Solubilization of phosphates in vitro by Aspergillus spp. and Penicillium spp.

Ecological Engineering 42 (2012) 85–89

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Short communication

Solubilization of phosphates in vitro by Aspergillus spp. and Penicillium spp. Flavia Paiva Coutinho a,∗ , Wagner Pereira Felix b , Adriana Mayumi Yano-Melo b a

Universidade Federal de Pernambuco, Centro de Ciências Biológicas, Departamento de Micologia, Rua Nelson Chaves s/n, CEP 50670-420, Recife, PE, Brazil Universidade Federal do Vale do São Francisco, Campus de Ciências Agrárias, Colegiado de Zootecnia, Rodovia BR 407, Km 12 Lote 453, Projeto de Irrigac¸ão Nilo Coelho C1, CEP 56300-990, Petrolina, PE, Brazil b

a r t i c l e

i n f o

Article history: Received 28 August 2011 Received in revised form 18 January 2012 Accepted 1 February 2012 Available online 1 March 2012 Keywords: Filamentous fungi P-solubilizing Single superphosphate Mono-ammonium phosphate Biomass

a b s t r a c t Phosphorus (P) is one of the most important nutrients for plant development and in most Brazilian soils the content of this element is low and relatively available to plants. Phosphate-solubilizing microorganisms play an important role in supplying P to plants, because of their ability to provide insoluble phosphates, added or existing in the soil, by the processes of acidification, chelation and ion-exchange reactions. The objective of this study was to evaluate the capacity and potential of ten fungal isolates in solubilizing simple superphosphate (SSP) and mono-ammonium phosphate (MAP) in vitro, in four periods (1st, 4th, 7th and 10th days after inoculation). It was found that 90% of these isolates showed potential for solubilization of SSP and MAP on the seventh day of evaluation, with average values of 23% and 22% higher than control, respectively, with a reduction thereafter. This reduction can be attributed to the increase in fungal biomass, which results in enhanced uptake of soluble phosphate for growth. All isolates, except for the PSF 94, solubilized the two sources of phosphate on the 7th day, but the isolated PSF 28 in MAP, and PSF 220 in SSP, stood out from others by having the highest values of soluble P (84 and 56 ␮g ml−1 , respectively). This is the first reported solubilization of single superphosphate and mono-ammonium phosphate in vitro by the Aspergillus and Penicillium species, demonstrating that these fungi can serve as phosphate-solubilizing these P sources, contributing to a better use of the SSP and MAP and reducing the cost of agricultural inputs and the impact caused by excess phosphorus. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Among the essential elements, phosphorus (P), followed by nitrogen (N), has a prominent place for living beings, in view of its structural and functional performance as well as energy transfer (Sharpley, 1995; Bissani et al., 2008). In general, Brazilian soils have low phosphorus appearance, being the soluble phosphorus content very low (0.03 mg P kg−1 ), requiring the application of phosphates in amounts far above the demands of the plants, due to the great reactivity and high retention rate of their anions to various constituents of soil (Mendes and Reis Júnior, 2003). Thus, the soluble forms are easily precipitated in insoluble complexes and are not efficiently absorbed by plants. This way, in order to overcome this obstacle, excessive doses of P are applied to achieve the production and economic return (Vassilev and Vassileva, 2003). On the other hand, several soil microorganisms, including bacteria, fungi and actinomycetes, have the ability to solubilize insoluble phosphates, converting them into soluble forms that

