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Procedia Engineering 138 (2016) 325 331

SYMPHOS 2015, 3rd International Symposium on Innovation and Technology in the Phosphate

Industry

Symbiotic Rhizobacteria for Improving of the Agronomic Effectiveness

of Phosphate Fertilizers
Oufdou K. a*, Bechtaoui N.a, El Alaoui A.a, Benidire L.a, Daoui K.c, Gttfert M.b
a

Laboratory of Biology and Biotechnology of Microorganisms, Faculty of Sciences Semlalia, University Cadi Ayyad, Marrakech, Morocco.
b
Technische Universitt Dresden, Institut fr Genetik, Helmholtzstr. 10, D-01069 Dresden Germany. c Institut National de la Recherche
Agronomique (INRA. Maroc)

Abstract
After nitrogen, phosphorus is the main element for plant growth. Most agricultural soils worldwide are deficient in phosphorus
and therefore require a contribution of phosphorus for the plant needs. There is a continuing need to improve soil fertility, to
increase yields and agricultural productivity. During the application of phosphate fertilizers, soluble phosphorus assimilated by
plants is rare because of its precipitation and then become unavailable to the plant. Rhizospheric bacteria including the plant
growth promoting rhizobacteria (PGPR) are of growing interest for their potential role in improving soil fertility and enhancing
an increase of crop yields and their nutrients contents. These bacteria make the insoluble phosphorus in soluble forms during the
application of phosphate fertilizers and make the phosphorus available to the plant. This work gives a review of methodology and
techniques used for the research of phosphate solubilization bacteria (PSB), their molecular characterization and the biochemical
mechanisms and genes tools involved in solubilization of phosphate and their relationships with symbiotic plants.
2015
2016 The
byby
Elsevier
Ltd.Ltd.
This is an open access article under the CC BY-NC-ND license

TheAuthors.
Authors.Published
Published
Elsevier
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review
under responsibility of the Scientific Committee of SYMPHOS 2015.
Peer-review under responsibility of the Scientific Committee of SYMPHOS 2015
Keywords: rhizobacteria; phosphate fertilizers; symbioses; phosphorus deficiency; agronomic effectiveness

* Corresponding author. Tel.: +212 668 61 08 87; fax: + 212 524 43 74 12.
E-mail address: oufdou@uca.ma

1877-7058 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the Scientific Committee of SYMPHOS 2015

doi:10.1016/j.proeng.2016.02.092

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K. Oufdou et al. / Procedia Engineering 138 (2016) 325 331

