Selection of Cocoa Tree (Theobroma cacao Linn) Endophytic Bacteria Solubilizing Tri-Calcium Phosphate, Isolated from Seedlings Grown on Soils of Six Producing Regions of Côte d’Ivoire

Phosphorus (P) is an essential macronutrient for the growth and development of cocoa tree (Theobroma cacao Linn). Most of the soils used for cocoa farming in Cote d’Ivoire are low in phosphorus. But cocoa farmers generally have a widespread reluctance to invest in chemicals fertilizers due to high costs and environmental associated risk. Phosphate Solubilizing Bacteria (PSB) are kwon to play an important role in supply of phosphorous to plants in a sustainable manner in P deficient soils. The aim of this research was to screen the endophytic bacteria of cocoa nurseries able to solubilize tri-calcium phosphate. Seedlings of two varieties of cocoa (P7 and NA32) and seedlings of an all-comer, were grown on eighteen (18) samples soils collected in six producing regions of Cote d’Ivoire. A total of 218 endophytic bacteria were isolated and tested on the Pikovskaya’s agar medium, containing Ca3 (PO4) 2. The colonies with clear zone around the microbial growth were suspected as phosphate solubilizing bacteria. Out of 218 bacteria, 90 (41.28%) showed a clear zone around colonies after 7 days of incubation. The Phosphate Solubilization Index (PSI) ranged from 20% to 200%. Bacterial isolated from the soils of Duekoue locality showed the highest mean index of 137.67%. Five PSB (CEBSP5, CEBSP6, CEBSP7, CEBSP8, and CEBSP9) from Duekoue soils and two PSB (CEBSP12 and CEBSP13) from Soubre soils have a PSI ranged from 150% to 200%. Further study in greenhouse and in field condition will confirm the use of these PSB as biofertilizer to increase the available P content in soils, reduce environmental pollution and promote sustainable agriculture.

Abstract Phosphorus (P) is an essential macronutrient for the growth and development of cocoa tree (Theobroma cacao Linn). Most of the soils used for cocoa farming in Côte d'Ivoire are low in phosphorus. But cocoa farmers generally have a widespread reluctance to invest in chemicals fertilizers due to high costs and environmental associated risk. Phosphate Solubilizing Bacteria (PSB) are kwon to play an important role in supply of phosphorous to plants in a sustainable manner in P deficient soils. The aim of this research was to screen the endophytic bacteria of cocoa nurseries able to solubilize tri-calcium phosphate. Seedlings of two varieties of cocoa (P7 and NA32) and seedlings of an all-comer, were grown on eighteen (18) samples soils collected in six producing regions of Côte d'Ivoire. A total of 218 endophytic bacteria were isolated and tested on the Pikovskaya's agar medium, containing Ca 3 (PO 4 ) 2 . The colonies with clear zone around the microbial growth were suspected as phosphate solubilizing bacteria. Out of 218 bacteria, 90 (41.28%) showed a clear zone around colonies after 7 days of incubation. The Phosphate Solubilization Index (PSI) ranged from 20% to 200%. Bacterial isolated from the soils of Duékoué locality showed the highest mean index of 137.67%. Five PSB (CEBSP5, CEBSP6, CEBSP7, CEBSP8, and CEBSP9) from Duékoué soils and two PSB (CEBSP12 and CEBSP13) from Soubré soils have a PSI ranged from 150% to 200%. Further study in greenhouse and in field condition will confirm the use of these PSB as biofertilizer to increase the available P content in soils, reduce environmental pollution and promote sustainable agriculture.

