Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an oxisol of the Brazilian Cerrado Biome

Soil Biology and Biochemistry 41 (2009) 1782–1787

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Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an oxisol of the Brazilian Cerrado Biome C.A. Oliveira a, V.M.C. Alves b, I.E. Marriel b, E.A. Gomes b, M.R. Scotti a, N.P. Carneiro b, C.T. Guimara˜es b, R.E. Schaffert b, N.M.H. Sa´ a, * a b

Federal University of Minas Gerais, Botany Department, CP 486, 31270-901 Belo Horizonte, MG, Brazil Embrapa Maize and Sorghum, CP 151, 35701-970 Sete Lagoas, MG, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 September 2007 Received in revised form 21 December 2007 Accepted 4 January 2008 Available online 6 May 2008

Many soil microorganisms are able to transform insoluble forms of phosphorus to an accessible soluble form, contributing to plant nutrition as plant growth-promoting microorganisms (PGPM). The objective of this work was to isolate, screen and evaluate the phosphate solubilization activity of microorganisms in maize rhizosphere soil to manage soil microbial communities and to select potential microbial inoculants. Forty-five of the best isolates from 371 colonies were isolated from rhizosphere soil of maize grown in an oxisol of the Cerrado Biome with P deficiency. These microorganisms were selected based on the solubilization efficiency of inorganic and organic phosphate sources in a modified Pikovskaya’s liquid medium culture containing sodium phytate (phytic acid), soybean lecithin, aluminum phosphate (AlPO4), and tricalcium phosphate (Ca3(PO4)2). The isolates were identified based on nucleotide sequence data from the 16S ribosomal DNA (rDNA) for bacteria and actinobacteria and internal transcribed spacer (ITS) rDNA for fungi. Bacteria produced the greatest solubilization in medium containing tricalcium phosphate. Strains B17 and B5, identified as Bacillus sp. and Burkholderia sp., respectively, were the most effective, mobilizing 67% and 58.5% of the total P (Ca3(PO4)2) after 10 days, and were isolated from the rhizosphere of the P efficient L3 maize genotype, under P stress. The fungal population was the most effective in solubilizing P sources of aluminum, phytate, and lecithin. A greater diversity of P-solubilizing microorganisms was observed in the rhizosphere of the P efficient maize genotypes suggesting that the P efficiency in these cultivars may be related to the potential to enhance microbial interactions of P-solubilizing microorganisms. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: P-solubilizing microorganisms (PSM) Phosphorus mineralization and solubilization Rhizosphere Zea mays

1. Introduction The Cerrado Biome is a large Brazilian ecosystem characterized by a mosaic of savanna, cropping and forest, where the soils are characterized by low pH, low phosphorus (P) content and high P fixation capacity (Marschner, 1995; Novais and Smyth, 1999; Hinsinger, 2001; Wakelin et al., 2004). The accumulated insoluble P, like total soil P, occurs in either organic or inorganic forms, unavailable to plants. Phosphate anions may be immobilized through precipitation with cations such as Ca2þ, Mg2þ, Fe3þ and Al3þ. Organic P in the soil generally accounts for around 50% of total insoluble soil P in soils with high organic matter content, such as the no-tillage management systems (Bayer et al., 2001; Gyaneshwar et al., 2002). A large proportion of the organic P is represented by inositol phosphates and lesser amounts of other phosphate esters as phospholipids (Richardson, 2001; Wakelin et al., 2004; Richardson * Corresponding author. Tel.: þ55 31 3499 2688; fax: þ55 31 3499 2673. E-mail address: [email protected] (N.M.H. Sa´). 0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2008.01.012

et al., 2005). The strategy of ameliorating the low P fertility production constraint in acid soils with corrective applications of P is limited economically and environmentally due to the high amounts of P fertilizer required and the high P fixing capacity of these soils (Hinsinger, 2001). Microbial populations are key components of the soil–plant continuum where they are immersed in a framework of interactions affecting plant development (Wakelin et al., 2004; Barea et al., 2005; Vassilev et al., 2006). P-solubilizing microorganisms (PSM) can solubilise and mineralize P from inorganic and organic pools of total soil P, and may be used as inoculants to increase P-availability to plants (Kucey et al., 1989; Richardson, 1994, 2001; Illmer et al., 1995; Whitelaw et al., 1999). This work may contribute in developing soil microbial community management schemes based on specific functions (P solubilization and mineralization), and selection of microorganisms as potential microbial inoculants (biofertilizers). These results could have significant impact on the more than six million hectares of maize under no-till cultivation in Brazil today. There is also a potential use of PSM in industrial bioprocessing of rock phosphate.

