Solubilization of Inorganic Phosphate by Endophytic Pseudomonas sp . from French Bean Nodules

Phosphorus (P) is one of the major essential macronutrient for plant growth. Phosphate solubilizing bacteria (PCB) is a group of heterotrophic bacteria capable of solubilizing organic and inorganic phosphorus from insoluble compounds, releasing phosphate available for plant uptake. In this study endophytic bacteria were isolated form nodules of French bean grown in different intercropping systems. Only 14 out of 158 endophytic isolates were able to solubilize tricalcium phosphate (TCP). None of them were observed to solubilize FePO4 and AlPO4. Eight isolates formed solubilization zones larger than 3 mm and were used for further test. The TCP solubilization was measured in liquid Pikovskaya medium and ranged from 14-182 μgml of soluble phosphorus. TCP solubilization was correlated with acidity and pH decreasing from 7.4 to 5.57 and 4.91 for the lowest and the highest solubilizer, respectively, were observed. The most efficient isolate MP18 was selected and identified as Pseudomonas sp. according to 16S rRNA gene analysis.


Introduction
Phosphorus (P) is an essential mineral nutrient most commonly limiting the growth of crops.The phosphate fertilization of soils has always been important because vast areas of agricultural land can be poor if the soil has a phosphate deficiency.Phosphorus is typically insoluble or poorly soluble in soils (Banerjee et al. 2010).Soil P dynamics is characterized by physicochemical (sorptiondesorption) and biological (immobilizationmineralization) processes (Khan et al. 2009).Anthropogenic addition to the soil can easily be precipitated into insoluble forms such as TCPtricalcium phosphate [Ca 3 (PO 4 ) 2 ], FePO 4 and AlPO 4 (Banerjee et al. 2010).Anthropogenic addition of P fertilizers can often have negative effects on ecosystems such as toxic effect on plants and freshwater eutrophication (Madigan & Martinko 2006).For these reasons, one very attractive approach for insoluble forms of phosphorus is the application of microorganisms.This group of bacteria can be found in the rhizosphere, at root surface and in association with roots.
Presence of plant growth promotion rhizobacteria (PGPR) can improve extent or quality of plant growth directly or indirectly (Mehta et al. 2010).PGPR are directly involved in solubilization of minerals such as phosphorus; stimulation of seedlings root length increased uptake of nitrogen; production of siderophores that chelate iron and original scientific paper/ originalni naučni članak Ratar.Povrt.51:2 (2014) 100-105 make it available to the plant root; synthesis of vitamins and phytohormones: indoleacetic acid (IAA), gibberellic acid, cytokinins, ethylene and efficient uptake of water (Meunchang et al. 2006, Mehta et al. 2010, Yasmin et al. 2012).Indirect promotion of plant growth occurs when PGPR lessen or prevent the deleterious effect of many pathogens, insects and nematodes through the production of antibiotics, lytic enzyme and hydrogen cyanide (Chaiahrn & Lumyion 2008, Yasmin et al. 2012).
The phosphate-solubilizing microorganisms (PSM) are capable of hydrolysing organic and inorganic phosphorus from insoluble compounds, releasing phosphate available for plant uptake (Ahmad et al. 2008, Chaiahrn & Lumyion 2008).Compared to fungi, phosphate solubilizing bacteria (PSB) are more effective in phosphate solubilization, which causes the wider potential uses of bacteria -they play a significant role in making phosphorus available to plants and enable the usage of less available sources of plant nutrients, such as rock phosphate.It is generally accepted that the major mechanism of mineral phosphate solubilization is the action of organic acid synthesized by soil microorganisms.Production of organic acid results in acidification of the microbial cell and its surroundings (Khan et al. 2009).
The aim of this study was to select endophytic bacteria from nodules of French bean grown in different intercropping systems with high ability of phosphate solubilization.

Materials and Methods
French bean was grown in agricultural field of Institute for Vegetable Crops, Smederevska Palanka (44 o 21'N, 20 o 57'E), central Serbia.Endophytic bacteria were collected from the nodules of French bean grown in different systems: sole crop and intercropped with red beet and radish.Nodules formation was induced by indigenous rhizobia in all intercropping systems.Nodules were sterilized and bacteria were isolated on Yeast Manitol Agar (YMA) ( Jošić et al. 2008).Pure cultures were obtained on YMA, King's B agar (KB) and Nutrient agar (NA) by restreaking several times (Djuric et al. 2011).
Taxonomic characterization of selected isolate MP18 was performed on the basis of Gram staining, biochemical properties using API 20 NE test (Biomerieaux, France) and genetic characterization of 16S rRNA gene.Total DNA were isolated and 16S rDNA amplicon were yielded using fD1/rD1 primer set (Weisburg et al. 1991, Jošić et al. 2012a).Sequence of obtained amplicon was compared to those in NCBI GenBank.

