture (Spiekermannm et al., 1999). All these stu-
dies used inoculation without any formulation of the culture medium or
formulated into macro-beads of alginate (2–3 mm in dia.) together with
Bacillus pumilus, AM fungi, and compost in screen houses (Bashan et al.,
2009a), or in the field (Bashan et al., 2012; Lopez-Lozano et al., 2016;
Moreno et al., 2017). However, long-term greenhouse and field ex-
periments (1–E12 years) did not measure the shorter net effect of for-
mulated A. brasilense for mesquite seedling production for a duration of
about six months, common for production of transplants in nurseries.
Formulation is an essential process because this bacterium is motile,
can be easily adsorbed by soil particles (Bashan and Levanony,
1988a,b), and has weak initial adsorption to roots (Bashan et al., 1986)
that later develops into a very strong attachment process (Levanony
et al., 1989).
A synthetic inoculant made of macrobeads of alginate (2–3 mm in
dia.) for PGPB was developed over 30 years ago (Bashan, 1986a), and is
used for wastewater treatment (de-Bashan et al., 2004), agricultural
experimentation worldwide (Bashan et al., 2014, 2016; Schoebitz et al.,
2013), studies of bacteria microalgae interaction (de-Bashan and
Bashan, 2008, de-Bashan et al., 2015) and bacterial diversity studies in
mesquite roots (Galaviz et al., 2018). A derivative of this inoculant,
microbeads of 100–200 µm, was developed for use in agriculture and
environmental re-vegetation (Bashan et al., 2002) but, so far, has lim-
ited experimental experience. The usefulness of this dry inoculant for
mesquite transplants production has not been tested, as well as some of
its inherent characteristics, such as long-term survival of bacteria after
long storage at ambient temperatures.
The two hypotheses of this study were: (1) it is feasible to construct
a dry, pulverized, synthetic inoculant of A. brasilense and use it suc-
cessfully to promote growth of mesquite transplants destined for en-
vironmental restoration, and (2) it is feasible to develop a reliable
technique to measure development of mesquite in nurseries without the
need to sacrifice plants for analytical measurements. To that end, we
checked survival and quantity of bacteria in dry inoculant for up to
210 days, measured the effect of inoculation on plant parameters,
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Applied Soil Ecology 129 (2018) 84–93
presence on roots, and content of photosynthetic pigments for 210 days,
a period common for transplant production in nurseries. We also de-
veloped a method of estimating biomass and aerial volume of the plant
without the need to uproot the plant, which is appropriate for mon-
itoring mesquite planted in reforestation areas.
2. Materials and methods
2.1. Bacterial strain, growth medium, and cultivation
The plant growth-promoting bacterium Azospirillum brasilense Cd
(DSM 1843, Leibniz-Institut DMSZ, Braunschweig, Germany) was used.
The bacterium was mass cultivated in BTB-1 medium (Bashan et al.,
2011) and incubated at 36 ± 2 °C at 120 rpm for 24 h. The culture was
centrifuged at 2817g for 5 min, the supernatant was discarded, and the
bacteria re-suspended in 200 mL sterile saline solution (0.85 NaCl, w/v)
at concentration of 109 cfu·mL−1.
2.2. Formulation of dry microbead inoculant
All procedures were done under sterile conditions. The bacteria
suspension (200 mL) was mixed with 800 mL of high viscosity sodium
alginate (MP Biomedicals, Santa Ana, Ca.) at a concentration of 1.4%
and viscosity of 280 mPa. This specific combination was chosen because
spraying of alginate is easy, it forms spherical beads, is consistent, and
keeps its shape once dried (described later, Fig. 1a). The two solutions
were mixed with a stirrer for 2 min and, once homogenized, connected
to a microbeads-producing device (Bashan et al., 2002). Droplets were
formed at room temperature at a pressure of 20 psi and deposited in a
tray containing 2 L of 2% CaCl2 under agitation of 40 rpm. Poly-
merization with calcium ions created beads of 200 µm average size.
After curing for 1 h in this solution, the beads were filtered with a paper
filter, washed three times with saline solution, and transferred to BTB-1
medium for 18 h at 30 °C and 120 rpm for one period or two periods.
This step was necessary to maintain the same concentration of bacteria
in the microbead that is reduced by the polymerization process. Then,
the beads were washed again, as described above.