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The objective of this research was to isolate phosphate solubilization and mineralization microorganisms in maize rhizosphere cultivated under conventional tillage and no-tillage management systems in the Brazilian Cerrado. Additionally, a new method to screening PSM using a P-Mehlich1 phosphorus extractor was also tested in this work. The knowledge of P-organic mineralization processes is expected to be useful in understanding seasonal Pcycling in the Brazilian oxisols under the no-till production system. 2. Materials and methods

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were inoculated on modified Pikovskaya’s agar medium containing organic forms of P, phytic acid or sodium phytate (P-phytate) and soybean lecithin (P-lecithin). P-Ca, P-Al, P-phytate and P-lecithin were added in Pikovskaya’agar medium at 5 g l1, 3.5 g l1, 10 g l1 and 15 g l1, respectively. Petri plates were incubated at room temperature for 10 days. Morphologically distinct colonies, both with and without halos were purified by repeated subculturing, maintained on potato dextrose agar (PDA) and incubated at room temperature. The isolates were grouped into filamentous fungi, actinobacteria or bacteria based on macro and microscopic observations.

2.1. Soil samples 2.3. Screening and phosphate evaluation Soil samples were taken from the rhizosphere of four maize cultivars contrasting in P efficiency; BRS3060 – P efficient hybrid, HS26  1113 – P inefficient hybrid, L3 – P efficient inbred line, and L22 – P inefficient inbred line developed by the Maize Breeding Program of Embrapa Maize and Sorghum (Parentoni et al., 2000) at Sete Lagoas, Minas Gerais, Brazil. These genotypes were growing in a conventional and no-tillage management system in an oxisol. The soils of conventional crop system were characterized by low P (3 mg P dm3), clay texture (55% clay, 11% silt, 34% sand) with pH 5.2 (soil/water ratio, 1:2.5 [w/v]) and 3% organic matter and 0.25, 2.29, and 0.36 cmolc kg1 of Al, Ca, and Mg, respectively, in a 1 N KCl extraction and 0.16 mg K dm3, in a Mehlich1 extraction (Mehlich, 1978). Samples of no-tillage maize rhizosphere were collected from oxisols at eight locations with 13–15 years of no-tillage management. Descriptions of each sample are provided in Table 1, including the location and the crops in rotation with maize. Samples were taken for each genotype and tillage system 60 days after planting, during the flowering stage. Each of three replicated samples was composed of the soil adhering to the roots of five maize plants. The roots were shaken carefully inside plastic bags in order to separate the soil from the roots. 2.2. Isolation of P-solubilizing microorganisms The rhizosphere soil samples from the maize plants in a conventionally managed low P environment were serially diluted and inoculated on modified Pikovskaya’s agar medium (Pikovskaya, 1948) containing insoluble inorganic forms of P, Ca3(PO4)2 or tricalcium phosphate (P-Ca), AlPO4 or aluminum phosphate (P-Al). The maize rhizosphere soil samples from the no-till management

