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Micología Aplicada International ISSN: 1534-2581 [email protected] Colegio de Postgraduados México

Pagano, M . C.; Scotti, M. R. A survey of the arbuscular mycorrhiza occurrence in paepalanthus bromelioides and Bulbostylis sp. in rupestrian fields, Brazil Micología Aplicada International, vol. 21, núm. 1, enero, 2009, pp. 1-10 Colegio de Postgraduados Puebla, México

Available in: http://www.redalyc.org/articulo.oa?id=68521101

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A s u r v e y o f t h e a r b u s c u l a r m y c o r r h i z a o c c u r r e n c e i n Pa e p a l a n t h u s b r o m e l i o i d e s a n d Bu l b o s t y l i s s p . i n r u p e s t r i a n f i e l d s , Br a z i l

M. C. Pa g a n o a n d M. R. Sc o t t i

Institute of Biological Sciences, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Pampulha, CEP: 31270-901, Belo Horizonte, MG, Brazil. Tel.: 55 31-34092680. Fax: 55 31-34092671. E-mail: [email protected]

Accepted for publication September 29, 2008

ABSTRACT

This study reports the percentage of mycorrhizal colonization, as well as the spore density of arbuscular mycorrhizal fungi in Paepalanthus bromelioides (Eriocaulaceae) and Bulbostylis sp. (Cyperaceae) in southeastern Brazil. Soil and root samples were collected in 2006. Spores of arbuscular mycorrhizal fungi were analyzed for species identification and roots samples for mycorrhizal colonization. Both plant species were mycotrophic showing arbuscular mycorrhizal fungi. Three genera of arbuscular mycorrhizal fungi found in rooting-zone soils were Glomus (two species), Acaulospora (one species) and Scutellospora (one species). Glomus was the dominant genus, and G. brohultii was the most common species. The average spore density of arbuscular mycorrhizal fungi was 77-139 per 100 g dry soil, and the richness was 3 to 4 species of arbuscular mycorrhizal fungi per sample. Both plant species showed high rhizosphere spore densities, and dominant hyphae and vesicle colonization. The diversity of arbuscular mycorrhizal fungi was found to be low. In P. bromelioides roots only hyphae and vesicles were observed.

Key words: Arbuscular mycorrhizal fungi, Brazil, Bulbostylis, Cyperaceae, Eriocaulaceae, highlands region, Paepalanthus bromelioides, rupestrian fields. Mi c o l . Ap l . In t ., 21(1), 2009, p p . 1-10 2 M. C. Pa g a n o a n d M. R. Sc o t t i

Es t u d i o d e l o s h o n g o s micorrízicos a r b u s c u l a r e s e n Pa e p a l a n t h u s b r o m e l i o i d e s y Bu l b o s t y l i s s p . e n c a m p o s r u p e s t r e s , Br a s i l

RESUMEN

Este estudio registra el porcentaje de colonización micorrízica, así como la densidad de esporas, de hongos micorrízico arbusculares de Paepalanthus bromelioides (Eriocaulaceae) y Bulbostylis sp. (Cyperaceae) en el sureste de Brasil. El muestreo del suelo y raíces de estas especies se realizó en 2006. Se identificaron las esporas de hongos micorrízico arbusculares y se evaluó la colonización en las raíces. Las dos especies vegetales mostraron micotrofía. Los tres géneros de hongos micorrízico arbusculares encontrados en los suelos rizosféricos fueron: Glomus (dos especies), Acaulospora (una especie) y Scutellospora (una especie). Glomus fue el género dominante y G. brohultii fue la especie más común. La densidad promedio de esporas fue 77-139 por 100 g de suelo seco, mientras que la riqueza específica fue de 3 a 4 especies de hongos micorrízico arbusculares por muestra. Las dos especies vegetales presentaron alta densidad de esporas en sus rizósferas, predominando la colonización por hifas y vesículas. La diversidad de especies de hongos micorrízico arbusculares fue baja. En las raíces de P. bromelioides sólo se observaron hifas y vesículas.

Palabras clave: Brasil, Bulbostylis, campos rupestres, Cyperaceae, Eriocaulaceae, hongos formadores de micorriza arbuscular, Paepalanthus bromelioides, regiones altas.

