ALVES, V. M. C.

COELHO, A. M.

GOMES, E. A.

MARRIEL, I. E.

NEVES, A. A.

OLIVEIRA-PAIVA, C. A.

RAPOSEIRAS, R.

SCHAFFERT, R. E.

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Mycorrhizal Diversity of Acaulosporaceae and Glomaceae Famílíesin the Sorghum Rhizosphere in Brazilian Acid SoilsIE Marrielh , R Raposeiras, EA Gomes! , AC Oliveira! , CA Oliveira", VMC Alves' ,AA Neves! , AM Coelho! and RE Schaffert'I. Embrapa Maize and Sorghum, CP 15\,35701-970 Sete Lagoas, MG, Brazil.2. University Center Sete Lagoas, Av Marechal Castelo Branco, 2765 Sete Lagoas, MG, Brazil.* Corresponding author , imarriel@ cnprns. embrapa. brABSTRACTThe genetic diversity of arhuscular mycorrhizal fungi (AMF) was characterized in the rhizosphere of two sorghum genotypescontrasting for tolerance to aluminum toxicity, using molecular techniques. The plants were cultivated in an oxisol of lheBrazilian cerrado with three levels of aluminum saturation (O, 20% and 40% ). DNA samples were amplified based on nestedPCR using fungal universal prirners and specific prirners for Glomaeeae and Acaulosporaceae families, DGGE profiles clearlyrevealed that the diversity (number of amplicons ) found was higher for Acaulosporaceae than Glomaceae. Both the sorghumcultivar and the level of aluminum saturation affected the AM fungi community structure.INTRODUCTIONTheexpansion of Brazilian agriculture has beenincreasing in the cerrado biome where there are extensiveareas of potential new agricultural land. Oxisol , the mostcommon soil type in this ecosystem , presents a set ofchernical constraint limiting plant growth and development,particularly , low pH, low levels of nutrients and highphosphorus adsorption capacity (Novais & Smith , 1999).Fertilizer applied to these soils , particularly phosphates ,presents low utilization efficiency by plants and ISresponsible for a significant portion of production cost.The use of acid soil tolerant sorghum cultivars providesan environmentally and eeonomical friendly componenteontributing to sustainable crop production. Plants havedeveloped several physiological and biochemical systems ofadaptation to Pi-deficiency stress, Marschner ( 1991) hasconcluded lha! the strategy of avoidance , such as: I) root-induced changes in the rhizosphere such as pH increase ,2) release of chelators for AI and increased acti vity ofectoenzymes (aeid phosphatases), and 3) increase in rootsurfaee area via mycorrhizae. Mycorrhyzal symbiosis , amutualistie plant-Iungus , is an essential feature of thebiologv arul «cology of most terrestrial plants , since itinfluences their growth 111 a multifunctional nature( Rosendahl, 2008; Nielson et al. , 2004; Smith and Read,1997; N,'wshalll et aI., 1995). Molecular studies haveheeu used with "UC"ASS to invcstigate the phylogeneticrelutionship and geneti« variation among the glomalean fungi( ~illl()lI, 1996; Sanders et aI. , 1996; Lanfraneo et a!. ,1998) and for ideutifving lhe preseu(;e of three families ofthe Glomales ( Clapp et al. , J 995; Rodecker, 2000) , usingribosomal RNA (rRNA) genes and polymerase chainreaction-PCRI denaturing gradient gel electrophoresis-DGGE( Muyzer and Smalla, 1998).Studies of the soil root interface are essential for a betterunderstanding of the mechanisms by which plants adapt toacid and mineral soils. The characterization andidentification of dominant groups of AMF and their role inthe rhizosphere of adapted rnaize and sorghum genotypes toBrazilian acid soil are currently poorly understood. Theobjective of this study was , 1) to analyze the AMFcomrnunities associated with sorghum plants in acid soils ,2) to compare the inter- and intra-family genetic diversity ofAMF in sorghum rhizosphere , and 3) to determine theinfluence of aluminum saturation in the soil on AMFcommunity structure in the sorghum rhizosphere of acidsoils.MATERIALS AND METHODSSamplingOxisol samples were taken from the rhizosphere of twosorghum lines contrasting in aluminum tolerance (G 1-tolerant aru] G2-sensible) developed by the Embrapa Maizeand Sorghum , Minas Gerais, Brazil, located at latitude 19"'28'S and longitude 44" 15'W. The genotypes were grown ina typical oxisol , with three levels of aluminum saturation (O,low , 20% , intermediate; 40%, high). The soil adheringto the roots of five plants/plot was pooled and considered asthe rhizosphere soi!. FOT control, soil without roots (bulksoil) was used, also collected 90 days aíter sorghumemergence. The samples were kept at - 20"C until DNA39extraction.Molecular and