A preliminary experiment was conducted to test the ability to produce soluble phosphate in liquid cultures by the isolates. Three replicate flasks containing modified Pikovskaya’s liquid medium were inoculated with each isolate using 8 mm mycelia disks of fungal and actinobacteria cultures, and bacterial suspension (108 cell ml1) taken from 10 days old cultures. Insoluble sources of P, Ca3(PO4)2, AlPO4, sodium phytate, and soybean lecithin, were added to the liquid medium at 1.5 g l1 (300 mg P l1), 1 g l1 (250 mg P l1), 1 g l1 (280 mg P l1), 15 g l1 (101.1 mg P l1), respectively. Three replicates of a control treatment were included in the experiment, each containing one P-insoluble source. The initial pH was adjusted to 6.0. The cultures were centrifuged (7000g, 10 min) after 10 days of incubation, at 27  C, with gentle shaking and 5 ml supernatant aliquots were filtered through Whatman n 42 filter paper to remove thick polysaccharide-like exudates. The filtrates were assayed for soluble P, using the Murphy and Riley’s (1962) colorimetric method. The amount of P solubilized was obtained by subtracting the soluble P of the inoculated sample from the corresponding sample uninoculated control (i.e. P released by autoclaving of the P suspension). 2.4. Evaluation for phosphate solubilization, pH and phosphatase activity The best 45 isolates from the preliminary screening were selected based on the amount of P solubilized in each medium and were again assayed for P solubilization as described above. The pH, phosphatase production and P-Mehlich1 solubilizing activity were also evaluated. The solution pH was measured after 10 days and

Table 1 Description of rhizosphere samples of maize cultivated in oxisols, production system, number of microorganisms (fungi, bacteria, actinobacteria) isolated from soil samples, and source of insoluble P; tricalcium phosphate (P-Ca), aluminum phosphate (P-Al), sodium phytate (P-phytate), and soybean lecithin (P-lecithin) Sample

City/statea

Crop management systemb

Total P inorganicsolubilizing isolatesc P-Ca

P-Al

P-phytate

P-lecithin

L3 L22 BRS3060 HS26  1113 CNPMS JSP CNPS MGO PGO GRGO SCRGO SMRGO

Sete Lagoas/MG Sete Lagoas/MG Sete Lagoas/MG Sete Lagoas/MG Sete Lagoas/MG Jardino´polis/SP Londrina/PR Morrinhos/GO Planaltina/GO Rio Verde/GO Rio Verde/GO Rio Verde/GO

CT/maize CT/maize CT/maize CT/maize NT/maize BRS3060 NT/maize NT/maize NT/maize (soybean) NT/maize (grassland/soybean) NT/maize (grassland) NT/maize (sugar cane) SM/NT/maize

34 23 28 21 – – – –

13 18 8 17 – – – –

– – – –

– – – – 21 25 10 22 20 15 11 22

Total a

(14) (6) (9) (7)

Total P organicsolubilizing isolatesc

(7) (4) (4) (6)

– – –

– – –

2 6 3 9 9 13 14 7

106

56

63

(1) (0) (1) (3) (1) (3) (10) (0)

(6) (3) (0) (7) (0) (0) (3) (5)

146

State’s acronym: MG, Minas Gerais; SP, Sa˜o Paulo; GO, Goia´s. b Production system: CT, conventional tillage; NT, no-tillage; SM, swine manure. In parentheses, the crop grown in rotation with maize. c Total number of isolates from each specific solid medium (number in parentheses represents the number of isolates showing P solubilization within 10 days in liquid culture).