1. Introduction
Given the increasing world population (more than 9 billion in 2050 provided by the UN), significant fertilizer
requirements including phosphorus is a major challenge for sustainable food security. Phosphorus is an essential
ingredient of all fertilizers, allowed soaring agricultural yields. Agricultural are often deficient in phosphorus and
therefore need a contribution of phosphorus for the plant requirements [1,2]. According to Hinsenger [3], 5.7 billion
hectares cultivated over the world are in phosphorus deficiency conditions. There is an urgent and continuing need
to improve soil fertility, to increase yields and agricultural productivity and feed. According to the African
Development Bank, Africa fertilizer consumption is estimated at 8 kg per year per hectare against 120 kg in the
world. Forecasts project of the "African Green Revolution" is to reach 50 kg / ha during the next years.
When phosphate fertilizer is applied to the soil, it quickly reacts with the soil components [4,5]. The resulting
products are sparingly soluble phosphate compounds and phosphorus adsorbed to soil particles. The absorption
phenomena, attachment and precipitation, occur much phosphorus brought by the fertilizer and it quickly becomes
impossible to assimilate by the plant [6].
Rhizospheric bacteria including that develop in harmony with agricultural crops are of growing interest for their
potential role in improving yields [7,8]. Among these symbiotic bacteria, rhizobia also have the advantage of fixing
atmospheric nitrogen. Annual levels of at least 35 million tons of nitrogen are attributed to rhizobia-legume
symbiosis [9] corresponding to a 25-30 % of global annual terrestrial nitrogen inputs. Rhizobacteria can make the
insoluble phosphorus soluble forms during the application of phosphate fertilizers and make the phosphorus
available to the plant [8,10].
During this paper, we report solutions by applying biotechnology made symbiotic rhizobacteria that promote
plant growth and improve the effectiveness of phosphate fertilizers. We will present firstly the methodology used for
the research of phosphate solubilization bacteria (PSB), the relationships between plants and PSB and the
biochemical mechanisms and genes tools involved in solubilization of phosphate by bacteria.
2. Methods and techniques used
Rhizobial strains are generally isolated from root nodules of legume cultures grown in the fields. Nodules of
legume plants are previously disinfected with diluted sodium hypochlorite (3 or 6 depending of the seed size) for
10 min and washed several times with sterile physiological water. The nodule is crushed in a sterile tube. The
suspension is streaked on Petri dishes containing Extract-Mannitol agar (YEM) medium agar with Congo Red. After
incubation for 48 h to 72 h at 28C, colonies of rhizobia, characterized by a gluey aspect and not uptaking Congo
red, are isolated on YEM medium [11]. The rhizobia strains are purified by repeated streaking on YEM agar with
Congo red. Pure isolates are checked for their nodulation of aseptic legume seedlings.
The isolated strains are tested for their capacity to solubilize phosphate using solid and liquid media with
different sources of phosphate and nitrogen such as: NBRIY, PVK, TCP NH 4Cl or TCP KNO3 supplemented with
Ca3(PO4)2, rock phosphate or other insoluble source of phosphate [12,13,14,15]. We use the drop plate method [13];
each part is inoculated with 7 L of the inoculums on medium agar containing insoluble phosphate. Inoculated
plates are incubated at 28C in dark. When occurring, the diameter of the clearing zone (halo) surrounding the
bacterial colony, corresponding to phosphate solubilization, is measured after 3, 10 and 15 days.
As for the other PGPR solubilizing phosphate, we isolate them from rhizospheric soils on solid media containing
insoluble source of phosphate. After incubation, the colonies with halo zone are isolated and purified and then
checked by the drop plate method to solubilize phosphate as described above for rhizobia stains.
The rhizobacterial strains are then tested for their ability to solubilize insoluble phosphate under liquid culture
medium conditions, to further confirm their phosphate-solubilizing activity and to quantify the mobilized
phosphorus by each strain.
After that, the isolated bacterial strains are tested for their infectiveness and effectiveness on the plants. The
inoculum is performed by growing the rhizobacterial strain in appropriate liquid medium at 28C for 2 to 3 days to
obtain an OD of 1 at 600 nm (approximately 10 9 colonies forming units (CFU)/mL) [16,17,18].

