Valverde Etal Plant and Soil (2006) 00:1–4

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3 Polyphasic characterization of phosphate-solubilizing bacteria isolated

4 from rhizospheric soil of the north-eastern region of Portugal

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6 A. Valverde, J.M. Igual & E. Cervantes1

7 Instituto de Recursos Naturales y Agrobiolog a de Salamanca, IRNA-CSIC, Cordel de Merinas, 40-52, 37008

8 Salamanca, Spain. 1Corresponding author*

9 Received j. Accepted in revised form j

10 Key words: phosphate solubilizing bacteria, Pseudomonas, rhizosphere, soil

11 Abstract

12 Phosphate-solubilizing microorganisms are often used as plant growth promoters. In the frame of a project

13 focused on the isolation of endosymbionts and ectorhizospheric bacteria associated withPhaseolus vulgaris

14 plants growing in a mountain soil at the north-east of Portugal, we obtained six bacterial strains with high

15 phosphate-solubilizing capability, as demonstrated by the formation of halos when plated in YED

16 medium supplemented with tricalcium-phosphate. These bacterial strains were characterized by a

17 polyphasic approach using both phenotypic (API 20 NE) and molecular assays (RAPD, with M13 primer,

18 TP-RAPD, and 16S rDNA sequencing). TP-RAPD yielded an identical band patterns in the six strains

19 indicating that they belong to the same bacterial species. The 16S rRNA sequence analysis of a

20 group-representative strain (P4-22) revealed a sequence similarity value of 99.27% with Pseudomonas

21 jessenii. Moreover, the RAPD fingerprints of these six strains did not show differences, indicating that they

22 form a highly homogenous group. This high homogeneity could be a consequence of the recurring agro-

23 nomical practices used in this region from the antiquity that include organic fertilization and monoculture

24 of Phaseolus vulgaris.

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2627 Introduction

28 The phosphorous is an essential plant nutrient

29 which is added to soil as soluble inorganic phos-

30 phates that, in a large portion, becomes insoluble

31 and, therefore, unavailable to plants (Singh and

32 Kapoor, 1994). Many species of bacteria are able

33 to solubilize phosphates in vitro and the most of

34 them live in the plant rhizosphere. At the present,

35 bacilli, rhizobia and pseudomonas are the most

36 studied P-solubilizers (Rodrguez and Fraga,

37 1999). Nevertheless, only a few number of species

38 belonging to the current genus Pseudomonas are

39known as P solubilizers. Within them, Pseudomo-

40nas putida (Kumar and Singh, 2001; Manna

41et al., 2001; Villegas and Fortin, 2002; Vivegan-

42andan and Jaurhi, 2000), P. aeruginosa (Musarrat

43et al., 2000), P. corrugata (Pandey and Palni,

441998), P. stuzteri (Va zquez et al., 2000) Pseudo-

45monas fluorescens are the most studied (Deubel

46et al., 2000; Di-Simine et al., 1998). Nevertheless,

47many rhizospheric phosphate solubilizing bacte-

48rial species remain unknown and more studies are

49needed to reveal the high biodiversity of these

50bacteria. Although the study of rhizospheric bac-

51teria is difficult because the high number of bacte-

52ria present in the soil, the characterization and

53identification of these bacteria are needed for

54wide ecological studies of the plant rhizosphere.* FAX No: +34-923219609.

E-mail: [email protected]

Journal : PLSO Dispatch : 26-6-2006 Pages : 4

Journal No. : 11104h LE h TYPESET

MS Code : 0108 h CP h DISK4 4

Plant and Soil (2006) 00:14 Springer 2006

DOI 10.1007/s11104-006-0108-y

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55 Materials and methods

56 Bacterial isolation and evaluation of their57 tricalcium-phosphate solubilizing capability

58 Phosphate-solubilizing bacteria were isolated

59 from rhizospheric samples by plating serial dilu-

60 tions of rhizospheric soil extracts in YED (yeast

61 extract 0.5%, glucose 1% and agar 2%) supple-

62 mented with 0.2% of tricalcium-phosphate

63 (YED-P). The plates were incubated at 28 C for

64 7 days. After this time the colonies surrounded

65 by a clear zone, that indicates the phosphate sol-

66 ubilization capability, were selected to obtain

67 pure cultures.

68 To measure the ability to solubilize trical-

69 cium-phosphate and to test the persistence of this

70 capability, each strain was plated in YED-P med-

71 ium and incubated for 7 days. The criterium for

72 strain selection was the size of clearing zone sur-

73 rounding the colonies of each strain and the sta-

74 bility of its phosphate-solubilizing capability after

75 five subcultures (Igual et al., 2001).