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150 ml centrifuged and filtrated aliquots were assayed for phosphatase activity of the isolates according to Freitas et al. (1997) modified protocol. Aliquots of each sample were added to 0.48 ml universal buffer 0.1 M, pH 6.5 or pH 11 for acid or alkaline phosphatase activity, respectively, and 0.12 ml of 0.05 M p-nitrophenyl phosphate (pNPP) solution, followed by 1 h incubation at 37  C. Control treatments containing only liquid medium were included in each experiment with pNPP added after incubation. The yellow color was measured at 410 nm (Tabatai and Bremmer, 1969). The effects of the treatments were analyzed by ANOVA and means were compared using Tukey’s test, at 5% significant level. 2.5. P-Mehlich1 solubilizing activity – phosphorus incorporated into the organic matrix of polysaccharides A simultaneous test was carried out using the same samples from the four experiments cited above to determine the amount of phosphorus incorporated into the organic matrix of polysaccharides formed during culture growth. After 10 days of incubation 10 ml aliquots were incubated for 24 h with 100 ml of P-Mehlich1 extractor solution (HCl 0.05 M þ H2SO4 0.0125 M), referred to as ‘‘double acid’’ extracting (Mehlich, 1978). After incubation, 5 ml supernatant aliquots were centrifuged, filtered, and assayed for soluble P using the same Murphy and Riley’s (1962) conventional method. The amount of P solubilized (P-Mehlich1) was obtained by subtracting the soluble P concentration of the inoculated from that of the corresponding uninoculated control culture. Factorial ANOVA was used to analyze the effects of the treatments and the phosphorus determination methods, whose means were compared using the Tukey test at 5% significance. 2.6. Identification of isolates The 45 most efficient isolates solubilizing P-phytate, P-lecithin, P-Ca, and P-Al were identified based on nucleotide sequence data from the rDNA – ITS for fungi and 16S rDNA for bacteria, including actinobacteria. Total DNA was extracted using BIO 101 kit protocols (FastDNA SPIN Kit, Bio 101 Inc., Vista, CA) from cultures recovered in a potato dextrose liquid medium (0.2 g of fresh weight of fungi and actinobacteria mycelium filtrated and 200 ml of bacterial suspension, 108 cell ml1), after 10 days growth at room temperature without shaking. The rDNA fragments were amplified using fungi universal primers, ITS1 and ITS4 or ITS5 and ITS2 (White et al., 1990) and the 16S rDNA bacteria and actinobacteria fragments were amplified using the F968 and R1401 primers (Nubel et al., 1996). PCR reaction was performed using 20 ng DNA, 50 mM of each dNTP, 2.5 mM of MgCl2, 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 0.2 mM of each primer and 1 unit of Taq DNA polymerase (Invitrogen, Carslbad, CA) in a final reaction volume of 50 ml. Amplifications were carried out using the temperature profiles: 94  C for 2 min, followed by 30 cycles at 94  C for 1 min, 55  C for 1 min, 72  C for 2 min, and a final extension step at 72  C for 10 min for bacteria (including actinobacteria) and 95  C for 4 min, followed by 30 cycles at 95  C for 30 s, 46  C for 60 s, 72  C for 30 s and primer extension at 72  C for 10 min for fungal isolates. PCR products were separated on a 1.5% (w/v) agarose gel, stained with ethidium bromide (1 mg ml1), and visualized under UV light using Eagle Eye II (Stratagene, La Jolla, CA). Amplified products were removed from the gel and purified using QIAquick Gel Extraction kit (Qiagen, Hilden, Germany) and sequenced using the ‘‘Big Dye Terminator v3.1. Cycle Sequencing’’ (Applied Biosystems, Foster City, CA) kit in an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Nucleotide sequence data were compared with GenBank data (http://www.ncbi.nlm.nih.gov/) using the BlastN search (Altschul et al., 1997).

