Abstract: The ability of thirty-one fungal strains isolated from Nigerian cultivated farmland to solubilize rock phosphate and tri-calcium phosphate (TCP) was investigated. pH, titratable acidity, available phosphorus, total phosphorus and organic acid released were analysed as a measure of solubilization ability in liquid based medium containing rock phosphate and TCP. Isolated fungal species belong mainly to the genera of Aspergillus, Penicillium, Trichoderma, Fusarium, Mucor, Ovularopsis, Tritirachium and Geotrichum. Apart from Geotrichum, all the isolates were able to solubilize phosphate rock and TCP. Phosphate solubilization was accompanied by a decrease in the pH of the medium by all the strains; however, this decrease differed significantly among isolates (p<0.05). The production of fumaric, acetic, gluconic, lactic and succinic acids accompanied solubilization of TCP, while citric, fumaric, malic and tartaric acid were detected in extracts of phosphate rock medium. Significant differences (p<0.05) were observed in the type and amount of organic acids produced by the fungi species Aspergillus terreus produced the highest amount of fumaric acid (264.45 mg/100 mL in TCP medium, while A. niger produced the highest amount of malic acid (18.20 mg/100 mL) in rock phosphate medium Succinic acid was the least produced of the acids.
INTRODUCTION
Apart from nitrogen, phosphorous is one major nutrient required for healthy growth by plants. Phosphorous is naturally present in soil but not all of it is readily available for plant use as some of it is transformed into insoluble complexes with soil constituents and therefore reducing the overall availability and efficiency of soil phosphorous (Vassilev, 2002). As such, in order to maintain the amount of phosphorous available in soil for plant use, large amount of phosphorous based fertilizer is added to soil (Omar, 1998), often, the bulk of which could also be converted to insoluble form. There is therefore a need for frequent application of soluble forms of inorganic phosphorous to soil. In Nigeria, inorganic fertilizer is not within the reach of peasant farmers as they are very expensive. In view of the recent interest in environmental friendly approach to agricultural techniques, more attention is being given to bio sourcing for plant nutrients and the use of rock phosphate as a source of phosphorous for plants is receiving attention.
Natural rock phosphate has been reported to be a source of phosphorous in a wide range of soil types (Chien and Meon, 1995). The rock phosphate has to be dissolved to be available for plant use. Many soil microorganisms have been reported to solubilize inorganic phosphates (Asea et al., 1988; Illmer and Schimer, 1992; Richardson, 2001; Nahas, 2004; Chuang et al., 2006; Alikhani et al., 2006).
In fact, rock phosphate dissolution by microorganisms directly affects soil fertility (Reyes et al., 2002). Solubilization of inorganic phosphate by microorganisms involve a wide range of processes involving the secretion of organic acids, lowering of pH as a result of acid production, ion chelation and exchange reactions (Molla and Chowdhary, 1984) which are considered to be part of the phosphorous cycle.
Several species of microorganisms isolated from soil particularly; fungi, bacteria and yeasts and actinomycetes have been isolated and studied for their phosphorous solubilization abilities in vitro (Whitelaw, 2000). Species of fungi particularly Aspergillus, Penicillum and some yeasts have been reported to be involved in the solubilization of inorganic phosphates. These fungal species are capable of producing citric acid and form non-ionizable association with calcium. These fungal species have been reported to possess better solubilizing abilities than bacteria (Nahas, 1996).
In practical terms, application of phosphorous solubilization microorganisms in the field has been reported to increase crop yield (Akinrinde et al., 2003).
Although studies on solubilization of inorganic phosphates have been reported for different soil types in different parts of the world, there are no reports on soil microorganisms native to Nigerian soils. This study was therefore carried out to investigate fungal species isolated from soils in south western Nigeria capable of solubilizing insoluble phosphates.