Once washed, the beads were transferred to a stainless steel tray
covered with paper towels and dried in a convection oven at 37 °C for
24 h. The dried bead clumps were pulverized with a hand mortar, pass
through 250 µm meshes to dust consistency, and maintained in sealed
serological flasks containing silica gel (Fig. 1b).
2.3. Counting A. brasilense in beads and quality control of the inoculant
This was done during formation of the inoculant and up to 210 days
later. For each sample, 100 mg of beads was dissolved by immersion for
∼30 min at ∼28 °C in 20 mL citrate buffer, containing (in mM): sodium
citrate (55), EDTA anhydride (30), and NaCl2 (150), and adjusted to pH
8 with NaOH. The solution was shaken at 120 rpm for 20 min and
centrifuged at 6000g; the supernatant was decanted, the pellet was
suspended in 1 mL 0.85% saline solution, serially diluted, and plated on
Nutrient Agar medium (Sigma-Aldrich, St. Louis, MO). After incubation
for 48 h at 35 °C, colony-forming units were counted. Further validation
of the viability of the inoculant were done at 120 and 210 days, using
the fluorescein diacetate method (Chrzanowski et al., 1984) and ana-
lyzed under a fluorescent microscope (Olympus BX41, Tokyo, Japan)
equipped with FITC filter (range 460–490 nm) and connected to an
image analyzer (Image Pro Plus 6.3.1.542). Verification of species
identity as A. brasilense was done by fluorescent in situ hybridization
(FISH), based on the method of Daims et al. (2005), and modified for A.
brasilense (Bashan et al., 2011, Bashan and de-Bashan, 2015), using the
specific probe Abras 1420 (Stoffels et al., 2001).
E.J. Gonzalez et al.
Fig. 1. Dry microbeads formulation of Azospirillum brasilense. (a) Individual microbeads, (b) inoculant in a sealed container used in survival trials, (c) verification of
purity of the inoculant by fluorescent in situ hybridization. (d–f) Three individual survival trials. Each analysis was done in triplicates. Curves or columns, in each
subfigure separately, denoted by different letter differ significantly by one-way ANOVA and then by Tukey’s post-hoc analysis at P < 0.05, using Statistica 10
software. Whiskers represent S.E.
2.4. Seed collection, disinfection and germination
Mesquite amargo seeds were collected from El Comitan area
(24°07′36″N, 110°25′48″W), 15 km west of La Paz, Baja California Sur,
Mexico in July 2014 from 10 mature mesquite trees randomly dispersed
to ensure plant diversity. We only used seeds that dried on the tree
inside the pods because they exhibited a high germination rate
of > 80%. Perforated pods infected with beetle larvae Acanthoscelides
obtectus (Say) were discarded. All pods collected from the different
plants were mixed together. About 1000 seeds were extracted manually
from the pods and stored at 4 °C in hermetically sealed, glass containers
with silica gel to reduce humidity (Leyva and Bashan, 2008).
Batches of 300 intact mesquite seeds were used in each experiment.
Seeds were slowly and constantly agitated (40 rpm) at room tempera-
ture (25–28 °C) in 5% sodium hypochlorite for 2 min. After decanting,
the disinfected seeds were thoroughly washed three times with sterile,
distilled water for 1 min each until the chlorine odor was eliminated.
Then, the seeds were placed in 200 mL of sterile, distilled water under
constant agitation (120 rpm) at room temperature (∼26–28 °C) for
seven hours. Seeds were later germinated in large Petri dishes on sterile
wet paper towels in the dark, at 35 ± 1 °C for three days. This tem-
perature is optimal for the mesquite species used in this study. Once
germination started, the plantlets were transferred to low light condi-
tions at 90 µmol photons·m2·s−1 until inoculation.
2.5. Inoculation procedure and soil type
Inoculation followed the recent guidelines established for such
studies (Bashan et al., 2016). After germination and appearance of one
true leaf, the roots of the seedlings were slightly rinsed with sterile
water and the dry alginate bead inoculant was adhered to the roots
(Supplementary material, Fig. S1a). Each plant was inoculated with
100 mg of this formulation containing 1.9 × 108 cfu plant−1. Each
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Applied Soil Ecology 129 (2018) 84–93
seedling was sown in a planting hole in the soil in black plastic con-
tainer (15 × 25 cm, 2 kg soil able to support mesquite growth for that
period and common in nurseries). Hand planting was preferred because
earlier studies showed 100% survival of the plantlets (Felker et al.,
1988). The soil used was highly a degraded desert alluvial haplic yer-
mosol containing (in mg kg−1 dry soil): Ca (3759); Na (790); Mg (750);
N (2300); P total (1000); orthophosphate (100); K (500); Cu (0.74); Zn
(0.77); Fe (3.85); Mn (55); total dissolved solids (12,000); conductivity
(2400 μS·cm−1), total C (35.3 g kg−1; inorganic C (8.6 g kg−1), ex-
changeable cations (20.6 mEq 100 g soil−1), at pH 6.78 (Bashan et al.,
2000; Carrillo-Garcia et al., 2000). Soil was maintained at field capacity
(irrigation of 125 mL of water per 2 kg of soil, when needed).
