REGULAR ARTICLE Mycorrhiza and soil bacteria influence extractable iron and manganese in soil and uptake by soybean M. A. Nogueira & U. Nehls & R. Hampp & K. Poralla & E. J. B. N. Cardoso Received: 10 April 2006 /Accepted: 1 August 2007 / Published online: 28 August 2007 # Springer Science + Business Media B.V. 2007 Abstract Excess manganese (Mn) in soil is toxic to crops, but in some situations arbuscular mycorrhizal fungi (AMF) alleviate the toxic effects of Mn. Besides the increased phosphorus (P) uptake, mycorrhiza may affect the balance between Mn-reducing and Mn- oxidizing microorganisms in the mycorrhizosphere and affect the level of extractable Mn in soil. The aim of this work was to compare mycorrhizal and non- mycorrhizal plants that received extra P in relation to alleviation of Mn toxicity and the balance between Mn-oxidizing and Mn-reducing bacteria in the mycor- rhizosphere. A clayey soil containing 508 mg kg-1 of extractable Mn was fertilized with 30 mg kg-1 (P1) or 45 mg kg-1 (P2) of soluble P. Soybean (Glycine max L. Merrill, cv. IAC 8-2) plants at P1 level were non- inoculated (CP1) or inoculated with either Glomus etunicatum (GeP1) or G. macrocarpum (GmP1), while plants at P2 level were left non-inoculated (CP2). Plants were grown in a greenhouse and harvested after 80 days. In the mycorrhizosphere of the GmP1 and GeP1 plants a shift from Mn-oxidizing to Mn-reducing bacteria coincided with higher soil extractability of Mn and Fe. However, the occurrence of Mn-oxidizing/reducing bacteria in the (mycor) rhizosphere was unrelated to Mn toxicity in plants. Using 16S rDNA sequence homologies, the Mn- reducing isolates were consistent with the genus Streptomyces. The Mn-oxidizers were homologous with the genera Arthrobacter, Variovorax and Ralstonia. While CP1 plants showed Mn toxicity throughout the whole growth period, CP2 plants never did, in spite of having Fe and Mn shoot concentrations as high as in CP1 plants. Mycorrhizal plants showed Mn toxicity symptoms early in the growth period that were no longer visible in later growth stages. The shoot P concentration was almost twice as high in mycorrhizal plants compared with CP1 and CP2 plants. The shoot Mn and Fe concentrations and contents were lower in GmP1 and GeP1 plants compared with the CP2 treatment, even though levels of extractable metals increased in the soil when plants were mycorrhizal. This suggests that mycorrhiza protected its host plant from excessive uptake of Mn and Fe. In addition, Plant Soil (2007) 298:273���284 DOI 10.1007/s11104-007-9379-1 Responsible Editor: Hans Lambers. M. A. Nogueira (*) CCB/Department of Microbiology, State University of Londrina, Lab. for Microbial Ecology, CP 6001, 86051-990 Londrina, PR, Brazil e-mail: nogueira@uel.br U. Nehls : R. Hampp University of T��bingen, Institute of Botany/Physiological Ecology of Plants, Auf der Morgenstelle 1, 72076 T��bingen, Germany K. Poralla Department of Biology, University of T��bingen, Auf der Morgenstelle 28, 72076 T��bingen, Germany E. J. B. N. Cardoso Department of Soil Science, University of S��o Paulo, ESALQ, CP 09, 13418-900 Piracicaba, SP, Brazil