3. Results 3.1. Isolation of P-solubilizing microorganisms and solubilization assay for inorganic and organic P sources Three hundred seventy-one morphotypes (116 fungi and 255 bacteria of which 126 were actinobacteria) were recovered from the rhizosphere of maize plants grown in a Cerrado Biome oxisol soil from both conventional and no-till planting systems (Table 1). A total of 36 isolates from P-Ca medium showed P-solubilizing in liquid culture medium solubilizing more than 80 mg P l1 (30% of total P), in 10 days, and of these, 14 (39%) were isolated from the P efficient maize inbred line, L3. In this study, 63 colonies were evaluated in P-phytate medium and 19 colonies showed P-solubilizing activity in 10 days, and of these colonies, 10 (53%) were from maize no-tillage rhizosphere in consortium with sugar cane (SCRGO). The numbers of P-lecithinsolubilizing isolates varied among samples, however, the highest number of isolates that presented P-solubilizing activity was found in no-tillage maize–soybean samples (Table 1). 3.2. Identification and evaluation of isolates for P-solubilizing activity, pH and phosphatase activity A total of 45 isolates screened for phosphate solubilizing efficiency in specific liquid medium were genetically identified using rDNA sequences in comparison with GenBank database (Tables 2–5). Morphological identification confirmed that the majority of the genera to the species identified by the molecular techniques. The strains of bacteria, fungi and most of the actinobacteria (Tables 2–5) were confirmed as PSM with high P solubilization rates, but the actinobacteria genera, Kitasatospora (Table 5), was identified as a new PSM. Among the bacteria isolates, B17 and B5, identified as Bacillus sp. (B17) and Burkholderia sp. (B5), were the most efficient P-solubilizing strains from P-Ca source culture solution (Table 2), solubilizing 67% and 58.5% of the total P, respectively. The B5 isolate also had the largest reduction in pH in the growth solution to 4.46. The actinobacteria most efficient in solubilizing P-Ca source was A4, identified as Streptomyces platensis (Table 2), according to morphological and molecular criteria. The most efficient isolates for aluminum phosphate solubilization were B58, F39 and F50, identified as Burkholderia cepacia and Aspergillus terreus, which were isolated from the inefficient maize genotypes, L22 and HS26  1113 (Table 3). No correlation was found between pH and P-Al solubilization (R ¼ 0.001; P < 0.001). The mineralization of organic P from acid phytic (P-phytate) is shown in Table 4. In general, no reduction in pH was observed. The species Talaromyces rotundus (F80, F102 and F105), A. terreus (F79) and B. cepacia (B116) were the most efficient in mineralizing P, but without significant difference. The strain of T. rotundus, F102 produced high levels of acid phosphatase. Among the microorganisms showing ability to mineralize P-lecithin, Penicillium citrinum (F95) and A. terreus (F93) were the most efficient solubilizing 44% and 42% of total P, respectively and were among the three best acid phosphatase producers (Table 5). 3.3. Quantification of P solubilization by P-Mehlich1 method P-solubilizing ability of microorganisms was also evaluated in liquid medium using P-Mehlich1 extractor (Mehlich, 1978). The amount of P solubilized varied significantly among the methods, with and without extractor, and among the isolates (Tables 2–5). In general, the values of soluble P detected by the method P-Mehlich1 for fungi, actinobacteria and bacteria were larger than for the conventional method of direct reading without extraction described by Murphy and Riley (1962). A large amount of a thick

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Table 2 Closest relatives using rDNA fragments, P solubilized, pH and phosphatase activity by microorganisms screened and isolated from samples of maize rhizosphere grown in liquid cultures containing P inorganic insoluble form tricalcium phosphate (P-Ca) Isolate (sample)a

Closest relatives database reference

Amount solubilized phosphate (mg P l1)b*

Species (accession number) – similarity index

Conventional methodc

P-Mehlich1 methodd

Acid

Alkaline

A4 (L3) A14 (L3) A19 (L22) A20 (L22) A26 (BRS3060) A29 (HS26  1113) B2 (L3) B5 (L3) B7 (L22) B17 (L3) B46 (BRS3060) B48 (HS26  1113) F14 (BRS3060)

Streptomyces platensis (AB163439.1) – 99% S. tumescens (AF346485.1) – 99% S. chartreusis (SCH399468) – 99% S. griseochromogenes (SGR310923) – 98% S. collinus (SCO306623) – 99% S. avermitilis (AB078897.2) – 98% Pantoea ananatis (AF364846.1) – 99% Burkholderia sp. (AY224513.1) – 97% Bacterium H3 (AY345547.1) – 97% Bacillus sp. (AF507879) – 89% Burkholderia cepacia (AY268142.1) – 90% B. cepacia (AY741360.1) – 96% Penicillium pinophilum (AF176660) – 98%

68.0 A abc 43.1 A abc 23.5 A abc 4.1 A ab 1.8 A a 3.7 A ab 81.8 A c 175.4 A d 77.1 A c 200.0 A d 70.6 A bc 64.3 A abc 25.9 A abc

39.8 A ab 29.6 A ab 37.3 A ab 30.5 A ab 9.4 A a 29.9 A ab 179.9 Bc 167.2 A c 63.0 A ab 211.1 A c 54.9 A ab 67.9 A b 8.9 A ab

1.88 a 0 0 0 0 0 2.62 a 0 0 0 0 0 0

0.59 a 1.89 a 0 0 0 0 7.24 a 0 0 6.15 a 18.71 a 70.98 b 0

Phosphatase activity (mg pNPP ml1 h1)*

*Means followed by the same letter did not differ significantly at 5% Tukey test. a Sample identification are according to Table 1. b Means followed by different higher case letter in each row represent the differences between the P-solubilizing isolates, and lower case letter in each column represents differences between the conventional method and the P-Mehlich1 method. c Amount of P solubilized by the Murphy and Riley’s (1962) procedure after 10 days of growth in a liquid medium containing 300 mg P l1 insoluble phosphate. d Amount of P solubilized extracted by the P-Mehlich1 procedure (Mehlich, 1978), after 10 days growth in a liquid medium containing 300 mg P l1 insoluble phosphate.