76 Phenotypic test

77 The selected strains were first subjected to the

78 commercial phenotypic assay API 20 NE. The79 API 20 NE test was carried out under the condi-

80 tions described by the manufacturer and identifi-

81 cation was done by the Apilab Plus v.3.3.3

82 software.

83 TP-RAPD and RAPD

84 The TP-RAPD profiles were obtained by using

85 the primers and conditions described by Rivas

86 et al. (2001). RAPD profiles were obtained by

87 using primer M13 (Schonian et al., 1993) under

88 the conditions previously described.

89 16S sequencing and analysis

90 DNA extraction was carried out as previously

91 described (Rivas et al., 2001). The amplification

92 of 16S rDNA and its sequencing was performed

93 according to the method already described (Ri-

94 vas et al., 2002). The sequence obtained was

95 compared with those from the GenBank using

96 the FASTA program (Pearson and Lipman,

97 1988).

98Mobilization of phosphorous in plants

99Experiments to study the P mobilization in com-100mon bean plants were carried out in plots con-

101taining vermiculate supplemented with insoluble

102tricalcium-phosphate (0.2% w:w) under green-

103house conditions. Six plots, containing six plants

104per plot, were inoculated as described below and

105other six plots were maintained as uninoculated

106controls.

107For inoculation, strain P4-22 was grown in

108Petri dishes with YED for 2 days. After that,

109sterile water was added to the plates to obtain a

110suspension with approximately 1011 cells ml)1

111and 1 ml of this suspension was added to each

112seedling.

113At harvest, 30 days after inoculation, shoot

114dry weights of common bean plants were deter-

115mined. Plant phosphate content was measured

116according to the A.O.A.C. methods (Johnson,

1171990). The data obtained were analysed by one-

118way analysis of variance, with the mean values

119compared using the Fishers Protected LSD

120(Least Significant Differences) (P = 0.05).

121Results and discussion

122Bacterial isolation and evaluation of their

123tricalcium-phosphate solubilizing capability

124Several bacterial strains were isolated from rhizo-

125spheric soil taken at Arcos de Valdevez (Portu-

126gal), where P. vulgaris is traditionally cultivated.

127Only six strains (P4-19 to P4-24) were able to

128solubilize actively phosphate in vitro and showed

129persistence of this capability after five or more

130subcultures. These phosphate-solubilizing strains

131were selected for further characterization.

132Phenotypic test

133The results of this test showed that all these bac-

134terial strains belong to the species Pseudomonas

135fluorescens. All strains isolated in this study uti-

136lize arabinose, mannose, mannitol, N-acethyl-D-

137glucosamine, gluconate, caprate, malate or citrate

138as sole carbon sources. By contrast, they do not

139grow in phenil-acetato nor adipate. This species

140actively produce urease, b-glucosidase, arginine

141dihydrolase and b-galactosidase. They do not

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142 reduce nitrate to nitrogen and not produce tri-

143 ptophan deaminase nor arginine dehidrolase.

144 TP-RAPD and RAPD

145 TP-RAPD yielded an identical band patterns in

146 the six strains indicating that they belong to the

147 same bacterial species. Moreover, the RAPD fin-

148 gerprints of these six strains did not show differ-

149 ences, indicating that they form a highly

150 homogenous group. Therefore, only the strain

151 P4-22 was selected for further experiments.

152 This high homogeneity could be a conse-

153 quence of the recurring agronomical practices

154 used in this region from the antiquity that in-155 clude organic fertilization and monoculture of

156 Phaseolus vulgaris.

157 16S rDNA sequence analysis

158 The strain P4-22 was identified at genus level

159 using 16S rDNA complete sequence. The com-

160 plete sequence of 16S rDNA was compared with

161 those from databanks using the FASTA program

162 (Pearson and Lipman, 1988). This comparison

163 showed a 99.27% whit P. jessenii (AF068259)

164 that is in disagreement with those results ob-

165 tained by using the commercial system API 20166 NE, due likely to the fact that this kind of iden-

167 tification systems have been designated for iden-

168 tification of clinical isolates but not for soil

169 bacteria.

170 Mobilization of phosphorous in plants

171 The results of the inoculation assays are shown

172 inTable 1. According to these results, no signifi-

173 cant differences in dry weight and total P were

174 observed between plants inoculated with strain

175 P4-22 and the uninoculated control plants.

176Moreover, it is observed a decrease in shoot dry

177weight and P content of the inoculated plants

178with respect to the uninoculated control plants179that may indicate some deleterious effects of this

180Pseudomonas strain on plant growth.