∗ Corresponding author. Tel.: +55 81 99214504; fax: +55 81 21268482. E-mail address: fl[email protected] (F.P. Coutinho). 0925-8574/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ecoleng.2012.02.002

are available to plants through different mechanisms, such as acidification, chelation and ion-exchange reactions, especially in this case, the production of acids (Sahu and Jana, 2000; Whitelaw, 2000). Due to the fact that these microorganisms are present in most soils, the solubilization of P through them can be a lower cost alternative to production in agriculture (Rajan et al., 1996; Mendes and Reis Júnior, 2003). One of the areas that require the most nutrients is fruit production, which by its intensive nature, consumes with fertilizers nearly 10% of total production costs (Albuquerque et al., 2009) which justifies the study of alternative practices that enable lower costs, without prejudice to productivity and product quality as well as the environment. Among the cultivated fruit in the Submédio region of the Vale do São Francisco (VSF), the vine (Vitis vinifera L.) is one of the most important due to the generation of employment and income, contributing with 99% (80,000 tons) of exports of grapes in the country (Silva et al., 2009). Besides the production of table grapes, the areas of grapes for wine and juice are expanding, justifying the need for alternatives to the efficient use of phosphate fertilizers. From these, mono-ammonium phosphate (MAP) and single superphosphate (SSP) are widely used in the culture of the vine in the VSF

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mainly because they contain, in addition to P, nitrogen (MAP) and calcium and sulfur (SSP). Although these phosphated sources are water soluble (44% MAP and 16% SSP water soluble), part of P can become adsorbed to the surface of colloids or converted into very poorly soluble compounds, thus studies in order to optimize the use of phosphated fertilizers added to soil through solubilization by fungi is desirable, contributing to the establishment of a sustainable agricultural system, based on greater efficiency in the use of non-renewable natural sources. The solubilization of P by fungi has been reported, especially in the Aspergillus species of the niger group (Aspergillus aculateus, Aspergillus awamori, Aspergillus niger and Aspergillus tubingensis) (Achal et al., 2007; Ahuja et al., 2007; Reddy et al., 2002; Vassilev et al., 2007) and some species of Penicillium (Oliveira et al., 2008; Saber et al., 2009; Vyas et al., 2007; Xiao et al., 2008). The increase in availability of P by the fungi may vary due to the source and fungal isolate. Vyas et al. (2007) reported that the rate of solubilized phosphate by Eupenicillium parvum ranged from 120.8 to 213.7 ␮g ml−1 , averaging 121 to 214% higher than the control. On the other hand, Gupta et al. (2010), assessing the ability of Aspergillus sp. as to the solubilization of rock phosphate (India), found variation from 45.2 to 54.4 ␮g ml−1 of soluble P, an increase from 63 to 105% compared to control. Some studies have reported the attempt to increase the availability of inorganic phosphate by inoculation with phosphatesolubilizing fungi (PSF), being the majority of experiments conducted in laboratories and greenhouses. Saber et al. (2009) evaluated A. niger and Penicillium sp. as inoculants in Vigna radiata (L.) R. Wilczek plants and found an increase of soluble P in the fertilized rhizosphere with tricalcium phosphate (211 ␮g ml−1 ), aluminum phosphate (104 ␮g ml−1 ), rock phosphate (99 ␮g ml−1 ), sodium phytate (89 ␮g ml−1 ) and iron phosphate (33 ␮g ml−1 ). Recently, Kapri and Tewari (2010) reported an increase in the dry weight of the aerial part (22–33%) and of the root (35–60%) of chickpea plants in the presence of Trichoderma sp. in soil fertilized with tricalcium phosphate, when compared to controls without inoculation of the fungus, demonstrating the potential application of these microorganisms. Studies indicate that the PSF may constitute a viable alternative to maximize the use of phosphorus. Although the use of PSF is extremely important or more relevant in natural phosphate sources, information about the solubilization in MAP and SSP do not exist and even being very soluble, PSF may contribute further to their solubilizations. Thus, the objective of this study was to evaluate the capacity and potential of ten fungal isolates, derived from the culture of the vine in solubilizing mono-ammonium phosphate and single superphosphate in vitro, analyzing the influence of the incubation period of such activity.