higher tissue P concentrations may have facilitated internal detoxification of Mn in mycorrhizal plants. The exact mechanisms acting on alleviation of Mn toxicity in mycorrhizal plants should be further investigated. Keywords Fe . Metal . Mn . Oxidation . Reduction . Toxicity Abbreviations AMF arbuscular mycorrhizal fungi CFU colony forming units CP1 non-mycorrhizal control at P level 1 (30 mg kg-1) CP2 non-mycorrhizal control at P level 2 (45 mg kg-1) GmP1 Glomus macrocarpum at P level 1 GeP1 Glomus etunicatum at P level 1 PCR polymerase chain reaction Introduction Manganese (Mn) is an essential trace element for all living organisms, but at high concentrations it is toxic, even for microorganisms (Nealson et al. 1988). The oxidized forms of Mn and iron (Fe) are insoluble, but solubility and availability in soil increase with increas- ing state of reduction (Marschner 1995). Mn oxidation in soil is a biological reaction, while Mn reduction may be either chemical or biological (Ghiorse 1984). In addition, Mn oxidation may support chemolitho- trophic growth because it is an exergonic reaction, although microbes may also oxidize Mn to detoxify a surplus of Mn2+ in the surrounding medium (Nealson et al. 1988). Pseudomonas fluorescens GB1 probably oxidizes Mn enzymatically (Okazaki et al. 1997), as does Leptothrix discophora for Fe oxidation (Corstjens et al. 1992). Other authors (Posta et al. 1994) found that bacteria from the genera Pseudomonas, Bacillus, and Actinomyces were capable of reducing Mn. Thus, some bacteria from the same genus, such as Pseudomonas, may act as either Mn-oxidizers or Mn-reducers. Besides bacteria, some soil-borne fungi may also oxidize Mn in soil (Thompson et al. 2005). Plants colonized by arbuscular mycorrhizal fungi (AMF) are frequently more resistant to excess Mn than non-mycorrhizal plants (Nogueira et al. 2002, 2004 Nogueira and Cardoso 2002 Yano and Takaki 2005). The improved plant P nutritional status may facilitate detoxification of Mn within the plant by cellular mechanisms (Cardoso 1996 Hall 2002 Nogueira et al. 2002). In addition, the formation of low-solubility complexes between P and Mn may also reduce Mn toxicity (Foy 1984). An increased growth of mycorrhizal plants may lead to decreased concen- trations of Mn in the plant tissue due to dilution (Nogueira et al. 2004). Moreover, mycorrhiza- induced changes in the balance between Mn-reducing and Mn-oxidizing bacteria in the mycorrhizosphere could have an impact on the availability of Mn (Marschner 1988) Kothari et al. (1991) observed a decrease of Mn-reducing bacteria in the rhizosphere of mycorrhizal plants, which coincided with a decrease of Mn concentration in plants. The aim of this work was to compare the alleviation of Mn toxicity either in mycorrhizal plants or in non-mycorrhizal plants that received extra P in a soil with a naturally excessive concentration of extractable Mn. In addition, we also assessed the mycorrhizal effect on bacteria active in Mn oxidation and reduction in soil. The hypothesis tested was that mycorrhiza affects the balance between Mn-oxidizing and Mn-reducing bacteria in the soil, what may affect the levels of extractable Mn. Materials and methods Experimental set up Soil samples were taken from the 0���20 cm depth of a Typic Rhodudalf (Soil Survey 1999) in an area previously cropped with soybean. After sieving (mesh size 4 mm), the soil was autoclaved for 2 h at 121��C to eliminate the native AMF. The chemical properties (Sparks et al. 1996) of the soil were: pH 6.1 (in 0.01 M CaCl2) organic matter: 23 g kg-1 P and S: 16 and 19 mg kg-1, respectively K, Ca and Mg: 3.3, 57 and 16 mmolc kg-1. Extractable Mn (Mehlich I���Mehlich 1953) was 43 mg kg-1 before autoclaving and increased to 508 mg kg-1 thereafter. The experimental design was completely randomized with four treatments and 20 replications. Pots were filled with 4 kg of soil and supplied with 100 mg kg-1 of K as KCl. Soluble P was added at two levels to the soil: 30 (P1) or 45 (extra P supply, P2) mg kg-1 as ground triple super phosphate (particle size0.25 mm). Pots 274 Plant Soil (2007) 298:273���284