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Seeds of tested plants are surface sterilized with diluted sodium hypochlorite for 10 min, rinsed several times with
sterile distilled water and germinated for two to four days at 25C in the dark. The germinated seedlings, selected for
uniformity, are placed during 30 min into a rhizobacterial inoculum in liquid medium in dark conditions.
The seedlings are then grown in pots. Nutrient solution is added to the trays. The trials are performed in the
greenhouse under natural climatic conditions or in preference at controlled conditions. Plants are grown either with
insoluble phosphate amendment as sole phosphorus source and inoculated by individual rhizobacterial test strains
(biofertilized plants) or with KH2PO4 (P-fertilized, non-inoculated plants) or non-inoculated plants and without any
sources of phosphorus. After the harvest of the plants, the lengths, biomasses and the phosphorus uptake are
determined in both shoots and roots of the plants in each treatment.
In addition, we can test the effect of the co-inoculation by rhizobia and other PGPR strains on the growth and the
yield of the plant and their phosphorus uptake.
3. Plant and Phosphate solubilizing bacteria
The diversification of plant protein resources and self-sufficiency in seeds widely used for human and animal
consumption are of major interest to the country's food security. In recent decades, the importance of grain legumes
and cereals in food and feed has increased significantly. The rhizobia and other PGPR are able to live in association
with legumes and non-legumes, respectively and improve their nutrient uptake and ameliorate the soil structure
[10,19]. These symbiotic bacteria are well known to promote plant growth by different direct and indirect
mechanisms including phytohormone synthesis and phosphate solubilization. Most known PGPRs with phosphate
solubilization capacity belong to rhizobia, actinomycetes and the genera Bacillus, Penibacillus, Pseudomonas,
Arthrobacter, Enterobacter, Rahnella, Serratia, Burkholderia etc [10,20,21,22]. Some of these bacteria were applied
for growth stimulation and nodulation of the plants [10,21,22].
The most important mechanisms for phosphate solubilization is the acid production by bacteria [8,23]. Bacteria
can produce acids like gluconic acid, citric acid, malic acid, oxalic acid, succinic acid, lactic acid, fumaric acid,
tartaric acid etc [10,24]. The acid production can lead to secretion of H+, anion exchange of PO 42- by acid anion, or
chelating iron, calcium and aluminum ions associated with phosphate [10].
Phosphorus can be released from organic compounds in soil by phytases which specifically cause phosphorus
release from phytic acid (phytate: a form of inositol phosphate), by non-specific phosphatases which perform
dephosphorylation of phosphoester or phosphoanyhdric bonds in organic matter and by phosphonatases and C-P
lyases that perform C-P cleavage in organophosphates [23,25]. The solubilization of phosphate by bacteria can also
be done with inorganic acids but they have generally less activity than that of organic acids at the same pH [26].
Bacteria may solubilize phosphate by other processes. Hamdali et al. [14] have reported that some rhizospheric
actinomycetes strains are able to produce siderophores chelating phosphorus, even they were not able to produce
halo zones on agar medium containing rock phosphate.
It is important to check if the in vitro phosphate solubilizing bacterium is able to benefit the plant for its
phosphorus uptake. Rhizobium and Sinorhizobium meliloti strains isolated from nodules of the legume plant
Phaseolus lunatus, exhibited good capacity to solubilize phosphorus in vitro but only Sinorhizobium could mobilize
it in the plant [27]. Valverde et al. [28] have reported that the bacterial in vitro phosphate-solubilization ability is not
always correlated to the plant phosphorus uptake. Otherwise, several studies have demonstrated that PSB increase
the growth and phosphorus uptake by the symbiotic plants [8,10,23].
The combined inoculation of atmospheric nitrogen rhizobia and other PGPR solubilizing phosphate may benefit
the symbiotic plant for phosphorus and also for nitrogen requirements, which are the two major plant nutrients [10].
Zaidi and Khan [29] have reported a beneficial effect of a PGPR alone and in combination with atmospheric fixer.
Vicia faba shoots and roots dry weights and phosphorus uptake were increased in the presence of Pseudomonas and
rhizobia (co-inoculation) in comparison to the non-inoculated plants [8].
The plant genotype could also influence on the phosphorus uptake released by PSB from insoluble phosphate.
Mandri et al. [30] have noted that the phosphorus uptake by Phaseolus vulgaris may be enhanced by selection of
both rhizobial strain and plant genotype most adapted to phosphorus limitation in soil. There are natural sources for
improving phosphorus nutrition of plants due to large genetic variation for plant traits that are associated with P