2.6. Setup of outdoors experiments and growth conditions in a shade house
The setup of each experiment was completely random
(Supplementary material, Fig. S2). The treatments were: (1) Plant in-
oculated with formulation containing A. brasilense Cd and alginate as a
carrier. (2) Non-inoculated plants (control). (3) Plants inoculated only
with alginate without A. brasilense Cd (control). Each treatment con-
tained 150 plants. Homogenous plants (∼8 cm height) were used for
collecting data. Data were collected from 120 detached plants (60, 120,
180, 210 days, 10 plants per treatment and 30 per sampling time) and
from 30 intact plants still growing during the experiment. To avoid
effect of space in the greenhouse after each sampling, the remaining
plants were re-organized (Supplementary material, Fig. S2). The out-
doors experiments were conducted in a shade house exposed to ambient
conditions of temperature and humidity with protection against her-
bivores, insects, gusts of wind, and reduced solar irradiation. Because of
the length of the outdoors experiments (∼7 months long each), the
temperature, humidity, and solar irradiation varied. The temperature
was an average of 30 °C and ranged between 18 and 32 °C,
reaching > 40 °C in the middle of summer (Carrillo-Garcia et al., 2000).
E.J. Gonzalez et al.
The relative humidity in the dry season was 57% (February to July) and
64% in the wet season (August to January). Solar radiation was con-
stant at ∼1000 µmol photons·m2·s−1.
2.7. Dry weight determination
Dry weight (DW) of plant segments was determined after 60, 120,
180, and 210 days. It was done after thoroughly and carefully washing
the root system. The roots were excised from the plants and placed on
paper towels. Each segment was placed in a pre-weighted paper bag,
dried at 80 °C for 8 h until no change in the weight was observed. DW
was determined on an analytical scale (Ohaus, Pioneer, Parsippany,
NJ). Biomass of the plants is the combined DW of the roots and shoots.
Biomass of uprooted plants was determined by calculating aerial
volume (described below) and density constant. Density constant
(g·cm−3) was measured by dividing average biomass of uprooted plant
by their average aerial volume in cm3. Therefore, biomass of uprooted
plant (in g) was:
B uprooted plant = Aerial volume × density constant
2.8. Aerial volume
Aerial volume of all the plants was measured at 60, 120, 180, and
210 days after planting. Aerial volume of the plants was calculated by
tree allometric equations (FAO) and by the GlobAllomeTree interna-
tional platform (http://globallometree.org, accessed February 1, 2018)
proposed for determining aerial parts of trees that are easy to measure
(Henry et al., 2013; Picard et al., 2012). In the case of mesquite, it was
done by measuring the specific volume of its internodes, using a digital
caliper (Digital Traceable® VWR, accuracy ± 0.0006 cm)
(Supplementary material, Fig. S1b). The volume of each internode in
mm3 is:
L
V= (S1 + 4 × (S2) + S3),
6 All inoculated mesquite plants had higher dry weight, either in the
where: L is the length of each internode and S is the cross section of
each segment. Cross sections were measured as:
S = π × r 2,
where r is radius of the section. The volume of the internode is mea-
sured by:
L
V= (π × r12) + 4(π × r22) + (π × r32)
6
and the total aerial volume (Vt) of a plant, in cm3, is the sum of the
volume of its internodes.
Vt = V1 + V2 + …+Vn.
2.9. Quantification of photosynthetic pigments
Pigments were analyzed by the HPLC method of Mantoura and
Repeta (1997), where leaf samples of 50 mg were collected from 10
intact plants for each treatment after 60 and 210 days of cultivation.
