Scholarly article on topic 'Symbiotic Rhizobacteria for Improving of the Agronomic Effectiveness of Phosphate Fertilizers'

Symbiotic Rhizobacteria for Improving of the Agronomic Effectiveness of Phosphate Fertilizers Academic research paper on "Agriculture, forestry, and fisheries"

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Abstract of research paper on Agriculture, forestry, and fisheries, author of scientific article — K. Oufdou, N. Bechtaoui, A. El Alaoui, L. Benidire, K. Daoui, et al.

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.

Academic research paper on topic "Symbiotic Rhizobacteria for Improving of the Agronomic Effectiveness of Phosphate Fertilizers"

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Procedía Engineering 138 (2016) 325 - 331

Procedía Engineering

www.elsevier.com/locate/procedia

"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, Göttfert M.b

aLaboratory of Biology and Biotechnology of Microorganisms, Faculty of Sciences Semlalia, University Cadi Ayyad, Marrakech, Morocco. bTechnische Universität Dresden, Institut für 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.

© 2016 The Authors.PublishedbyElsevierLtd. 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

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

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 28°C, 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 NH4Cl 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 28°C 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 28°C for 2 to 3 days to obtain an OD of 1 at 600 nm (approximately 109 colonies forming units (CFU)/mL) [16,17,18].

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 25°C 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 PO42- 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

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].

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.

References

[1] M.D.A. Bolland, K.H.M. Siddique, R.F. Brennan, Grain yield responses of faba bean (Viciafaba L.) to applications of fertilizer phosphorus

and zinc, Austr. J. Exp. Agric. 40 (2000) 849-857.

[2] J. Matula, Relationship between phosphorus concentration in soil solution and phosphorus in shoots of barley, Plant Soil Environ. 57 (2011)

307-314.

[3] P. Hinsinger Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review, Plant Soil 237

(2001) 173-195.

[4] M.A. Whitelaw, Growth promotion of plants inoculated with phosphate solubilizing fungi, Adv. Agron. 69: 99-151.

[5] P. Gyaneshwar, G.N. Kumar, L.J. Parekh, P.S. Poole, Role of soil microorganisms in improving P nutrition of plants, Plant Soil 245 (2002)

83-93.

[6] S.A. Wakelin, R.A. Warren, P.R. Harvey, M.H. Ryder, Phosphate solubilization by Penicillium spp. closely associated with wheat roots, Biol. Fertil. Soils 40 (2004) 36-43.

[7] R. Srinivasan, A.R. Alagawadi, S. Mahesh, K.K. Meena, A.K. Saxena, Characterization of phosphate solubilizing microorganisms from

salt-affected soils of India and their effect on growth of sorghum plants [Sorghum bicolor (L.). Moench], Annals Microbiol. 62 (2012) 93105.

[8] S. Demissie, D. Muleta, G. Berecha, Effect of phosphate solubilizing bacteria on seed germination and seedling growth of faba bean (Vicia faba L.), Intern. J. Agric. Res. 8 (2013) 123-136.

[9] J.R.J. Freire, Fixa§ao do nitrogenio pela simbiose Rhizobium/Leguminosas, In: E.J.B.N. Cardoso, S.M. Tsai, M.C.P. Neves, (Cords.). Microbiologia do solo. Campinas: Sociedade Brasileira de Ciencia do Solo, 1992, pp. 121-140

[10] M.S. Khan, A. Zaidi, P.A. Wani, Role of phosphate-solubilizing microorganisms in sustainable agriculture - A review, Agronomy for Sustainable Development 27 (2007) 29-43.

[11] J.M. Vincent, A manual for the practical study of root nodule bacteria. IBP. Handbook, 15. Blackwell Scientific Publications, Ltd., Oxford, England, 1970.

[12] C.S. Nautiyal, An efficient microbiological growth medium for screening of phosphate solubilizing microorganisms. FEMS Microbiol. Lett. 170 (1999) 265-270.

[13] H.A. Alikhani, N. Saleh-Rastin, H. Antoum, Phosphate solubilizing activity of rhizobia native to Iranian soils, Plant Soil 287 (2006) 3541.

[14] H. Hamdali, B. Bouizgarne, M. Hafidi, A. Lebrihi, M. Virolle, Y. Ouhdouch, Screening for rock phosphate solubilizing actinomycetes from Moroccan phosphate mines. Applied Soil Ecology 38 (2008) 12-19.