MATERIALS AND METHODS
Phosphate Sources
Ogun phosphate rock used in this study was collected from an uncultivated
farmland in Oja-Odan, Ogun State, southwestern Nigeria, while tri-calcium phosphate
(Ca3(PO4)5) was obtained from the soil science
laboratory of the University of Agriculture, Abeokuta, Nigeria. Ogun rock phosphate
was pulverized into fine powder and further sieved in a 63 mm mesh to remove
coarse particles.
Isolation of Soil Fungi
Fungal species used for the experiment were isolated from Teaching and Research
farms of the University of Agriculture, Abeokuta after serial dilution of soil
solution; pour plate technique was used for the isolation using a basal medium
comprising of 10 g glucose, 0.5 g (NH4)2SO4;
0.2 g NaCl; 0.1 g MgSO4, 7H2O; 0.2 g KCl; 0.5 g yeast
extract; 0.2 g FeSO4.7H2O; 3 g Ca3(PO4)2;
0.2 g MnSO4 and 15 g agar in 1 L distilled water. Plates were incubated
at room temperature for 2-5 days. Colonies were purified by repeated sub-culturing;
pure cultures were maintained on Potato Dextrose Agar (PDA) slants at 5±2°C.
Fungi isolates were identified by their colony characteristics and microscopic
features particularly their vegetative and reproductive structures including
conidia and sporangia with reference to Bernett (1960) and Gilman (1975). The
nine most effective fungi isolates that solubilized phosphorus in both phosphate
rock and tri-calcium were used to assay for the organic acids.
In vitro Phosphate Solubilizing Test
The isolates were tested for phosphate solubilizing ability using the basal
medium in which the phosphorus source was replaced by 2 g phosphate rock or
10 g tri-calcium phosphate. An aliquot of 100 mL of the basal medium were measured
into 250 mL Erlenmeyer flasks. NaOH was added to adjust the pH to 7 prior to
sterilization at 121°C for 15 min. The flasks were inoculated with 4 mm
mycelial discs of actively growing fungal isolate. Control flasks were not inoculated.
The flasks were incubated at 20°C in a shaker incubator for 14 days. After
the incubation period, the cultures were harvested by filtration. The filtrates
were analysed for pH, titratable acidity (Nahas et al., 1990), soluble
phosphate (Ames, 1966), total phosphorus (Singh and Amberger, 1991). Organic
acids were determined by spectrophotometric method as described by AOAC (1990).
The experiments were done in triplicates.
Data Analysis
Anova and Duncan’s multiple Range tests were employed for comparing
means using SAS version 6.0. (SAS, 1989).
RESULTS
The thirty one fungi species isolated and used for the experiments are mainly
species of Aspergillus, Penicillium, Mucor, Trichoderma,
Fusarium, Ovulariopsis, Tritirachium and Geotrichum
(Table 1). Compared to the control, pH of inoculated media
decreased, this is an indication of acid production, there were however significant
differences in the pH recorded for the different fungal species. Differences
were also observed on the pH, titratable acidity, soluble phosphorous and total
phosphorous in media supplemented with phosphate rock and tri-calcium phosphate
of the test isolates (Table 1 and 2). Fusarium
oxysporium (CL) effected the least pH change (3.0) in phosphate rock medium,
while Penicillium digitatum effected the least pH change (2.23) in tri-calcium
phosphate medium. The titratable acidity recorded in rock phosphate ranged between
0.40-18.30%, while in tri-calcium phosphate it ranged between 0.63-26.73%.