2.10. Presence of A. brasilense on mesquite roots during the experiment
It was done to verify that the inoculated bacteria are residing on the
root surfaces. Five roots of inoculated plants per treatment were placed
in Erlenmeyer flasks containing 50 mL distilled water and 5 glass beads
(6 mm in dia.). This assembly was constantly agitated at 200 rpm for
15 min at 30 °C (New Brunswick, series 25, Edison NJ). Roots and glass
beads were discarded and the solution was centrifuged at 9500g for
6 min. The pellet was suspended in 1 mL saline solution. Detection and
identification of A. brasilense was done by FISH, as described above.
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Applied Soil Ecology 129 (2018) 84–93
2.11. Statistical analyses
Each plant experiment was independently repeated twice, where
survival of A. brasilense in dry microbeads was performed three times.
Data from each laboratory experiment were analyzed separately. Data
from each treatment from the two independent outdoors experiments
were combined for statistical analysis. All data sets were analyzed first
by one-way ANOVA and then by Tukey’s post-hoc analysis at P < 0.05,
employing Statistica 10 software (Tibco Statistica, Palo Alto, CA).
3. Results
3.1. Formulation of synthetic inoculant of A. brasilense
The inoculant made of A. brasilense Cd and Na-alginate formulated
into microbeads of < 200 µm bead size and dry (Fig. 1a). Ten g (total
DW) of microbeads for both experiments were sealed in serological
bottles (Fig. 1b). This dry inoculant contained initially
1.9 × 109 cfu·g−1 to 1011 cfu·g−1, depending on the trial. The inoculant
was manufactured similarly three times. Each formulated batch was
maintained in a sealed container on the shelf at room temperature of
26–30 °C for different length of time, from 90 days and up to 210 days.
During these periods, two analyses were routinely done at each sam-
pling time, counting of viable cell by the FDA method and verification
that the counted bacteria are A. brasilense by FISH. The number of CFU
declined with time in all batches. In the longest test, the amount of
bacteria immediately after formulation was 1.9 × 109 cell·g dry mi-
crobead−1 declining to 6.6 × 106 cell·g dry microbead−1 after 210 days
(Fig. 1f). In the two shorter trials, the same phenomenon was observed
(Fig. 1d,e). During these periods, the inoculant was not contaminated
and only contained A. brasilense Cd (Fig. 1c).
3.2. Effect of dry synthetic inoculant of A. brasilense on mesquite seedling
growth
roots or shoot. This was apparent after 120 days (Fig. 2e) and sig-
nificantly apparent after 210 days of cultivation, when the seedlings
were of shipping size (Fig. 2a,d). In addition, inoculation created longer
and more voluminous root systems with more secondary roots (Fig. 2b,
c). When A. brasilense was washed from roots after 210 days, its pre-
sence on the roots was identified by FISH. Biomass (DW of roots and
shoot) of uprooted mesquite (Fig. 3a), as well as the biomass calculated
for intact mesquite seedlings (Fig. 3b) showed statistical difference
from 180 days of cultivation onward.
3.3. Effect of dry synthetic inoculant of A. brasilense on aerial volume of
uprooted and intact mesquite seedlings
Aerial volume was measured in uprooted mesquite seedlings
(Fig. 4b) and calculated for intact mesquite seedlings (Fig. 4a). The two
independent measuring systems yielded almost identical results, having
significantly larger aerial volume of inoculated mesquite after 180 days
of cultivation and onward, but were visible as early as 120 days of
cultivation (Fig. 2e). When the two sets of data were combined and
analyzed together, the differences remained, as for each measuring
system (Fig. 4c).
3.4. Effect of dry synthetic inoculant of A. brasilense on photosynthetic
pigments
Five photosynthetic pigments were measured after 60 and 210 days
of cultivation. Inoculation with A. brasilense Cd significantly increased
chlorophyll a and b content after 210 days of cultivation (Fig. 5a, b), but
none of the photo-protective photosynthetic pigments, violaxanthin,
lutein, and β-carotene were enhanced (Fig. 5c–e). No enhancement of
E.J. Gonzalez et al. Applied Soil Ecology 129 (2018) 84–93

Fig. 2. Effect of inoculation with A. brasilense on dry weight of roots (a) and shoots (d) of mesquite. (b, c) Photographs of the roots at 120 and 180 days. (e)
Photograph of the shoots at 120 days. Each experiment was conducted in 50 replicates, where a single plant served as a replicate. Samples for analysis at each
sampling time were taken from 10 plants and analyzed together. Curves in each subfigure separately, denoted by different letter differ significantly by one-way
ANOVA and then by Tukey’s post-hoc analysis at P < 0.05, using Statistica 10 software. Whiskers represent S.E.