data analysisSoil samples of 500 mg were used for total DNAextraction using BIO 101 Kit protocols, following themanufacturer's recommendations. AMF community structureswere examined by PCR-DGGE. The small subunit (SSU)ribosomal RNA gene was amplified from a total soil DNAextract based on nested PCR using fungal universal primers(NS5 and 'ITS4) and ITS/Glomaceae and ITS/Acaulosporaceae specific primers attached with the sequenceclamp, ITS2/GLOM131OCG and ITS2/ACAU1660 CG,respectively (Redecker, 2(00). PCR products were loadedby electrophoresis in a 6% polyacrilamide gel composed ofdenaturing gradients between 45% and 70% of urea-formamide gels (DGGE) and run for 16 h in a BIO-RADDcode System, VA, USA. Cluster analysis was performedusing the Jaccard's coefficient of similarity and theunweighted pair group method (UPGMA) using the NTSYSsoftware package (version 2. 02) .RESUL TS AND DISCUSSIONThe plant exudation pattem affects the structural andfunctional diversity of rhizosphere microbial communities( 12 ) . Based' on DGGE pattems, the data allow thecomparison of variation in the AM fungal communitycomposition among plants genotypes, between the presenceand absence of plants and 'between levels of aluminumsaturation. There was a strong shift in the mycorrhizalcommunities due to AI saturation, principally in the bulk soilsamples and in the rhizosphere soil for the Acaulosporaceaefamily, with reduced diversity in the high AI saturationenvironment. An apparent paradoxical increase in AMFrichness was noted in the tolerant sorghum genotype, with ahigh diversity AMF at the high AI saturation level forGlomaceae. The results also revealed a higher diversity forthe Acaulosporaceae family than the Glomaceae family. Thecluster analysis identified two principal groups forGlomaceae; group I included samples of soil without roots,and group II included the rhizosphere soil samples. Threeprincipal groups encountered for Acaulosporaceae , group I,rhizosphere soil samples without aluminum; group Il, bulksoil without plants, and group III, samples from rhizospheresoil in the presence of Al3 +. The data show the influencealuminum saturation and sorghum genotype on the40composition of the AMF studied. The high diversity andvariation of the AMF observed in this study indicate theirimportance ecologically and thus may have the potential toinfluence adaptation of sorghum cultivars to acid soil, Thereis evidence that AMF may be functionally distinct inphosphorus acquisition and overall benefit to the host plant(Sanders et alo , 1996). This study showed that the fungalcommunity composition in the rhizosphere is affected by acomplex interaction between sorghum plant genotypes, AMfungi genotypes and aluminum saturation; and that there is ahigher diversity of Acaulosporaceae than Glomaceae in thesorghum rhizosphere of an acid soil.REFERENCESClapp JP, Young JPW, Merryweather JW, et alo 1995.Diversity of fyngal symbionts in arbuscular mycorrhizasfrom a natural community. New Phytol, 130: 259 -265.Grayston SJ, Wang S, Campbell CD, et alo 1998. Selectiveinfluence of plants species on microbial diversity in therhizosphere. Soil Biol Biochem, 30 (3) : 369 - 378.Lynch JM, Whipps JM. 1990. Substrate flow in therhizosphere. Plant Soil, 129: 1 -10.Muyzer G, Smalla K. 1998. Application of denaturinggradient gel electrophoresis (DGGE) and temperaturegradient gel electrophoresis (TGGE) in microbialecology. Antonie van Leeuwenhoek, 73: 127 - 141. .Newsham KK, Filter AH, Watkinson AR. 1995a. Multi-functionality and biodiversity in arbuscular mycorrhizas.Trends Ecol Evol, 10: 407 -41l.Redecker D. 2000. Specific PCR prmers to identifyarbuscular mycorrhizal fungi within colonized roots.Mycorrhiza, 10: 73 - 80.Rosendahl S. 2008. Communities, populations andindividuals of arbuscular mycorrhizal fungi. New Phytol,178 (2): 253 -266.Simon L. 1996. Phylogeny of the glomales , Deciphering thepast to understand the present. New Phytol, 133: 95 -1Ol.Smith SE, Read DJ. 1996. Mycorrhizal Symbiosis 2nd edn.Academic Press, san Diego.Smith SE, Read DJ. 1997. Mycorrhizal symbiosis. London,UK: Academic Press.Wang B. and Qiu YL. 2006. Phylogenetic distribution andevolution of mycorrhizas in land plants, 16 (5): 1432-1890.

Mycorrhizal diversity of acaulosporaceae and glomaceae families in the sorghum rhizosphere in Brazilian acid soils.

Mycorrhizal diversity of acaulosporaceae and glomaceae families in the sorghum rhizosphere in Brazilian acid soils.

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