Phosphate solubilization potential of rhizosphere fungi isolated from satoimo taro plant

Phosphorus is an essential nutrient required by plants for their growth and development. Phosphates bioavailability in nature is approximately 95-99% but insoluble, therefore the plant can not simply use them. The alternative to overcome phosphate deficiency is by utilizing phosphate solubilizing fungi. This research is aimed at selecting and characterizing phosphate solubilizing fungi in rhizosphere of saitomo taro (Colocasia esculenta var antiquorum). The test on isolate ability to solubilize phosphate was conducted through qualitative and quantitative. Qualitative was carried out by counting index of phosphate solubilization on solid Pikovskaya while quantitative was through counting concentration of phosphate solubilization through spectrophotometry at wave length 693 nm. The result of qualitative test showed that the highest index of phosphate solubilisation was 1.08 at isolate RTP25 while quantitative demonstrated that isolate RTP16 released highest concentration of phosphate solubilization with the amount 12.78 µgl−1. Isolate RTP 25 (by Trichoderma sp) and RTP16 (by Aspergillus sp) be potential to be developed as phosphate biofertilizer.


Introduction
Phosphorus is important nutrient for plants to grow and develop.The soil provides high phosphorus around 95-99% but remain insoluble, preventing plant to be able to perform direct adsorption [1,2].The strong bound among phosphate with iron and aluminum oxide hinders the plants, accordingly they just can merely take less than 1% phosphorus in the soil [3].One of the efforts in increasing absorbable phosphorus supply is by applying phosphate fertilizer, but it will then be turned slightly into insoluble form.Consequently, this matter obstruct plants in absorbing phosphate and finally triggers continuous application inflicting high production cost for farmers.
The other solution to accelerate the efficiency of phosphates fertilization in eliminating their deficiency is by taking advantage of phosphate solubilizing microorganisms, they possess ability to extract phosphate from insoluble substance into soluble ones through secretion of amino acids : acetate, propionate, glutamate, lactic acid, malate and etc [4].These organic acids will further react with phosphate bonding substances like -Al 3+ , Fe 3+ , Ca 2+ , Mg 2+ creating stable organic chelate, hence they are capable to break phosphate bonds that have been bounded by organic compound to boost phosphate adsorption by plants or mineralizing phosphates from their organic bound into dissolved inorganic phosphates [5,6] Obtained dissolved inorganic phosphates are utilized by microbes for their activity and new cells configuration, evoking phosphate bonding (immobilization).Those Soil microbes as following: Pseudomonas, Bacillus, Escherichia, and Xanthomonas bacteria; Aspergillus, Penicillium and 1230 (2023) 012072 IOP Publishing doi:10.1088/1755-1315/1230/1/012072 2 Curvularia fungi; Streptomyces which belongs to actinomycetes , have the ability to solubilize insoluble in organic phosphate by secreting amino acids [7].As phosphate solubilizing fungi play role in promoting absorbable phosphorus, it needs to isolate and test the ability of rhizosphere fungi in solubilizing phosphate to be potential biofertilizer.This research is aimed at selecting and characterizing phosphate solubilizing fungi in rhizosphere of saitomo taro (Colocasia esculenta var antiquorum).

Isolation of Rhizosphere Fungi
Soil sample was taken compositely from taro plant rhizosphere at 5 cm from the bottom of the roots in the 0-20 cm depth, mixed together and put into paper sample.10 grams were transferred into 100 ml aquades to be suspended.Then 1 ml of suspension was poured into 9 ml sterile aquades in reaction tube, stirred until homogenous (dillution 10 -1 ), the same process repeated until dillution 10 -6 .The dillution 10 -1 -10 -6 results were taken respectively for 1 ml into petri dish filled with PDA (Potato Dextrosa Agar) media, evened by spatula then further incubated at room temperature for 2-3 days.The growing fungal colonies at dillution 10 -1 -10 -6 were purified by moving one colony to new sterile PDA medium.

Qualitative Method of Phosphate Solubilizing Test
Test on ability of rhizosphere fungal isolates as phosphate solubilizing fungi was set up through Pikovskaya [8].Pure fungal isolate was inoculated at solid Pikovskaya medium with composition 10 g glucose, 5 g Ca3PO4, 0,5 g (NH4)2SO4, 0.2 g KCl, 0.1 g MgSO4.7H2O,0.01 g MnSO4.H2O, 0.5 g yeast extract, 0.01 g FeCl3.6H2O, 15 g agar in one liter solution at pH 7.0.The next step is incubating the isolates which grew at testing media for 7 days .The colony that grew and formed clear zone was indicated as phosphate solubilizing isolate.Based on the clear zone, Index of phosphate solubilization can be known, since it was obtained from the comparison between clear zone diameter divided by diameter of colony [9].