2. Materials and methods Ten specimens of PSF, eight of Aspergillus (PSF 9, 28, 39, 57, 145, 198, 212 and 220) and two of Penicillium (PSF 94 and 169), were isolated from rhizospheric soil of the vine (V. vinifera L. cv. Cabernet Sauvignon) in the region of the Submédio of the Vale of São Francisco (Farm Planaltina, ViniBrasil), Petrolina, Brazil (08◦ 59 49 S, 40◦ 16 19 W) using the technique of suspension in series (1:1000, v/v) of soil. These PSF were grown in erlenmeyers containing 50 ml of GL medium (Sylvester-Bradley et al., 1982) liquid supplemented with 0.52 g 50 ml−1 of mono-ammonium phosphate (MAP) or 1.39 g 50 ml−1 of single superphosphate (SSP), corresponding to 0.25 g P2 O5 50 ml−1 , with medium pH adjusted to 6.5. One milliliter

of spore suspension of each PSF specimen, representing about 107 spores ml−1 , was added as inoculum. The erlenmeyers were incubated in a BOD (biochemical oxygen demand) at 30 ◦ C for a period of 10 days. Erlenmeyer flasks with the same medium, without inoculation, corresponding to the control group were maintained. Evaluations of P in the solution were carried out after 1 (T1), 4 (T2), 7 (T3) and 10 (T4) days after inoculation, withdrawing 5 ml of the culture of each flask. The cultures were centrifuged at 10,000 × g for 5 min and the supernatant was filtered (Whatman paper No. 40). Phosphorus in the solution (filtrated) was determined by spectrophotometry (660 nm) according to procedure described by Tedesco et al. (1995). The pH of the supernatant was determined by pH meter and the number of spores was quantified in a Neubauer chamber. At the end of the incubation period, the mycelium of each treatment was washed with distilled water and dried at 70 ◦ C for 72 h or until constant weight for estimation of the dry biomass. The experimental design was the completely randomized type with the factorial of 11 (10 isolated and control) × 4 periods (T1, T2, T3 and T4) in three repetitions. The variables analyzed were soluble P, pH and number of spores. Analyses of simple correlations were made between soluble P, pH and number of spores. Data were submitted to analysis of variance (ANOVA) and averages were compared by the Tukey test at 5% probability, using the program Statistica 5.0 (Statsoft, 1997). The data of soluble P was used to calculate the increase (Weber et al., 2004) provided by the PSF, using the formula 100[(X − Y)/Y], where X represents the inoculated treatment with the PSF and Y, control treatment.

3. Results and discussion There was an effect of the factors and the interaction between isolates and evaluation periods for all analyzed variables, for both sources of phosphate. On the first and fourth days of assessment (T1 and T2), there was no difference between control treatment and the isolates; the only isolate that showed small potential for solubilization in both sources of P and remained statistically equal to control in T3 and T4 was PSF 94. It was found that 90% of the isolates showed potential for solubilization of single superphosphate and mono-ammonium phosphate on the seventh day of evaluation (T3), with average values 23% and 22% higher than control, respectively, with a decrease after this period of time (Tables 1 and 2). According to Barroso and Nahas (2008), this reduction in the availability of P is related to the increase in fungal development, which leads to enhanced uptake of soluble phosphate by the fungus for its own growth, both vegetative and reproductive. On the 10th day (T4) the number of spores was higher (Tables 1 and 2), making it probable that the PSF tested have also mobilized part of the source of phosphate in its mycelium, contributing to the decrease in the amount of soluble P. Clearly, as seen by the results of PSF 94, which presented the greatest number of spores, in contrast, the concentration of soluble P did not differ from control. However, PSF 39 (MAP and SSP) and PSF 220 (SSP) also had higher soluble P in T4, which may be related to the metabolism of growth of these isolates. All the isolates, except PSF 94, solubilized P from the two phosphate sources in T3, although the isolated PSF 28 and FSP 220 stood out from others by having the highest values of soluble P in MAP and SSP (84 and 56 ␮g ml−1 ), respectively, being the rates of increase in comparison to control 29% and 44%. Inoculation of PSF in experiments which took place in the greenhouse or field could reduce