polysaccharide-like compound was observed in some flasks of bacteria and fungi, which could have functioned as an organic matrix adsorbing the P liberated by the microorganisms in the culture solution. 4. Discussion The frequency of isolates varied among the maize genotypes evaluated (Table 1). A greater number of microorganisms were isolated from the rhizosphere of the maize genotypes L3 and BRS3060, both considered P efficient by the Embrapa Maize and Sorghum Breeding Program (Parentoni et al., 2000). The most efficient P-Ca solubilizing isolates, identified as Bacillus sp. (B17), Burkholderia sp. (B5), and Streptomyces platensis (A4) were isolated from the rhizosphere of L3 maize. This result supports the hypothesis that these microorganisms could contribute to the efficiency of these P efficient maize genotypes in acquiring P from the soil. Several studies have indicated that the presence of different plant species or genotypes influence the microbial community due to the differential response of these organisms to different root signaling and exudation patterns, especially when plants are under environmental stress (Lynch and Whipps, 1990; Richardson, 1994) for example phosphorus stress (Hinsinger, 2001).

The aluminum phosphate solubilization rates were lower than the P-Ca mobilization (Table 3), in agreement with Narsian et al. (1994) that found lower rates of solubilizing from P-Al than P-Ca among eight microorganisms tested in tricalcium and aluminum phosphate. Similar results were also found by Whitelaw et al. (1999) and Barroso and Nahas (2005) that described this phenomenon is probably due to the higher solubility of the Ca phosphates in culture solutions. Some authors have pointed out that the phosphorus liberated from clay minerals with AlPO4 can increase the level of toxic Al3þ in solution (Illmer et al., 1995) and could possibly explain the suppression of the P-solubilizing activity of the PSM that was observed in the P-Al medium. A low correlation was observed between pH reduction and an increase of soluble P in the liquid culture after 10 days of growth indicating that the decline of the concentration of insoluble phosphorus might have been influenced by other factors (Whitelaw et al., 1999; Barroso and Nahas, 2005). In this study, the fungi were the most efficient in the liberation of P from P-phytate. Several authors have reported similar results, affirming that the genus Aspergillus was the most efficient in solubilizing phytate (Yadav and Tarafdar, 2003). A strain of Talaromyces (F102) producing high levels of acid phosphatase was isolated in this study and is the first report of this genus with the

Table 3 Closest relatives using rDNA fragments, P solubilized and phosphatase activity by microorganisms screened and isolated from samples of maize rhizosphere grown in liquid cultures containing P inorganic insoluble form, aluminum phosphate (P-Al) Isolate (sample)a

B52 (L3) B53 (L3) B56 (L22) B58 (L22) F39 (HS26  1113, L22) F40 (L22) F50 (HS26  1113)

Closest relatives database reference

Amount solubilized phosphate (mg P l1)b*

Species (accession number) – similarity index

Conventional methodc

P-Mehlich1 methodd

Acid

Pantoea ananatis (AF364846.1) – 96% Pantoea agglomerans (AY924376.1) – 89% Paenibacillus elgii (AY090110.1) – 98% Burkholderia cepacia (F3AY509957.1) – 95% Aspergillus terreus (AY373871.1) – 98% Penicillium pimiteouiense (AF037436.1) – 96% A. terreus (AJ413985.1) – 95%

10.3 13.3 0.03 20.3 14.9 12.1 13.7

10.8 14.6 16.0 38.4 16.3 20.8 18.3

3.57 0.36 0 0 48.1 57.9 50.2

A A A A A A A

b b a c bc b bc

Aa A ab B ab Bc A ab Bb A ab

Phosphatase activity (mg pNPP ml1 h1)* Alkaline a a

b b b

19.5 50.1 53.1 64.8 0 0 0

a b b b

*Means followed by the same letter did not differ significantly at 5% Tukey test. a Sample identification are according to Table 1. b Means followed by different higher case letter in each row represent the differences between the P-solubilizing isolates, and lower case letter in each column represents differences between the conventional method and the P-Mehlich1 method. c Amount of P solubilized by the Murphy and Riley’s (1962) procedure after 10 days of growth in a liquid medium containing 250 mg P l1 insoluble phosphate. d Amount of P solubilized extracted by the P-Mehlich1 procedure (Mehlich, 1978), after 10 days growth in a liquid medium containing 250 mg P l1 insoluble phosphate.