[15] A. Sagervanshi, P. Kumari, A. Nagee, A. Kumar, Media optimization for inorganic phosphate solubilizing bacteria isolated from Anand argiculture soil, Internat. J. Life Sci. Pharma Res. 2 (2012) 24-255

[16] F. El Khalloufi, K. Oufdou, M. Lahrouni, M. Faghire, A. Peix, M.H. Ramírez-Bahena, V. Vasconcelos, B. Oudra, Physiological and antioxidant responses of Medicago sativa-rhizobia symbiosis to cyanobacterial toxins (Microcystins) exposure, Toxicon, 76 (2013) 167177.

[17] M. Lahrouni, K. Oufdou, F. El Khalloufi, M. Baz, A. Lafuente Pérez, M. Dary, E. Pajuelo, B. Oudra, Physiological and biochemical defense reactions of Vicia faba L.-Rhizobium symbiosis face to cyanotoxins chronic exposure, Environ. Sci. Pollut. Res. 20 (2013) 54055415.

[18] K. Oufdou, L. Benidire, L. Lyubenova, K. Daoui, Z.A. Fatemi, P. Schroder, Enzymes of the glutathione-ascorbate cycle in leaves and roots of rhizobia-inoculated faba bean plants (Vicia faba L.) under salinity stress, Eur. J. Soil Biol. 60 (2014) 98-103

[19] C. Santaella, M. Schoue, O. Berge, T. Heulin, W. Achouak, Role of exopolysaccharide produced by Rhizobium sp. YAS34 in the colonization of Arabidopsis thaliana and Brassica napus and biofilm formation on roots, Environ. Microbiol. 10 (2008) 2150-2163.

[20] H. Rodriguez, G.M. Rossolini, T. Gonzalez, L. Jiping, B.R. Glick, Isolation of a gene from Burkholderia cepacia IS-16 encoding a protein that facilitates phosphatase activity, Curr. Microbiol. 40 (2000) 362-366.

[21] D.K. Malik, S.S. Sindhu, Production of indole acetic acid by Pseudomonas sp.: Effect of coinoculation withMesorhizobium sp. Cicer on nodulation and plant growth of chickpea (Cicer arietinum), Physiol. Mol. Biol. Plants 17 (2011) 25-32.

[22] S. Savci, An agricultural pollutant : Chemichel fertilizer, Internat. J. Environ. Sci. Dev. 3 (2012) 77-80.

[23] H. Rodriguez, R. Fraga, T. Gonzalez, Y. Bashan, Genetics of phosphate solubilization and its potential applications for improving plant growth-promoting bacteria, Plant Soil 287 (2006) 15-21.

[24] S.A. Omar, The role of rock phosphate solubilizing fungi and vesicular arbuscular mycorrhiza (VAM) in growth of wheat plants fertilized with rock phosphate, World J. Microbiol. Biotechnol. 14 (1998) 211-219.

[25] R.T. Maougal, A. Brauman, C. Plassard, J. Abadie, A. Djekoun, J.J. Drevon, Bacterial capacities to mineralize phytate increase in the

rhizosphere of nodulated common bean (Phaseolus vulgaris) under P deficiency, Eur. J. Soil Biol. 62 (2014) 8-14.

[26] K.Y. Kim, D. Jordan, G.A. Mc Donald, Solubilization of hydroxyapatite by Enterobacter agglomerans and cloned Escherichia coli in culture medium, Biol. Fert. Soils 24 (1997) 347-352.

[27] E. Ormeno, R. Toress, R. Mayo, R. Rivas, A. Peix, E. Velazquez, D. Zuniga, Phaseolus lunatus is nodulated by phosphate solubilizing strain of Sinorhizobium melilotti in Peruvian soil, First International Meeting on Microbial Phosphate Solubilization, E. Velazquez and C. Rodriguez-Barrueco (eds), (2003), pp. 143-147.

[28] A. Valverde, J.M. Igual, E. Cervantes, Polyphasic characterization of phosphate solubilisating bacteria isolated from rhizospheric soil of the North-Eastern region of Portugal. First International Meeting on Microbial Phosphate Solubilization, E. Velazquez and C. Rodriguez-Barrueco (eds), (2007), pp. 273-276.

[29] A. Zaidi, M.S. Khan Interactive effect of rhizospheric microorganisms on growth, yield and nutrient uptake of wheat, J. Plant Nutr. 28 (2005), 2079-2092.