Quantitative Method of Phosphate Solubilizing Test
This method was carried out by culturing fungal isolates on liquid pikovskaya media and shaking them for 7 days.Then, isolate suspension was further filtrated by filtration paper and centrifuged for 15 minutes at speed 10000 rpm. 5 ml supernatant were placed into reaction tube, added with 0.5 ml dense P reactor (112 g ammonium moblibdat, 0.277 g calcium antimol tartrat), stirred for 5 minutes and left for 30 minutes.Filtrate solution pH and the adsorption were measured using spectrophotometer with λ 693 nm.Titrisol curve standard (PO4) was created from dilution with concentration ranges from 0 -2.5 mgl -1 in equation of regression Y = 0.191 x + 0.04, R 2 = 0.957 filtrate concentration get determined based on the adsorption which is compared to curve standard.

Identification
Isolate with highest dissolved phosphate concentration value was characterized both in macroscopic and microscopic way.Macroscopically was done through observing the morphology of colony on Potato Dextrosa Agar (PDA) media while microscopically by observing sporangium, sporangiophore and spore of fungal isolate through microscope with magnification 40x

Results and Discussion
Qualitative test showed two isolates from ten isolates had marked the presence of clear zone on pikovskaya media.Those are isolate RTP25 and RTP29 in which RTP25 appeared the highest phosphate solubilizing index was 1.08 (Figure 1).Identification of sample RTP25 in microscope showed the isolate is Trichoderma sp.Trichoderma sp potential for solubilization of phosphate present in the soill, enhancing soil fertility and plant growth The ability in solubilizing phosphate is marked by the forming of clear zone in inoculated fungi on Pikovskaya media containing tricalcium phosphate (Ca3(PO4)2.Clear zone was formed around colony, indicating that isolate was able to solubilize bounded phosphate on medium containing Ca3(PO4)2.It happened since the released organic acids could substitute phosphate anion and chelate cations such as Ca, Al and Fe creating complex compound [10].
Based on the quantitative test result, the ability of fungi in solubilizing phosphate was various.10 of rhizosphere fungal isolates which had been quantitatively tested using spectrometer at 693 nm showed varied concentration of soluble phosphate from 3.84 µgl -1 until 12.78 µgl -1 in which 12.78 µgl -1 was the highest value produced by isolate RTP16 (Figure 2) The ability of phosphate solubilizing fungi is widely diverse depending on their genetic characteristics [11] and types of media [12] .The amount of yielded organic acids gives direct impact as well [13].Obtained organic acids will react with phosphate binding substance such as Al 3+ , Fe 3+ , Ca 2+ , Mg 2+ forming stable organic chelate.It contributes a help to break phosphate bound on organic compound, provoking higher adsorption of phosphorus by plant.Phosphate solubilizing microorganisms can convert insoluble form into soluble mostly through acidification process, chelation and ion exchange reaction as well as polymer substance forming [14,15] Phosphate test results based on the clear zone diameter and concentration of phosphate solubilizing presented a trend, the wider the diameter of the clear zone the higher concentration obtained.But not all of the tested isolates showed the same since there was isolate with high phosphate solubility but low index due to a condition where the diameter of clear zone and phosphate solubilizing concentration really depend on colony growth.The bigger the growth the higher the concentration achieved, besides, acidification level of Pikovskaya media contributed as well [16] Identification of sample RTP16 in microscope showed the isolate is Aspergillus sp (Figure 3).Aspergillus genus have highest ability in solubilizing phosphate in Amaranthus spinosus [17].Some of fungal soil especially Penicillium and Aspergillus genus, are capable of altering phosphate insoluble soil to be soluble by releasing weak organic acids:formate, acetate, propionate, lactic, gluconate, glonat, fumarate and succinic acids [18].Penicillium sp was able to solubilize 26% until 40% of Ca3(PO4)2, while Aspergillus sp was 18% of Ca3(PO4)2.

Conclusion
The ability of fungi in solubilizing phosphate were 3.84 µgl -1 until 12.78 µgl -1 in which the highest value produced by isolate RTP16.It is potential to be developed as phosphate biofertilizer.

Figure 2 .
Figure 2. The ability of rhizosphere fungi isolates to dissolve phosphates (Quantitatively tested)