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Table 1 Average values of soluble P, pH and number of spores in liquid GL culture medium supplemented with mono-ammonium phosphate (MAP) and inoculated with isolates of phosphorus-solubilizing fungi (PSF) at different sampling times. Isolates

Soluble P (␮g ml−1 )

Control PSF 9 PSF 28 PSF 39 PSF 57 PSF 94 PSF 145 PSF 169 PSF 198 PSF 212 PSF 220

65 aA 71 aC 71 aC 68 aB 65 aC 66 aB 69 aC 68 aC 72 aB 67 aC 68 aB

T1

I (%) 09 09 05 00 02 06 05 11 03 05

T2 71 aA 77 aB 77 aB 73 aB 75 aB 74 aA 75 aB 71 aBC 79 aB 71 aB 73 aB

CV (%)

NS (×107 spores ml−1 )

pH I (%)

T3 65 bA 80 aA 84 aA 80 aA 80 aA 69 bB 80 aA 81 aA 83 aA 79 aA 77 aA

08 08 03 06 04 06 00 11 00 03

I (%) 23 29 23 23 06 23 25 28 22 18

T4 65 cA 74 abB 71 bC 79 aA 75 aB 62 cB 74 abB 75 aB 74 abB 73 abB 71 bB

I (%) 14 09 22 15 −05 14 15 14 12 09

T1

T2

T3

T4

T1

T2

T3

T4

6.2 abA 5.8 bcA 5.6 cdeA 5.2 fA 5.7 cdeA 6.2 abA 5.3 efA 6.3 aA 5.4 defB 5.6 cdefA 6.0 abcA

6.1 aA 4.8 bcC 3.5 dB 3.8 cdB 3.8 cdB 5.1 abB 4.6 bcdB 5.3 abA 5.3 abB 4.8 bcB 5.6 abB

6.1 aA 5.4 bB 3.5 cB 3.5 cC 3.9 cB 6.0 abA 5.6 abA 5.3 bA 5.5 abAB 5.6 abA 5.6 abB

6.2 aA 5.4 bB 3.5 cB 3.7 cBC 3.9 cB 5.9 abA 5.8 abA 5.8 abA 5.8 abA 5.6 abA 5.7 abAB

0.0 dA 1.8 bD 1.2 bcC 1.2 bB 0.5 cdD 4.3 aD 1.8 bC 1.8 bC 4.9 aC 1.3 bB 5.5 aD

0.0 fA 5.7 cC 1.6 deC 1.6 deB 1.3 eC 8.4 abC 2.6 dB 2.4 deBC 6.8 bcB 2.2 deB 8.9 aC

0.0 eA 8.9 cB 2.7 dB 2.1 dB 2.3 dB 35.3 aB 3.1 dB 3.7 dB 8.6 cB 3.2 dA 19.7 bB

0.0 fA 23.0 cA 4.1 eA 3.0 eA 3.1 eA 96.6 aA 3.8 eA 4.9 eA 10.9 dA 3.7 eA 47.0 bA

3.369

0.349

0.670

Means followed by the same letter, lower case in the column and capital letters in the line, did not differ significantly by Tukey test (p ≤ 0.05). NS = number of spores; I = increment {100[(X − Y)/Y]}, X = treatment inoculated with PSF and Y = control treatment; CV = coefficient of variation. T1 = 1, T2 = 4, T3 = 7 and T4 = 10 days after inoculation.