Introduction
Phosphorus is one of the major nutrients for plant growth, and involved in the early stages of plant development [1]. It plays a key role in roots growth, tissue rigidity, formation of inflorescences and fruits [2]. It is also essential in the synthesis of living plant matter, in resistance to cold and diseases [3]. Despite its content is relatively high in soil, not all phosphorus is present in an available form for the roots [4]. Otherwise, plants are only able to assimilate phosphorus in its soluble forms primary and secondary orthophosphate ions 2 4 H PO − and 2 4 HPO − [5]. Then, to avoid deficiencies, phosphate fertilizer inputs may be recommended. But added 75% to 95% of phosphate fertilizer is precipitated by metals complexes existing in the soils [6]. In addition, these chemical fertilizers This could reduce production costs, minimize the misuse of chemical fertilizers, and increase soil fertility and crop yields over the long term [11]. In view of this property, these microorganisms are an ecological and economic interest for a sustainable agriculture. Recently, phosphate solubilizing bacteria (PSB) have attracted the attention of agriculturists for their use as biofertilizers to improve plant growth and yield [12].
In Côte d'Ivoire, 81% of the soils in the forest zone where cocoa (Theobroma cacao Linn) cultivation is practiced are low in phosphorus [13]. In addition, the cost of chemical fertilizers remains high for most farmers who are forced to resort to organic fertilizers such as poultry manure and sometimes cocoa pod compost. But this can pose a risk of plant infections with fungal agents such as Phytophthora, which causes brown pod rot [14]. Thus, it is necessary to investigate other sustainable and less expensive ecological alternatives to the chemical fertilization of cocoa. There is no data on the cocoa tree endophytic bacteria solubilizing the insoluble forms of soil phosphates into available forms for plants.
The aim of this research was to screen the endophytic bacteria of cocoa nurse-

Plants Samples
The plant material was consisted of young cocoa seedlings (Theobroma cacao Linn) from a 45 day after sowing cocoa nursery, of two varieties (P7 and NA32) and an all-comer (Tv) grown on the collected soils. The seeds of the two varieties

Bacteria Strains
A total of two hundred and eighteen (218) endophytic bacteria were isolated from the organs (roots, stems, leaves) of cocoa seedlings were tested in vitro to solubilize tri-calcium phosphate. The isolates considered as efficient were coded "CEBSP" (Cocoa Endophytic Bacteria Solubilizing Phosphate Tri-Calcium).

Soils Sampling
Soil samples were removed aseptically in the upper layers about 0 -20 cm deep under cocoa trees. Before any sampling, the dead leaves of cocoa are released.
Each sample (10 kg) of soil is obtained by mixing five samples of 2 kg of soil, taken at the four ends and at the center of a plot of at least one hectare of cocoa in production, after each sampling of materials are disinfected with alcohol.

Phosphate Solubilizing Test
The test was carried out on the solid PVK medium [15]. Before the test all bacte-

Statistical Analysis
All data obtained were analyzed using R version 3.5.1 software [17]. All data reported were means of at least three replicates. One-way analysis of variance and

Endophytic Bacteria of Cocoa Solubilizing Phosphate
Bacteria solubilizing tri-calcium phosphate Ca 3 (PO 4 ) 2 were characterized by the appearance of a clear zone around colonies on solid PVK medium after 7 days of incubation at 28˚C ± 2˚C (Figure 2). A total of 90 bacteria among 218% or 41.28% of the collection were able to solubilize the tri-calcium phosphate on solid PVK medium. The solubilization indices were ranged from 20% to 200%. Twenty-two isolates have a solubilization index greater than or equal to 100%. Five isolates (CEBSP5, CEBSP6, CEBSP7, CEBSP8, and CEBSP9) from cocoa plants grown on Duékoué soils and two isolates (CEBSP12 and CEBSP13) from Soubré soils have a solubilization index ranged from 150% to 200% (Table 1).

Comparison of the Means Indices of Tri-Calcium Phosphate Solubilization
In general, the means solubilization indices of tri-calcium phosphate of cocoa (Theobroma cacao Linn) endophytic bacteria were significantly different (p < 0.001) depending on the localities from which the soil originate. Those prove that soil composition significantly influences the ability of bacteria to solubilize phosphate.
A difference was observed between the means of the phosphate solubilization indices of cocoa endophytic bacteria from different soils of the six regions ( Figure 3). Bacteria isolated from cocoa plants grown on Duékoué soils were the most efficient and have the highest phosphate solubilization index ranged from 100% to 200% with an average index of 137.67% followed by those from Soubré and Aboisso soils. The lows solubilization indices were observed with the bacteria from Divo and Abengourou soils.
Student-Newman-Keuls test showed a significant difference (p < 0.001) between the means indices of bacteria isolated from Duékoué soils and those of Soubré, Aboisso, Daloa Abengourou, and Divo. According to this test the means with the same letter are not significantly different (Figure 3).
There was no significant difference (p = 0.0985) between the medians of phosphate solubilization indices of bacteria populations from different varieties P7, NA32 and all-comers (Figure 4(a)) on the one hand and those from different organs of cocoa plants (p = 0.4685) leaves, stems, and roots on the other hand (Figure 4(b)).