| Table 1: | Final pH, Titratable Acidity (TA), Soluble Phosphorus (SP) and Total Phosphorus (TP) of the culture media supplemented with rock phosphates and inoculated with fungi isolated from soil |
| Values followed by different alphabets within columns are significantly different at p<0.05 | |
| Table 2: | Final pH, Titratable Acidity (TA), Soluble Phosphorus (SP) and Total Phosphorus (TP) of the culture media supplemented with Tri-calcium phosphates (TCP) and inoculated with fungi isolated from soil |
| Values followed by different alphabets within columns are significantly different at p<0.05 | |
| Table 3: | Organic acid produced by the most solubilized soil fungi in basal medium modified with Phosphate rock (R) at 14 days of incubation |
| `Values followed by different alphabet within columns are significantly different at p< 0.05 | |
Results of the study showed that soluble phosphate levels in culture medium was higher in medium containing tri-calcium (1.6-5.55 mg 100 mL-1) compared to rock phosphate medium (0.30-4.20 mg 100 mL-1. Some of the fungi species did not release soluble phosphorous rather; they utilized available phosphate. There were significant differences in the organic acids produced by the fungal species in the modified media. More acetic acids, gluconic, glutaric, lactic and succinic acids were produced by the fungi in tri-calcium modified medium, while more citric, fumaric, maleic, malic and tartaric acids were produced by the fungal species in phosphate rock medium. Aspergillus niger produced the highest malic acid (138.20 mg 100 mL-1) in phosphate rock medium and Aspergillus terreus produced the highest fumaric acid (264.45 mg 100 mL-1) in tri-calcium medium (Table 3).
DISCUSSION
With the exception of Geotricum sp. all the fungal isolates tested were able to solubilize phosphate rock and tri-calcium phosphate in vitro. Kucey and Paul (1982) recognised and reported the ability of several species of Aspergillus and Penicillium to solubilize insoluble phosphate. However, it is pertinent to note that other soil microorganisms are capable of solubilizing insoluble soil phosphate. In addition to some bacteria species (Nahas, 2006), soil chytrids have been reported to possess solubilizing ability (Midgley et al., 2006). Also, Rhizobia were reported to possess the ability to solubilize soil phosphate (Alikhani et al., 2006) Significant decrease in pH accompanied the release of phosphorous from TCP in culture supernatants. This confirms the implication of organic acid production in phosphorous solubilization by fungi. The increase observed in titratable acid is responsible for the observed decrease in pH. This effect was pronounced in media supplemented with tri-calcium phosphate. This is an indication that solubilization was more on tri-calcium phosphate medium.
Observations on the type of organic acids produced by the nine selected fungal
isolates (Table 4) showed that more acids were produced in
tri-calcium medium than in medium supplemented with phosphate rock. Succinic
acid was not produced by fungal isolates on phosphate based medium but some
amount of the acid was produced on tri-calcium based medium.
| Table 4: | Organic acid produced by the most solubilized soil fungi in basal medium modified with tri-calcium Phosphate (TCP) at 14 days of incubation |
| Values followed by different alphabets within columns are significantly different at p< 0.05 | |
Perhaps this is an indication that solubilizing ability may have a relationship with the type of organic acids produced by the fungi species rather than the quality of acid (Singh and Amberger, 1998).
In view of the fact that natural soil environment is more complex than in vitro setting, it is possible that several other factors may be at play in determining solubilizing ability of a fungi species. Pradhan and Sukla (2005) observed diverse levels of phosphate solubilization activity in the presence of various carbon and nitrogen sources.
Generally, within organisms a significant strain effect was observed, indicating that the ability of strains to solubilize phosphate varies significantly. Based on observations in this study a relationship seems to be established between the pH values, titratable acidity, the type of fungi species and solubilizing ability. This may imply that these parameters constitute the factors that are likely to affect solubilization of insoluble phosphates by Nigerian soil fungi.
As earlier reports have established that calcium in soil are dissolved by acidification. It is possible that strains of fungi that exhibit good ability to acidify its external medium as observed in this study will show some degree of phosphorous solubilization.
In conclusion, results of this study have shown that several naturally occurring fungi species isolated from Nigerian soil are capable of producing organic acids that aid the solubilization of insoluble phosphates. As this is a preliminary report, investigations are on going on other details of the solubilizing ability of the fungal species.