the doses of MAP and SSP applied, allowing the reduction of costs of agricultural inputs. This maximum solubilization was obtained seven days after inoculation, confirming results obtained by Achal et al. (2007) and Mittal et al. (2008), which showed an increase of soluble P from the dissolution of tricalcium phosphate and rock phosphate/India (4.7 and 248 ␮g ml−1 , respectively) for Aspergillus spp. Likewise, Saber et al. (2009) reported maximum solubility of P from rock phosphate/Egypt soluble for A. niger (67 ␮g ml−1 ) and Penicillium sp. (46.2 ␮g ml−1 ) also on the 7th day of the experiment. Although factors such as immobilization of P in the fungal mycelium and the use of P for growth and reproduction of fungi may explain the results obtained on day 7 of this work and the others, more studies are needed to elucidate the factors responsible for such response. Most isolates in T3, for both sources of phosphate, showed a high rate of P solubilization and low number of spores (Tables 1 and 2), while the FSP 94 specimen presented in both moments (T3 and T4), increased spore numbers and lower concentrations of soluble P, but there was no correlation (r2 = −0.05 MAP; r2 = −0.01 SSP, n = 132, p < 0.05) between the number of spores and soluble P. Reyes et al. (1999) and Yadav and Tarafdar (2003) registered a negative correlation between biomass (vegetative and reproductive structures) and solubilization of P, suggesting the occurrence of a pump mechanism of H+ involved in the solubilization of small amounts of phosphate by the fungus, allowing the development the greater biomass. These authors also suggest that isolates with low biomass

may translocate carbon to produce different types and greater amounts of organic acids to solubilize more P, which is reinforced by the data, in T4, between dry biomass and number of spores, where the increase in biomass was accompanied by the growing number of spores, the isolated PSF 39 had the lowest rates of these variables and a high rate of soluble P, while the PSF 94 showed the greatest values in number of spores, dry biomass and low solubility, indicating that greater development and sporulation are not related to the greater solubility of P (Fig. 1 and Tables 1 and 2). The results suggest that isolates may have different metabolic strategies, reflecting the lack of correlation and different responses in relation to solubilization and production of mycelium and spores. It is possible to point out the need for more studies on the physiology of these fungi in order to select efficient microorganisms. Previous reports have shown a negative correlation between the development and reproduction of fungi and the pH of the medium due to the affinity of these microorganisms to grow in acidic environment (Yadav and Tarafdar, 2003; Barroso and Nahas, 2005). However, this study did not report a correlation between these variables (r2 = 0.14 MAP; r2 = −0.01 SSP, n = 132, p < 0.05), although there were increased numbers of spores and decreased pH. The reduction of the pH of the medium in this study (Tables 1 and 2), suggests an effect of the production of organic acids on phosphate solubilization, which is confirmed by analysis of correlation between pH and amount of soluble phosphate (r2 = −0.47 MAP; r2 = −0.68 SSP, n = 132, p < 0.05). Reports have demonstrated

Table 2 Average values of soluble P, pH and number of spores in liquid GL culture medium supplemented with single superphosphate (SSP) and inoculated with isolates of phosphorussolubilizing fungi (PSF) at different sampling times. Isolates

Soluble P (␮g ml−1 ) T1

Control 39 aA PSF 9 39 aC 38 aC PSF 28 38 aC PSF 39 44 aB PSF 57 43 aAB PSF 94 PSF 145 39 aC 38 aC PSF 169 PSF 198 45 aAB 37 aC PSF 212 44 aB PSF 220 CV (%)

I (%) 00 −03 −03 13 10 00 −03 15 −05 13

T2 42 aA 45 aB 46 aB 43 aB 44 aB 45 aA 41 aB 44 aB 47 aA 43 aB 45 aB

NS (×107 spores ml−1 )

pH I (%) 07 10 02 05 07 −02 05 12 02 07

T3 39 cA 47 bA 50 abA 48 bA 51 abA 40 cB 47 bA 51 abA 47 bA 48 bA 56 aA

2.366

I (%)

T4

21 28 23 31 03 21 31 21 23 44

40 cA 45 bB 47 bB 52 aA 47 bB 39 cB 44 bB 46 bB 43 bB 44 bB 52 aA

I (%) 13 18 30 18 −03 10 15 08 10 30

T1

T2

T3

T4

3.6 aA 3.4 aB 3.5 aA 4.0 aA 3.2 aC 3.5 aC 3.3 aB 3.5 aB 3.0 aC 3.4 aB 3.1 aC

3.5 abA 3.0 dC 2.9 dB 2.9 dC 3.4 abBC 3.4 bcC 3.7 aA 3.0 dC 3.7 abB 3.0 dC 3.1 cdC