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acquisition efficiency. P-efficient genotypes integrate different traits and mechanisms that contribute to adaptation to
low P availability and are therefore more tolerant to P deficiency as compared to P-inefficient genotypes.
Adaptations to low phosphorus availability comprises more and longer adventitious roots, basal roots, increased root
hair density and length, increased organic acid exudation, more high-affinity P transporters and greater formation of
aerenchyma. Therefore, the soil volume explored by P-efficient genotypes is much larger compared to P-inefficient
genotypes [31,32].
Some plant genotypes may also themselves solubilize the insoluble phosphate by secreting phosphatases in root
exudates [33,34].
Magoual et al. [25] have demonstrated that Phaseolus vulagris plants grown under phosphorus deficiency can
increase the bacterial phytase activities in their rhizosphere and can degrade phytate which is one of the most
abundant sources of organic phosphorus in Mediterranean soils.
4. Molecular characterization of PSB
The extraction of genomic DNA of rhizobacterial strains can be carried out according to the protocol of Dhaese
et al. [35]. The genetic diversity can be analyzed through RAPD-PCR fingerprinting, a strain dependent technique
suitable in analyzing the intraspecific diversity of rhizobial populations [36,37,38]. The RAPD fingerprinting is
done in order to cluster the isolates and the representative strains will be identified by sequencing of the 16S
ribosomal gene to classify strains within a phylogenetic group and generally gives rhizobacterial classification at the
genus level [39,40,41].
The identification at species level is realized by research of housekeeping genes such as recA and atpD genes [39].
The housekeeping genes are involved in basic metabolic functions needed for maintenance of the cell and are
encoded in the chromosome. The identification at the species level is based on core gene analysis, whereas
symbiovar identification is based on nodulation genes. The nodA gene [42,43], nodB gene [44] and the nodC gene
[41,45, were used to define symbiovars. The analysis of symbiovars is as imperative as the analysis of species in
rhizobia, because rhizobial host range, legume promiscuity, coevolution and biogeography studies should be based
on symbiovars rather than on species [41,46].
The sequences obtained were compared to those held in GenBank by using the BLASTN program. Phylogenetic
analysis was conducted with MEGA version 6 [47].
Mineral phosphate-solubilizing genes have been determined in their relationship with phosphate solubilizing
bacteria. The gluconic acid biosynthesis is carried out by the glucose dehydrogenase enzyme and the co-factor,
pyrroloquinoline quinone (PQQ). The first gene involved in mineral phosphate-solubilizing, was cloned from
Erwinia herbicola [48]. Expression of this gene allowed production of gluconic acid in Escherichia coli HB101 and
conferred the ability to solubilize hydroxyapatite.
The gene gabY was identified from Pseudomonas cepacia, the expression of this gene in E. coli JM109 led to
production of gluconic acid and solubilizes mineral phosphate [49].
Pyrroloquinoline quinone biosynthesis gene pqqC is encoding the pyrroloquinoline quinone synthase C applied in
the phosphate-solubilization by bacteria. The pqq C gene is considered as a molecular marker for studying the
phylogeny and diversity of phosphate-solubilizing Pseudomonads [50]. The cofactor PQQ is also related to other
multiple plant beneficial effects: growth-promoting factor for bacteria and plants, antioxidant properties [51],
production of antimicrobial substances [52] as well as to the induction of systemic plant defenses [53].
Several acid phosphatase genes have been also isolated and characterized [54]. These cloned genes represent an
important source of material for the genetic transfer to PGPR strains. The acpA gene isolated from Francisella
tularensis expresses an acid phosphatase with optimum action at pH 6, with a wide range of substrate specificity
[55]. The genes encoding non specific acid phosphatases class A (PhoC) and class B (NapA) were isolated from
Morganella morganii [56].
Phytase genes (phy) has been cloned from Bacillus strains [57,58]. Acid phosphatase/phytase genes from E. coli
(appA and appA2 genes) have also been isolated and characterized [59,58].

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5. Conclusion
Phosphate fertilization may constitute a valuable alternative source of phosphorus, when a biotechnological
process could be applied to promote its solubilization and make phosphorus more available to the plants. The
selection of rhizobia and other PGPR strains solubilizing phosphate and able to promote plant growth in stressful
conditions will be a great challenge to improve the productivity of plants. Rhizobia and other PGPR-plant
associations diverging in their symbiotic performances (effective PSB and P-efficient genotypes), may exhibit
differences in their tolerance to phosphorus deficiency conditions, and can constitute a very important pathway to
increase soil fertility and quality and can improve the performances of phosphate fertilizers.
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