any pigment was detected after 60 days of incubation (Supplementary
material, Fig. S3).
4. Discussion
To compensate for the continuous, massive harvesting of wild
mesquite trees for the charcoal industry in Mexico (Taylor, 2006), es-
tablishment of nurseries with solid protocols for growth are necessary,
if this approach is accepted by the environmental authorities. This
study presents experimental data in this direction. When the goal is
producing commercial cultivation of mesquite trees, there are faster
methods, based on selection of desired clones, than propagation from
seeds (listed in Section 1). Yet, these faster methods lose the natural
diversity of the plant, useful for environmental restoration.
Mesquite is known to respond positively to inoculation with A.
brasilense, using either liquid bacterial cultures without formulation or
macro-bead formulation (details in Introduction). While there are sev-
eral potential polymers allowing construction of bead bacterial for-
mulation (Bashan et al., 2014; Nussinovitch, 2010; Schoebitz et al.,
2013), formulations from alginate are so far the most useful for many
practical reasons (Bashan et al., 2014; Yabur et al., 2007), especially
dry formulations having extended shelf life (Bashan and Gonzalez,

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E.J. Gonzalez et al. Applied Soil Ecology 129 (2018) 84–93

Fig. 3. (a) Total biomass of uprooted mesquite. (b) Total biomass calculated for intact mesquite seedlings. Each experiment was conducted in 50 replicates, where a
single plant served as a replicate. Samples for analysis at each sampling time were taken from 10 plants and analyzed together. Data of intact plants was collected
from the same plants at different time intervals. Curves in each subfigure separately, denoted by different letter differ significantly by one-way ANOVA and then by
Tukey’s post-hoc analysis at P < 0.05, using Statistica 10 software. Whiskers represent S.E.

1999). The latter is an attractive option when used in arid soils with
limited water. This study advanced this knowledge and tested the less
studied microbead formulation. The original microbeads formulation
was limited to testing for viability and effectiveness for a few weeks on
tomato plants (Bashan et al., 2002). Our current study extended its
usefulness to seven months, a period sufficient to establish a commer-
cial nursery for mesquite without significantly losing the quantity of
live bacteria after this period. It is well established that inoculation with
Azospirillum species requires ∼106 cfu g−1 inoculant (Kapulnik et al.,
1985; Bashan, 1986b). The dry inoculant produced in this study
surpassed this threshold even after 7 months and was more than suffi-
cient at inoculation time in all trials. The faster loss of viability in the
shorter survival trials can be explained by the monthly opening of the
sealed containers, allowing more moist air to enter, compared to the
months with a long period of sealing in the longer trial.
A main concern in production of seedlings of any plant species in
nurseries destined for transplantation is the proper development of
cultivation, where seedlings are usually valued by size, the better de-
veloped, the more expensive. This is especially critical for mesquite
amargo that develops a voluminous thorny seedling, where sampling is

89
E.J. Gonzalez et al.
Fig. 4. (a) Aerial volume calculated for intact mesquite seedlings. (b) Aerial
volume in uprooted mesquite seedlings. (c) Combined aerial volume of intact
and uprooted. Each experiment was conducted in 50 replicates, where a single
plant served as a replicate. Samples for analysis at each sampling time were
taken from 10 plants and analyzed together. Data of intact plants was collected
from the same plants at different time intervals. Curves in each subfigure se-
parately, denoted by different letter differ significantly by one-way ANOVA and
then by Tukey’s post-hoc analysis at P < 0.05, using Statistica 10 software.
Whiskers represent S.E.
difficult and requires the sacrifice of large numbers of seedlings;
therefore, it is time consuming. Evaluating the size and weight of
plantation-grown mesquite trees was developed for 2 years-old (Felker
et al., 1983), ∼3 years-old (Felker et al., 1989) and large mature trees
(El Fadl et al., 1989). The analysis of these field studies was based on
90
Applied Soil Ecology 129 (2018) 84–93
regression equations that related basal stem diameter or area with ei-
ther biomass (expressed mostly as fresh weight) or canopy diameter.
Other mathematical methods to estimate the growth of the plants in
term of biomass and aerial size, based on a few simple measurements
done on intact trees were proposed and implemented (Henry et al.,
2013; Picard et al., 2012). This study aimed to develop such a method
for mesquite seedlings, based on the specific characteristics of the plant.