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Table 4 Closest relatives, P solubilized and phosphatase activity by microorganisms screened and isolated from samples of maize rhizosphere grown in liquid cultures containing P organic insoluble form, sodium phytate (P-phytate) Isolate (sample)a

Closest relatives database reference

Amount solubilized phosphate (mg P l1)b*

Species (accession number) – Similarity index

Conventional methodc

P-Mehlich1 method

Acid

Alkaline

B116 B118 B119 B121

Burkholderia cepacia (AY741355.1) – 98% Arthrobacter sp. (AF408967.1) – 99% Bacillus megaterium (AY167865.1) – 98% Uncultured gamma prokaryote (AJ575715.1) – 94% Pantoea agglomerans (AY924376.1) – 90% Arthrobacter sp. (AF408952.1) – 99% Aspergillus terreus (ATE413985) – 96% Talaromyces rotundus (AF285115) – 96% T. rotundus (AF285115) – 97% T. rotundus (AF285115) – 96%

52.7 A de 28.0 A abc 19.5 A ab 4.9 A a

92.8 B f 28.2 A ab 37.3 B bc 1.5 A a

0 0 46.4 bc 0

9.7 a 23.6 de 21.6 cd 20.9 cd

25.5 30.5 46.5 57.5 48.6 47.4

24.9 A ab 31.4 A b 9.1 A cd 67.6 A e 54.2 A cde 60.3 B de

0 0 26.4 ab 12.4 a 62.6 c 45.4 bc

31.9 20.7 10.8 13.4 12.0 12.2

(MGO) (MGO) (PGO) (GRGO)

B122 (GRGO) B124 (GRGO) F79 (SCRGO) F80 (SCRGO) F102 (SCRGO) F105 (SCRGO)

A A A A A A

abc bcd cde e cde cde

Phosphatase activity (mg pNPP ml1 h1)* d

e bcd a abc a ab

*Means followed by the same letter did not differ significantly at 5% Tukey test. a Sample identification are according to Table 1. b Means followed by different higher case letter in each row represent the differences between the P-solubilizing isolates, and lower case letter in each column represents differences between the Conventional method and the P-Mehlich1 method. c Amount of P solubilized by the Murphy and Riley’s (1962) procedure after 10 days of growth in a liquid medium containing 280 mg P l1 insoluble phosphate. d Amount of P solubilized extracted by the P-Melich1 procedure (Mehlich, 1978), after 10 days growth in a liquid medium containing 280 mg P l1 insoluble phosphate.

potential to mineralize P-phytate. The ability of soil microorganisms to solubilise various forms of inorganic P is well documented (Kucey et al., 1989; Richardson, 1994; Whitelaw et al., 1999; Goldstein et al., 2003); however, there have few reports regarding the potential of soil microorganisms to increase the availability of P from phytin. The results of this work clearly demonstrate the efficiency of newly identified microorganisms able to release P from phytate (P-phytin) and other organic P sources. The fungus A. terreus has been cited in the literature as an important phytase producing fungus (Mitchell et al., 1997). In this study, A. terreus was also able to solubilise P from aluminum phosphate, phytate and lecithin (Tables 3-5). T. rotundus was one of the highest phosphatase producers in the phytate (Table 4) and lecithin (Table 5) media, indicating that this fungus represents an important species for producing phytase and phospholipases. The bacteria Pantoea agglomerans and Burkholderia sp. identified in this study produced phosphatase and mineralized phytate (Table 4). P. agglomerans also solubilized P-Al (Table 3) and