Results and Discussion
Total of 42.4% (67 out of 158) of endophytic bacteria isolated from French bean nodules belongs to indigenous rhizobia.All of 6 rhizobia that formed halo zones on PVK medium were concerned as poor TCP solubilizers, showing zones between 1 and 2 mm.Other bacterial genera were represented by 57.6% of isolates (91) and only 8.79% among them showed ability to solubilize TCP in PVK medium.Two out of 8 selected isolates were isolated from nodules of French bean grown as a sole crop and two from nodules of French bean intercropped with red beet, as shown in Table 1.Four isolates originated from nodules of French bean near radish rhizosphere.
No growth was observed on NBRIP supplemented with FePO 4 and AlPO 4 and PVK supplemented with AlPO 4 .Control strains and 3 isolates grew on PVK supplemented with FePO 4 , but no halos were observed around the colonies.Since the direct measurement of phosphate solubilization in broth assay is likely to give more reliable results than a regular plate assay, the eight phosphate-solubilizing strains were further tested for their ability to solubilize TCP in PVK broth.The values of P-solubilization zones, pH values and concentration of solubilized TCP in liquid PVK medium are shown in Table 2.
The selected isolates showed a wide range of TCP solubilization after four days: 14-182 μgml -1 .МP18, isolated from nodules of French bean intercropped with radish, showed the highest phosphate P-solubilization by MP18 was accompanied by a decrease in pH of the culture supernatants from 7.40 to 4.91.Similarly, the pH value of 4.53 was observed after 4 days of cultivation for Pseudomonas sp.Q4 strain.Similar pH decrease (to 4.8) was observed for numerous taxonomically different rhizospheric fluorescent pseudomonads (BMC) (Naik et al. 2008).Although phosphate solubilization is not necessarily correlated with acidity, a basic principle is related to the production of organic acids and the releasing of protons (Lin et al. 2006).It is correlated to our results for MP3 isolate where the lowest pH decreasing of medium (to 5.57) and the lowest TCP solubilization was recorded.The decrease in pH in relationship to release of soluble phosphates is also reported for free-living soil bacteria (Rajkumar & Freitas 2008) and rhizobia (Sridevi et al. 2007).The phosphate solubilization depends not only to the quantity of organic acid secreted, but to the structural complexity and particle size of phosphates and other mechanisms (Nautiyal et al. 2000).
Characterization of selected isolate MP18, as the most efficient, was based on biochemical characteristics obtained by API 20NE test and 16S rRNA gene sequence analysis.No fluorescent pigment production was observed on KB medium.Isolate MP18 was Gram-negative, motile, rod shaped, non-fluorescent, positive for cytochrome oxidase test and able to grow at 4°C and 42°C.Comparison with similar sequences in NCBI GenBank showed 99% similarity to several Pseudomonas sp. and allowed to characterize it as Pseudomonas sp.
The bacteria belonging to Bacillus, Achrobacter, Aerobacter, Acinetobacter, Enterobacter, Mesorhizobium, Rhizobium, Klebsiella and Micrococcus, have been reported as phosphate solubilizers.However, strains belonging to Pseudomonas, especially fluorescent strains, are considered as efficient phosphate solubilizers (Villegas & Fortin 2002).Among endophytic bacteria isolated from root nodules in various leguminous plants (alfalfa, clover, soybean, pigeon pea) are Agrobacterium, Bacillus, Pseudomonas, Burkholderia and Enterobacteria (Zakhia et al. 2006, Kan et al. 2007, Geetha et al. 2008, Li et al. 2008).The results from our study showed the most efficient phosphate solubilizing endophytic isolate MP18 belongs to Pseudomonas genera, according to phenotypic characters and 16S rRNA gene sequence.The plant growth promoting traits of different endophytic (Stajković et al. 2011) and rhizospheric Pseudomonas spp.from Serbia is already reported and most of them are able to solubilize TCP (Djuric et al. 2011, Josic et al. 2012b).Besides being vital for photosynthesis, energy transfer and formation of sugars, P also promotes nitrogen fixation in legumes (Gaur 1990).Low levels of phosphorus reduce the population of legume-nodulating microorganisms and affect rhizobia-legume symbiosis (Moreira et al. 2010).Phosphate solubilization occurred by rhizobia isolated from Crotalaria sp.(Sridevi et al. 2007).Only rhizobia able to solubilize TCP in very low rate were observed in our study.The natural simultaneous presence of rhizobia and Pseudomonas sp.MP18 in French bean nodule may be explained by low P-solubilizing capacity of indigenous rhizobia and possibility of MP18 to improve availability of phosphorus for microsymbiont and host-plant.
Numerous studies confirmed beneficial effects of legumes in dual inoculation with rhizobia and PSB.Rudresh et al. (2005) reported the increase in nodulation and biological nitrogen fixation due to synergistic effect of rhizobia and PCB comparing to their individual application.Significant response of dual inoculation was observed in chickpea crop, increasing yield and leghemoglobin content in the nodular tissues (Barea et al. 2005).The dual inoculation of endophytic strains P. chlororaphis Zong1 and Mesorhizobium sp.SQ1 cause synergistic or addictive effect and improve not only phosphate solubilization, but also siderophores production, organic acid production, IAA production and in vitro antifungal activity, compared to single inoculation (Zhao et al. 2013).

Conclusions
The selected endophytic Pseudomonas sp.MP18 isolate reached high phosphate solubilizing efficiency comparing to positive control.For the agricultural production it could be added to some microbial fertilizers and used especially on soils containing high amounts of insoluble TCP.Also, it could be suitable for organic production and other sustainable production systems.According to the obtained results, it will be further investigated for other PGP traits and synergistic effect with indigenous rhizobia.

Table 1 .
Isolates from French bean nodules able to solubilize TCP and control strains used in this study