3.6 bcdA 3.5 cdAB 3.3 dA 3.5 cdB 3.9 abA 3.8 abcB 3.9 abA 3.9 abA 4.1 aA 3.9 abA 4.1 aB

3.6 bcA 0 eA 3.7 bA 34 bC 3.3 cA 17 cdD 3.9 abA 17 cdC 3.6 bcB 13 deC 4.0 abA 63 aD 3.9 abA 19 cdC 3.8 bAB 28 bcC 4.3 aA 24 bcdC 3.9 abA 14 dD 4.3 aA 17 cdD

0.187

T1

T2

T3

0 eA 49 bC 34 bcdC 34 bcdB 19 dcC 78 aC 40 bcBC 48 bC 44 bBC 24 cdC 31 bcdC

0 fA 108 bB 60 cdB 45 dA 33 eB 141 aB 62 cdB 98 bB 85 bcB 38 eB 50 cdB

T4 0 fA 235 bA 77 deA 49 eA 52 eA 387 aA 115 cdA 161 cA 153 cA 49 eA 98 cdA

4.294

Means followed by the same letter, lower case in the column and capital letters in the line, did not differ significantly by Tukey test (p ≤ 0.05). NS = number of spores; I = increment {100[(X − Y)/Y]}, X = treatment inoculated with PSF and Y = control treatment; CV = coefficient of variation. T1 = 1, T2 = 4, T3 = 7 and T4 = 10 days after inoculation.

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Fig. 1. Mean values of dry biomass (g 50 ml−1 ) and number of spores (×1010 spores ml−1 ) form isolates of phosphorus-solubilizing fungi (PSF) grown in liquid GL medium supplemented with (a) mono-ammonium phosphate (MAP) and (b) single superphosphate (SSP) at 10 days (T4) of incubation.  = dry biomass;  = number of spores.

similar negative correlation between these factors (Alam et al., 2002; Pradhan and Sukla, 2005; Kang et al., 2008). However, other studies mention lack of correlation, suggesting that the amount of acids produced by fungi could be equal, but the effectiveness of solubilization may be different due to the type of acid produced (Barroso and Nahas, 2005; Mittal et al., 2008). Besides, the decrease in pH of the culture medium may be the result of selective ion uptake by fungi, and not a direct effect of organic acid production, according to Cerezine et al. (1988). It is still noted that the solubilization may be due to the proton H+ excretion and depend on the type of microorganisms and phosphate (Illmer et al., 1995). 4. Conclusions This is the first reported solubilization of mono-ammonium phosphate and single superphosphate in vitro by Aspergillus spp. and Penicillium spp., indicating that these isolates can serve as solubilizers for these types of phosphates, when inoculated in soils where the MAP or SSP are used as fertilizers. The combined use of PSF with these sources optimizes the output of P and reduces costs with agricultural inputs and impacts generated by the excess of phosphorus, since even if MAP and SSP have water solubility, these phosphates can quickly become fixed or adsorbed into the

soil, because of their physical properties such as aeration, water retention, compaction, structure and timing of application of P. During the incubation period, the seventh day evaluation showed the greatest potential for solubilization of both sources of phosphate. It is worthy to report, however, the need for further studies on the metabolism of these isolates, which substances are responsible for the solubilization of P, as well as factors that may affect this production. Such answers may contribute to select and offer the PSF inoculants for agricultural areas, as well as to better manage the indigenous population in order to reduce the use of phosphates through a better use of existing P or added to soil and formed by applying phosphated sources. Acknowledgments The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for PhD (Coutinho) and PQ (YanoMelo) scholarships, the Fundac¸ão de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) for research assistance, the Empresa Brasileira de Pesquisa Agropecuária (Embrapa Semiárido) and the Farm Planaltina (ViniBrasil) for research support, and Leonardo Costa for reviewing the English.

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