Burkholderia sp. solubilized P-Ca (Table 2). According to Nacamulli et al. (1997), B. cepacia represents one of the predominant bacterial species among the microorganisms occurring in maize rhizosphere. Even though this is the first report of B. cepacia being capable of solubilizing unavailable sources of phosphorus in maize, several studies have shown that B. cepacia is able to compete with the indigenous microflora, survive, colonize roots of various maize cultivars (Nacamulli et al., 1997; Bevivino et al., 2000), enhance the yield of several crop plants (Rodrigues and Fraga, 1999; Baldani et al., 2001), antagonize and repress the major soil-borne fungal pathogens of maize (Bevivino et al., 2000). The mechanism or mechanisms involved in enhanced phosphorus solubilization by this bacteria need to be further investigated to utilize it as a biofertilizer in maize production systems. The utilization in this study of the P-Mehlich1 extractor to measure the quantity of P released indicates that the P solubilization estimated in part of this work and in other studies may have been underestimated and may have resulted in discarding isolates

Table 5 Closest relatives, P solubilized and phosphatase activity by microorganisms screened and isolated from samples of maize rhizosphere grown in liquid cultures containing P organic insoluble form, soybean lecithin (P-lecithin) Isolate (sample)a

A62 (CNPMS) A65 (CNPMS) A68 (JSP) A80 (MGO) A83 (MGO) B62 (CNPMS) B65 (JSP) B70 (MGO) B76 (SMRGO) B86 (JSP) B104 (SCRGO) F87 (MGO) F93 (SCRGO) F94 (SCRGO) F95 (SCRGO)

Closest relatives database reference

Amount solubilized phosphate (mg P l1)b*

Species (accession number) – similarity index

Conventional methodc

P-Mehlich1 methodd

Acid

Alkaline

Kitasatospora putterlickiae (AY189976.1) – 98% K. paracochleatus (U93328) – 99% Streptomyces ipomoeae (AY207593.1) – 98% S. hygroscopicus (SH16SRR) – 98% S. hygroscopicus (SH16SRR) – 98% Arthrobacter sp. (AF408967.1) – 99% Methylobacterium sp. (D32232.1) – 98% Arthrobacter sp. (AF408967.1) – 99% Uncultured bacterium (P reactor) (AF527601.1) – 96% Arthrobacter sp. (AY177350.3) – 97% Uncultured Bacillus sp. (AY242537.1) – 93% Acremonium strictum (AY138846.1) – 93% Aspergillus terreus (AF516138.1) – 98% Talaromyces rotundus (AF285115) – 95% Penicillium citrinum (AY373904.1) – 99%

14.9 A ab 17.4 A ab 19.0 A abc 2.1 A a 3.4 A a 2.1 a 0 0.6 A a 0 0.9 A a 6.7 A ab 29.8A bcd 42.9 B cd 24.0 A abcd 44.0 B d

13.2 A ab 24.9 A bc 22.4 A bc 24.4 B bc 34.3 B c 0 36.5 c 15.5 B ab 24.8 bc 24.4 B bc 7.1 A ab 23.8 A bc 24.7 A bc 20.3 A bc 20.2 A bc

76.1 cde 67.1 cde 53.9 abcd 66.1 bcde 64.6 bcde 5.1 ab 1.1 a 16.4 abc 0 26.0 abc 4.9 ab 77.5 cde 0 117.6 e 95.8 de

0 0 0 0 0 0.7 a 0.7 a 8.2 b 8.1 b 4.2 ab 7.9 b 19.4 c 0 0.8 a 0

Phosphatase activity (mg pNPP ml1 h1)*

*Means followed by the same letter did not differ significantly at 5% Tukey test. a Sample identification are according to Table 1. b Means followed by different higher case letter in each row represent the differences between the P-solubilizing isolates, and lower case letter in each column represents differences between the Conventional method and the P-Mehlich1 method. c Amount of P solubilized by the Murphy and Riley’s (1962) procedure after 10 days of growth in a liquid medium containing 101.1 mg P l1 insoluble phosphate. d Amount of P solubilized extracted by the P-Mehlich1 procedure (Mehlich, 1978), after 10 days growth in a liquid medium containing 101.1 mg P l1 insoluble phosphate.

C.A. Oliveira et al. / Soil Biology and Biochemistry 41 (2009) 1782–1787

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