[30] B. Mandri, J. Drevon, A. Bargaz, K. Oufdou, M. Faghire, C. Plassard, H. Payer, C. Goulam, Interactions between common bean genotypes and rhizobia strains isolated from Moroccan soils for growth, phosphatase and phytase activities under phosphorus deficiency conditions, J. Plant Nutr. 35 (2012) 1477-1490.

[31] K. Ranathunge, E. Steude, R. Lafitte, Control of water uptake by rice (Oryza sativa L.), role of the outer part of the root, Planta 217 (2003) 193-205.

[32] L. Ramaekers, R. Remans, I.M. Rao, M.W. Blair, J. Vanderleyden, Strategies for improving phosphorus acquisition efficiency of crop plants, Field Crops Res. 117 (2010) 169-176.

[33] N.A. Tejera Garia, M. Olivera, C. Iribane, C. Lluch, Partial purification and characterization of a non-specific acid phosphatase in leaves and root nodules of Phaseolus vulgaris, Plant Physiol. Biochem. 42 (2004) 585-591.

[34] M. Nuruzzaman, H. Lambers, D.A. Bolland Mike, E.J. Veneklaas, Phosphorus benefits of different legume crops to subsequent wheat grown in different soils of Western Australia. Plant Soil 271 (2005) 175-187.

[35] P. Dhaese, H. De Greve, H. Decraemer, J. Schell, M. Van Mongatu, Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens, Nucleic Acids Res. 7 (1979) 1837-1849.

[36] G. Moschetti, A. Peluso, A. Protopapa, M. Anastasio, O. Pepe, R. Defez, Use of nodulation pattern, stress tolerance, nodC gene amplification, RAPD-PCR and RFLP-16S rDNA to discriminate genotypes of Rhizobium leguminosarum biovar viciae, Syst. Appl. Microbiol. 28 (2005) 619-631.

[37] D. Mulas, P. García-Fraile, L. Carro, M.H. Ramírez-Bahena, P. Casquero, E. Velázquez, F. González-Andrés, Distribution and efficiency of Rhizobium leguminosarum strains nodulating Phaseolus vulgaris in Northern Spanish soils: selection of native strains that replace conventional N fertilization, Soil Biol. Biochem. 43 (2011) 2283-2293.

[38] R. Rivas, E. Velázquez, J.L. Palomo, P. Mateos, P. García-Benavides, E. Martínez-Molina, Rapid identification of Clavibacter michiganensis subspecies sepedonicus using two primers random amplified polymorphic DNA (TP-RAPD) fingerprints, Eur. J. Plant Pathol. 108 (2002) 179-184.

[39] M.W. Gaunt, S.L. Turner, L. Rigottier-Gois, S.A. Lloyd-Macgilp, J.W.P. Young, Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia, Int. J. Syst. Evol. Microbiol. 51 (2001) 2037-2048.

[40] R. Rivas, A. Peix, P.F. Mateos, M.E. Trujillo, E. Martinez-Molina, E. Velázquez, Biodiversity of populations of phosphate solubilizing rhizobia thatnodulate chickpea in different Spanish soils, Plant Soil 287 (2006) 23-33.

[41] R. Rivas, M. Laranjo, E. Velázquez, P.F. Mateos, S. Oliveira, E. Martínez-Molina, Strains of Mesorhizobium amorphae and M. tianshanense carrying symbiotic genes of common chickpea endosymbiotic species constitute a novel biovar (ciceri) able to nodulate Cicer arietinum, Lett. Appl. Microbiol. 44 (2007) 412-418.

[42] M.C. Villegas, S. Rome, L. Mauré, O. Domergue, L. Gardan, X. Bailly, J.C. Cleyet-Marel, B. Brunel, Nitrogen-fixing sinorhizobia with Medicago laciniata constitute a novel biovar (bv. medicaginis) of S. meliloti, Syst. Appl. Microbiol. 29 (2006) 526-538.

[43] K.G. Nandasena, G.W. O'Hara, R.P. Tiwari, A. Willlems, J.G. Howieson, Mesorhizobium ciceri biovar biserrulae, a novel biovar nodulating the pasture legume Biserrulapelecinus L, Int. J. Syst. Evol. Microbiol. 57 (2007) 1041-1045.

[44] C. Silva, P. Vinuesa, L.E. Eguiarte, V. Souza, E. Martínez-Romero, Evolutionary genetics and biogeographic structure of Rhizobium gallicum sensu lato, a widely distributed bacterial symbiont of diverse legumes, Mol. Ecol. 14 (2005) 4033-4050.

