CORE Metadata, citation and similar papers at core.ac.uk Provided by CONICET Digital Brazilian Journal of Microbiology 45, 2, 585-594 (2014) Copyright © 2014, Sociedade Brasileira de Microbiologia ISSN 1678-4405 www.sbmicrobiologia.org.br Research Paper Arbuscular mycorrhizal fungi in saline soils: Vertical distribution at different soil depth Alejandra Becerra1, Norberto Bartoloni2, Noelia Cofré1, Florencia Soteras1, Marta Cabello3 1Instituto Multidisciplinario de Biología Vegetal, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Córdoba, República Argentina. 2Facultad de Agronomía, Universidad de Buenos Aires, Buenos Aires, República Argentina. 3Instituto Spegazzini, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata, República Argentina. Submitted: March 7, 2013; Approved: March 14, 2014. Abstract Arbuscular mycorrhizal fungi (AMF) colonize land plants in every ecosystem, even extreme condi- tions such as saline soils. In the present work we report for the first time the mycorrhizal status and the vertical fungal distribution of AMF spores present in the rhizospheric soil samples of four species of Chenopodiaceae (Allenrolfea patagonica, Atriplex argentina, Heterostachys ritteriana and Suaeda divaricata) at five different depths in two saline of central Argentina. Roots showed medium, low or no colonization (0-50%). Nineteen morphologically distinctive AMF species were recovered. The number of AMF spores ranged between 3 and 1162 per 100 g dry soil, and AMF spore number de- creased as depth increased at both sites. The highest spore number was recorded in the upper soil depth (0-10 cm) and in S. divaricata. Depending of the host plant, some AMF species sporulated mainly in the deep soil layers (Glomus magnicaule in Allenrolfea patagonica, Septoglomus aff. constrictum in Atriplex argentina), others mainly in the top layers (G. brohultti in Atriplex argentina and Septoglomus aff. constrictum in Allenrolfea patagonica). Although the low percentages of colo- nization or lack of it, our results show a moderate diversity of AMF associated to the species of Chenopodiaceae investigated in this study. The taxonomical diversity reveals that AMF are adapted to extreme environmental conditions from saline soils of central Argentina. Key words: arbuscular mycorrhiza, saline environments, soil profile, vertical distribution, mycor- rhizal status. Introduction the characteristics of marginal lands for agriculture and In central Argentina 9% of the area is occupied by livestock and no stable human population within it, have fa- halophytic vegetation (Cabido and Zak, 1999). This type of cilitated the preservation of this pristine ecosystem. Within vegetation grows in habitats that are rare worldwide, since these saline habitats, the distribution patterns of plant com- only approximately 7% of the global land surface is cov- munities are defined by the salt gradient, with plant cover ered with saline habitats (Ruíz-Lozano and Azcón, 2000). inversely proportional to the presence of salt. At sites where Central Argentina presents some conspicuous salt flats: the Salinas Grandes and the Salinas de Ambargasta, which to- plant life is still possible, the most characteristic plant com- gether occupies an area of approximately 600,000 hectares. munity is the halophytic shrub or jumeal, composed of spe- The environmental isolation, the harsh climatic conditions, cies of the Chenopodiaceae family (Cabido and Zak, 1999). Send correspondence to A. Becerra. Instituto Multidisciplinario de Biología Vegetal, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, C.C. 495. 5000 Córdoba, República Argentina. E-mail: [email protected]. 586 Becerra et al. According to Juniper and Abbott (1993), high salinity the understory vegetation is represented by Larrea in soils has adverse effects on plant colonization by arbus- divaricata Cav. and some halophytes. cular mycorrhizal fungi (AMF). However, there are reports Our studied area, the edge of the salt flat, shows in the literature from all over the world that plants of salt heavy constraints to the developing of any type of plant marshes can be colonized by AMF (Hildebrandt et al., cover, being extremely open and scarce with the only pres- 2001; Juniper and Abbott, 1993; Landwehr et al., 2002; ence of four species adapted to harsh environment: Smith and Read, 2008; Wang et al., 2004). Even in families Allenrolfea patagonica (Moq.) Kuntze, Atriplex argentina that are generally considered non-mycorrhizal, such as Speg., Heterostachys ritteriana (Moq.) Moq. and Suaeda Chenopodiaceae (Gerdemann, 1968; Hirrel et al., 1978; divaricata Moq. (Cabido and Zak, 1999; Cabido et al., Mohankumar and Mahadevan, 1987; Peterson et al., 1985), 2006). Sampling was made in an area of approximately 50 x the most salt-tolerant Salicornia sp. and Suaeda maritima 50 m in the two sites in end of the growing season (March, can be colonized (Kim and Weber, 1985; Rozema et al., Summer) because during this period the plants present their 