This calculated data was compared to data of uprooted seedlings that
were sacrificed and their exact data was taken. The method we present
here gave almost identical results for both uprooted plants and intact
plants, making it a useful tool for mesquite cultivation in nurseries.
Desert trees of several species respond to inoculation with PGPB, as
did the mesquite seedlings in our study. These PGPB probably use di-
verse mechanisms for promoting growth of plants (Lugtenberg and
Kamilova, 2009). Specifically, for desert trees, these mechanisms in-
clude nitrogen fixation in desert mangroves (Bashan et al., 1998; Toledo
et al., 1995), improving development of lateral roots of cacti (Bashan
et al., 1999), and enhanced nutrient uptake in the cardon cactus
(Bacilio et al., 2006). While Azospirillum spp. possess over 20 different
mechanisms to enhance plant performance (Bashan and de-Bashan,
2010), the most common is by providing a variety of growth hormones,
mainly indole-3-acetic acid (IAA) that interacts with many basic me-
tabolic pathways of the plant; one of those enhances photosynthesis in
some crops inoculated with this PGPB (Panwar et al., 1990; Tsimilli-
Michael et al., 2000), and also when this PGPB interacts with uni-
cellular Chlorella sp. (Amavizca et al., 2017; de-Bashan et al., 2002).
The same strain of PGPB used in this study enhanced all photosynthetic
pigment production in wheat (Bashan et al., 2006). This study showed
that, following inoculation, an increased in chlorophyll a and b oc-
curred, but not in auxiliary photoprotective photosynthetic pigments at
the end of the growth period (7 months). This indicates that the growth
conditions of the mesquite plant performed in this study in an outdoors
nursery, while extreme for crops, are normal for mesquite trees and that
the plants were not stressed. Naturally, when cultivation started in the
colder period to allow full development of the seedlings during the hot
summer, these effects were not apparent at the earlier evaluation time.
Even though metabolism of the inoculated plants was not specifically
evaluated, the data we found indicate a potential involvement of hor-
mones manifesting as improvement in root growth and improvement in
production of chlorophyll.
Finally, it is well established that Azospirillum spp. creates synergism
with rhizobia for many agricultural crops by enhancing nodulation and
nitrogen fixation (for review of cases, Bashan et al., 2004, Table B1).
Mesquite nodules are commonly found at depth of 100–250 cm
(Johnson and Mayeux, 1990). Yet, A. brasilense used in this study can
migrate vertically on roots to depths of at least 50 cm (Bashan and
Levanony, 1987), being passively transferred by the root tips (Bashan
and Levanony, 1989). None of this was shown for indigenous rhizobia
of mesquite inoculated with A. brasilense. Nonetheless, the possibility
that it will occur is feasible.
In summary, this study shows: (1) in production of mesquite
transplants from seeds, incorporation of alginate microbeads containing
a PGPB into the protocol is beneficial for the plants for the entire
duration of the growth period, and (2) it is possible to measure devel-
opment of the plants in long-term growth in nurseries without sacrifi-
cing part of the population, using noninvasive measurements that yield
the same data as true samplings.
Acknowledgments
At CIBNOR Mexico, we thank Manuel Moreno for statistical ana-
lysis, Blanca Lopez, Edgar Amavizca, and Francisco Choix for many
helpful discussions, Francisco Hernández for pigment analysis, Carlos
Soto for solving issues with alginate bead production, and Carlos
Palacios for helping in healthy seed selection. At CICIMAR Mexico, we
thank Gustavo Hernandez Carmona for analyzing alginate viscosity.
E.J. Gonzalez et al.
Fig. 5. Effect of inoculation with Azospirillum brasilense on pigment content of mesquite leaves after 210 days. (a) chlorophyll a, (b) chlorophyll b, (c) lutein, (d)
violaxanthin, (e) β-carotene. Samples were collected from 10 intact plants. Each HPLC analysis was done in triplicates. Columns in each subfigure separately, denoted
by different letter differ significantly by one-way ANOVA and then by Tukey’s post-hoc analysis at P < 0.05, using Statistica 10 software. Whiskers represent S.E.
Writing of this manuscript was supported by the Bashan Foundation,
USA. This is contribution 2018-026 of the Bashan Institute of Science,
USA.
Conflict of interest
None.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.apsoil.2018.04.011.
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