[45] B. Mnasri, M. Mrabet, G. Laguerre, M.E. Aouani, R. Mhamdi, Salt tolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2-fixation with Phaseolus vulgaris constitute a novel biovar (bv. mediterranense) of Sinorhizobium meliloti, Arch. Microbiol. 187 (2007) 79-85.

[46] M. Faghire, B. Mandri, K. Oufdou, A. Bargaz, C. Ghoulam, M.H. Ramirez-Bahena, E. Velázquez, A. Peix, Identification at the species and symbiovar levels of strains nodulating Phaseolus vulgaris in saline soils of the Marrakech region (Morocco) and analysis of the otsA gene putatively involved in osmotolerance, Syst. Appl. Microbiol. 35 (2012) 156-164.

[47] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: Molecular evolutionary genetics analysis Version 6.0, Mol. Biol. Evol. 30 (2013) 2725-2729.

[48] A.H. Goldstein, S.T. Liu, Molecular cloning and regulation of a mineral phosphate solubilizing gene from Erwinia herbicola, Biotechnol. 5 (1987), 72-74.

[49] S. Babu-Khan, C. Yeo, W.L. Martin, M.R. Duron, R. Rogers, A. Goldstein, Cloning of a mineral phosphate-solubilizing gene from Pseudomonas cepacia. Appl. Environ. Microbiol. 61 (1995) 972-978.

[50] J.B. Meyer, M. Frapolli, C. Keel, M. Maurhofer, Pyrroloquinoline quinone biosynthesis genepqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing Pseudomonads, Appl. Environ. Microbiol. 77 (2011) 7345-7354.

[51] O. Choi, J. Kim, J.G. Kim, Y. Jeong, J.S. Moon, C.S. Park, I. Hwang, Pyrroloquinoline quinone is a plant growth promotion factor produced by Pseudomonas fluorescens B16, Plant Physiol. 146 (2008) 657-668.

[52] Y.B. Guo, J. Li, L. Li, F. Chen, W. Wu, J. Wang, H. Wang, Mutations that disrupt either the pqq or the gdh gene of Rahnella aquatilis abolish the production of an antibacterial substance and result in reduced biological control of grapevine crown gall, Appl. Environ. Microbiol. 75 (2009) 6792-6803.

[53] S.H. Han, C.H. Kim, J.H. Lee, J.Y. Park, S.M. Cho, S.K. Park, K.Y. Kim, H.B. Krishnan, Y.C. Kim, Inactivation of pqq genes of

Enterobacter intermedium 60-2G reduces antifungal activity and induction of systemic resistance, FEMS Microbiol. Lett. 282 (2008) 140146.

[54] G.M. Rossolini, S. Shipa, M.L. Riccio, F. Berlutti, L.E. Macaskie, M.C. Thaller, Bacterial non-specific acid phosphatases: physiology, evolution, and use as tools in microbial biotechnology, Cell Mol. Life Sci. 54 (1998) 833-850.

[55] T.J. Reilly, G.S. Baron, F. Nano, M.S. Kuhlenschmidt, Characterization and sequencing of a respiratory burst inhibiting acid phosphatase from Francisella tularensis, J. Biol. Chem. 271 (1996) 10973-10983.

[56] M.C. Thaller, G. Lombardi, F. Berlutti, S. Schippa, G.M. Rossolini, Cloning and characterization of the NapA acid phosphatase/phosphotransferase of Morganella morganii: identification of a new family of bacterial acid phosphatase encoding genes, Microbiology 140 (1995) 147-151.

[57] K.Y. Kim, D. Jordan, H.B. Krishnan, Expression of genes from Rahnella aquatilis that are necessary for mineral phosphate solubilization in Escherichia coli, FEMS Microb. Lett. 159 (1998) 121-127.

[58] J. Kerovuo, M. Lauraeus, P. Nurminen, N. Kalkinen, J. Apajalahti, Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis, Appl. Environ. Microbiol. 64 (1998) 2079-2085.

[59] S. Golovan, G. Wang, J. Zhang, C.W. Forsberg, Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities, Can. J. Microbiol. 46 (2000) 59-71.

[60] E. Rodriguez, Y. Han, X.G. Lei, Cloning, sequencing and expression of an Escherichia coli acid phopshatase/phytase gene (appA2) isolated from pig colon, Biochem. Biophys. Res. Comm. 257 (1999) 117-123.