1986; Sengupta and Chaudhuri, 1990). full splendor (with flowers). Soil samples were randomly Ecological studies on the community structure of and carefully taken with a metal corer (3 cm of diameter) AMF are generally restricted to the top 20 cm of soil, where from under the canopy of five plants of each species to con- most of the root biomass is concentrated (Brundrett, 1991). firm connection between roots and shoots. The five soil Only a few studies included the subsoil. Mycorrhizal colo- samples per species were considered replicates. nization (Jakobsen and Nielsen, 1983; Rillig and Field, Samples were collected from 0 to more than 40 cm in 2003), infective propagules (Ann et al., 1990), extra-radical depth, at 10-cm intervals (at 0-10, 10-20, 20-30, 30-40, and mycelium (Kabir et al., 1998) and AMF spores (Oehl et al., 40-50 cm depths) in each site. Samples from each layer 2005) decrease with increasing soil depth. Few studies have (620 cm3 soil volume) of each replicate were placed in indi- documented what happens with AMF diversity along soil vidual plastic bags and stored at 4 °C. profile. Cooke et al. (1993), Oehl et al. (2005), Cuenca and To characterize the soil from each site, four soil sam- Lovera (2010) and Wang et al. (2004) have published the ples per depth level were taken and the following parame- species diversity and distribution across the soil profile in ters were measured: electrical conductivity (mmhos/cm), salt marsh grasses in the United States, in cultivated soils of extractable P determined with the method of Bray and Central Europe, tropical soils of Venezuela and in the Yel- Kurtz I (Jackson, 1964), pH in water (1:2.5), organic matter low River Delta of China, respectively. Until now, nothing content (Nelson and Sommers, 1982), carbon: nitrogen ra- has been reported about vertical distribution of AMF com- tio and soil texture. Total nitrogen was determined using munities in natural saline soils of Central Argentina. the micro-Kjeldhal method (Bremner and Mulvaney, In the present work we report for the first time the 1982). mycorrhizal status and the vertical fungal spores in four species of Chenopodiaceae (Allenrolfea patagonica (Moq.) AMF colonization and spores Kuntze, Atriplex argentina Speg., Heterostachys ritteriana Fresh roots were rinsed with water, cleared with 10% (Moq.) Moq. and Suaeda divaricata Moq.) in two saline KOH (15 min at 90 °C) and bleached with 30% H2O2 soil of central Argentina. Species of Chenopodiaceae are (10 min, room temperature), acidified with 1% HCl (1 min, the only plants able to growth in such extremophilus condi- room temperature) and stained for 5 min in 0.05% trypan tions and in Argentina ecosystems have not been previ- blue (Phillips and Hayman, 1970). To confirm mycorrhizal ously examined for AMF presence. structure, in a second stage we then mounted the roots on glass slides for examination under a Kyowa 4-100x micro- Materials and Methods scopic. The presence of arbuscules, vesicles, hyphal coils and intra- and intercellular hyphae without septa were used Study area and sample collection to designate AM associations. Quantification of AM root The study was conducted in two saline “Salinas de colonization was estimated visually as the proportion of the Ambargasta” -SA- (64º18’ W, 29º27’ S) and “Salinas root which was colonized and characterized using five Grandes” -SG- (64º31’ W, 29º44’ S), in the north of Cór- classes: very high (> 80%), high (60-79%), medium (40- doba Province, central Argentina. The climate in both sites 59%), low (20-39%), and very low (1-19%), following is dry and warm, with a mean annual precipitation below Zangaro et al. (2002). 500 mm and mean temperature of 19.9 °C. The highest ar- AMF spores were extracted from 100 g (dry weight) eas (170 masl, with low salt concentration) are occupied by of each soil sample by wet sieving and decanting (Ger- a xerophytic forest of Aspidosperma quebracho-blanco demann and Nicolson, 1963), and the supernatant was cen- Schltdl., Prosopis flexuosa DC., Cercidium australe trifuged in a sucrose gradient (Walker et al., 1982). The Johnst., Mimozyganthus carinatus (Grisseb.) Bukart, procedure included passage through 500-, 125-, and 38-mm Ziziphus mistol Griseb., Prosopis torquata (Cav. ex Lag.) sieves. The 500-mm sieve was checked for large spores, DC., and Stetsonia coryne (Salm-Dyck) Britton & Rose; spore clusters, and sporocarps. The contents of the 125- and Arbuscular mycorrhizal fungi in saline soils 587 38-mm sieves were layered onto a water-sucrose solution intraradical aseptate hyphae, intracellular hyphal coils and (70% [wt/vol]) gradient and centrifuged at 900 x g for intracellular vesicles in both sites at all soil depths. 2 min. Only apparently healthy spores (those that contained Arbuscules were not detected. AM colonization in plant cytoplasm, with no collapsed surface and no evidence of species was very low to low (5-31% and 2-37%) in A. parasitism) were counted under stereomicroscope directly. patagonica and S.