181In conclusion, the phosphate-solubilizing abil-

182ity in vitro is not always correlated with phos-

183phate mobilization to plant. In order to obtain

184efficient inoculants it is very important to identify

185correctly the target strain and to test its perfor-

186mance with the selected crops to avoid potential

187deleterious effect on plant growth.

188References189Deubel A, Gransee A and Merbach W 2000 Transformation of190organic rhizodepositions by rhizosphere bacteria and its191influence on the availability of tertiary calcium phosphate. J.192Plant Nutr. Soil Sci. 163, 387392.193Di-Simine C D, Sayer J A and Gadd G M 1998 Solubilization194of Zinc phosphate by a strain ofPseudomonas fluorescens195isolated from a forest soil. Biol. Fertil. Soils 28, 8794.196Igual J M, Valverde A, Cervantes E and Vela zquez E 2001197Phosphate-solubilizing bacteria as inoculants for agriculture:198use of updated molecular techniques in their study. Agron-199omie 21, 561568.200Jonhson F J 1990 Detection method of nitrogen (total) in201fertilizers. In Methods of Analysis of the Association of202Official Analytical Chemists. Ed. K Elrich. pp. 1719.

203Association of Official Analytical Chemists, USA. 204Kumar V and Singh K P 2001 Enriching vermicompost by205nitrogen fixing and phosphate solubilizing bacteria. Biore-206sour. Technol. 76, 173175.207Manna M C, Ghosh P K, Ghosh B N and Singh K N 2001208Comparative effectiveness of phosphate-enriched compost209and single superphosphate on yield, uptake of nutrients and210soil quality under soybeanwheat rotation. J. Agric. Sci. 137,2114554.212Musarrat J, Bano N and Rao R A K 2000 Isolation and213characterization of 2,4-dichlorophenoxyacetic acid-catabo-214lizing bacteria and their biodegradation efficiency in soil.215World J. Microbiol. Biotechnol. 16, 495497.216Pandey A and Palni L M S 1998 Isolation of Pseudomonas217corrugata from Sikkim Himalaya. World J. Microbiol.218Biotechnol. 14, 411413.219Pearson W R and Lipman D J 1988 Search for DNA220homologies was performed with the FASTA program. Proc.221Natl. Acad. Sci. USA 85, 24442448.222Rivas R, Vela zquez E, Valverde A, Mateos P F and Martnez-223Molina E 2001 A two primers random amplified polymor-224phic DNA procedure to obtain polymerase chain reaction225fingerprints of bacterial species. Electrophoresis 22, 10862261089.227Rivas R, Vela zquez E, Willems A, Vizcano N, Subba-Rao N S,228Mateos P F, Gillis M, Dazzo F B and Martnez-Molina E2292002 A new species of Devosia that forms a nitrogen-fixing230root-nodule symbiosis with the aquatic legume Neptunia231natans (L. f.) Druce. Appl. Environ. Microbiol. 68, 52172325222.

Table1. Total P per plot

Strain Dry weight

(mg plot)1)

Total P

(mg plot)1)

P4-22 6115a 3.04a

Control 6305a 3.56a

Values followed by the same letter are no significantly different

from each other at P = 0.05 according to Fishers Protected

LSD (Least Significant Differences).

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233 Rodrguez H and Fraga R 1999 Phosphate solubilizing bacteria234 and their role in plant growth promotion. Biotechnol. Adv.235 17, 319339.236 Schonian G, Meusel O, Tietz H J, Meyer W, Graser Y, Tausch237 I, Presber W and Mitchell T G 1993 Identification of clinical238 strains of Candida albicans by DNA fingerprinting with239 polymerase chain reaction. Mycoses 36, 171179.240 Singh S and Kapoor K K 1994 Solubilization of insoluble241 phosphates by bacteria isolated from different sources.242 Environ. Ecol. 12, 5155.243 Va zquez P, Holguin G, Puente ME, Lo pez-Cortez A and244 Bashan Y 2000 Phosphate solubilizing microorganisms

245associated with the rhizosphere of mangroves in a semiarid246coastal lagoon. Biol. Fertil. Soils 30, 460468.247Villegas J and Fortn J A 2002 Phosphorous solubilization and248pH changes as a result of the interactions between soil249bacteria and arbuscular mycorrhizal fungi on a medium250containing NO3

) as nitrogen source. Can. J. Bot. 80, 571251576.252Viveganandan G and Jaurhi K S 2000 Growth and survival of253phosphate-solubilizing bacteria in calcium alginate. Micro-254biol. Res. 155, 205207.

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