Discussion
The results obtained in vitro showed that 90 isolates in our collection showed a clear zone around the colonies on the solid PVK medium and the diameter of this zone varied from one isolate to another. This proved that these cocoa (Theobroma cacao Linn) endophytic bacteria are capable of producing enzymes to mineralize the soluble insoluble tri-calcium phosphate in primary and secondary ions orthophosphates 2 4 H PO − and  According to many researches, the solubilization of inorganic phosphate by microorganisms occurs mainly by producing organic acids such as gluconic acid, oxalic acid, citric acid, lactic acid and aspartic acid [19]. Thus, any isolate of this study surrounded by a clear zone on the PVK medium is capable to produce organic acids to mineralize the tri-calcium phosphate. However, this selection criterion is not infallible because some bacteria do not form a transparent halo in PVK solid medium, but they can solubilize tri-calcium phosphate in a liquid broth [20]. This aspect of the result can be explained by the weak diffusion in the solid medium of the acids produced by these bacteria during their growth [21].
In addition, the solubilization index of tri-calcium phosphates of cocoa endophytic bacteria vary from one soil to another. Bacteria isolated from plants These results are similar to those obtained by Amadou [21], who isolated natural phosphate solubilizing bacteria from three soils in Mali. In this case, Solubilization index for phosphate rock varied from one soil to another. This difference can be explained by the fact that the microorganisms present in a soil low in soluble phosphate will provide more energy to mineralize insoluble phosphate reserves. Therefore, these microorganisms will be equipped with an enzymatic system more efficient than other microorganisms present in a soil rich in soluble phosphorus. The study of Balliah et al. [19] revealed that the population level of PSB was varied in different rhizospheres soils. This is mainly due to the abiotic factors of the soils. This was supported by Kucey [22], who demonstrated that PSB have been found in almost all soils tested, although the number varies with soil, climatic and cropping history. This large variation in the distribution of PSB in different soils may be due to the differences in organic carbon content of the soil [23]. It has been also reported that the percentage of phosphobacteria or Rhizobacteria may be accepted by soil physical properties, organic matter content, types and fertility and the way of farming [24]. Moreover, a study by Kassin et al. [13] who made the soil diagnosis in Côte d'Ivoire, showed that Duékoué soils are poorer in soluble form of phosphorus than the soils of Soubré, Divo, Aboisso and Abengourou localities which are more or less rich in phosphorus.
No difference was found between the solubilization indices of bacterial isolates from different varieties and organs of cocoa plants. This is explained by the fact that phosphorus is assimilated in soluble form and entered in the biological processes once inside the plant. There is no data about the endophytic bacteria of cocoa solubilizing phosphate. However, endophytic bacteria isolated from the roots and stems of the carob tree (Ceratonia siliqua L.) in Morocco showed a similar activity of solubilization of tri-calcium phosphate [25]. Various species of rhizospheres' bacteria, mycorrhizal fungi, actinomycetes and algae such as cyanobacteria have also been reported to show similar activity [26]. Moreover, in Côte d'Ivoire, preliminary studies conducted by Konate et al. [27] showed that endophytic bacteria isolated from cacao plants mainly belong to the genera of Bacillus, Pseudomonas and group of Actinomytes. These bacteria solubilizing insoluble phosphate are interesting potential agents for the biofertilization of cocoa. Thus, in the literature, these bacteria solubilizing phosphate, also called phosphate bacteria, are cited as a sustainable ecological solution in plant fertilization [11].

Conclusion and Perspectives
This study showed that cocoa trees (Theobroma cacao Linn) harbor endophytes bacteria capable of solubilizing tri-calcium phosphate. A total of 90 bacteria among 218% or 41.28% of the collection were able to solubilize the tri-calcium phosphate on solid PVK medium. Solubilization index varied between 20% and

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.