INTRODUCTION In AMF, spores can be identified to species level. Spore density and species di- Arbuscular mycorrhizal fungi (AMF) are versity of AMF in soil are variable31. AMF ecologically obligate symbionts of a wide structures, i.e. arbuscules, hyphal coils, range of plants, and can establish long- vesicles, and non-septate hyphae, in plant term compatible interaction, improving roots may have various functions related to plant nutrient acquisition in infertile soils. both the fungus and the host31. Arbuscular They also have ameliorative effects modi- mycorrhizae (AM) have been divided into fying water relations31 and soil structure28. two classes, Arum-type and Paris-type, ac- AMF can be influenced by environmental cording to fungal structures in roots7. factors, such as: climate conditions, soil Plant species that belong to the family chemical and physical properties, and host Cyperaceae usually form mycorrhizae be- plant species including their age and vari- cause of their occurrence under wet con- ety31. In the tropics, AMF are significant ditions31. There are reports on the myc- for soil fertility and constitute an important orrhizal status of its members as greatly biological resource enhancing soil sustain- influenced by environmental conditions25. ability3. Though mycorrhizal associations have

Mi c o l . Ap l . In t ., 21(1), 2009, p p . 1-10 Ar b u s c u l a r m y c o r r h i z a i n r u p e s t r i a n l a n d s c a p e 3 been noted in many species of Cyperaceae, association in Paepalanthus bromelioides their ecological role is not clear and their and Bulbostylis sp., species that co-occur role on plant growth and nutrient uptake or in rupestrian fields (rocky montane grass- non-nutritional benefits has yet to be fully land). In relation to the conservation of the ascertained. flora from rupestrian fields and the lack of There have been several reports of myc- reports on AM interactions, this research orrhizal association in the Cyperaceae, pre- work was carried out in order to describe dominantly AM and a few species showing AM root colonization and AM fungal diver- ectomycorrhizal associations25. Current in- sity (density and richness) of AM spores in terest is driven by the widespread observa- Paepalanthus bromelioides and Bulbosty- tion of mycorrhizae in the Cyperaceae. The lis sp., which grow at Serra do Cipó, Minas review of Muthukumar et al.25 summarized Gerais, Brazil. the available information on mycorrhizal association in sedges, and highlighted potential mechanisms involved in low my- MATERIALS AND METHODS corrhizal incidence. Intraradical vesicles and hyphae are AMF structures frequently Area of study. This area is located at the reported in sedge roots, but reports on ar- Serra do Cipó, southeastern Brazil (43°30’ buscule occurrence are limited25. W, 19°10’ S). Serra do Cipó is at the south- Information available for 221 species of ernmost portion of Espinhaço Mountains, Cyperaceae shows that 40% are mycor- a predominantly quartzitic range extending rhizal, 11% are facultatively mycorrhizal, for 1,100 km in central Brazil29. Several and 49% are non-mycorrhizal (NM)25, plants were collected in rupestrian fields whereas the mycorrhizal association can from Serra do Cipó, south-eastern Brazil. either be restricted to a short period during This region is characterized by quartzitic the growing season20 or it may be found mountains with altitudes varying between throughout the growing season1. For ex- 1,000-1,400 m. Regions above 1,000 m sup- ample, Fontenla et al.9 found arbuscular port highly xerophytic vegetation with high mycorrhiza (AM) colonization in only one plant species diversity and endemism29. out of nine Cyperaceae species. Wang and These areas, termed rupestrian fields, have Qiu36, in a recent review, summarized the shrubby, tortuous and sclerophyllous veg- status of six species of Cyperaceae: Bul- etation or open grasslands29, and follow the bostylis barbata (Rottb.) C. B. Clarke: fac- cerrado (savanna) vegetation from around ultative AM; B. capillaris (L.) Kunth ex C. 900-1,000 m altitude13. Climate is charac- B. Clarke: NM, AM, facultative AM; B. terized by dry winters (3-5 months) and cf. conifera C. B. Clarke, B. densa Hand.- rainy summers with an average annual rain- Mazz., and B. paradoxa Nees: AM; and B. fall of 1,500 mm and mean temperature of puberula C. B. Clarke: NM. They also dis- 17.4-19.8 C11. According to Köppen, the cli- cussed the potentially important ecological mate of the region is Aw type (tropical). In role of these associations. the rupestrian landscape, soils are shallow, In Venezuela, Aristizábal et al.2 showed acid, nutrient-poor, and have excessively the AM colonization in decomposing drained sands that are highly erodible10. leaves of Paepalanthus sp. In Brazil, there Plant species. Paepalanthus bromelio- is no record of the establishment of AM ides Silveira (Eriocaulaceae), a plant com-

Mi c o l . Ap l . In t ., 21(1), 2009, p p . 1-10 4 M. C. Pa g a n o a n d M. R. Sc o t t i monly found in the region of the Cipó Mg, and Ca were determined by atomic- Mountain range, Minas Gerais State, absorption spectrometry using 1 N am- Brazil, is probably endemic to the high al- monium acetate as extracting solution32. titude rocky-substrate vegetation. It has a Exchangeable Al was extracted with 1 M clumped distribution, occurring in small KCl solution, and determined by titration patches of oligotrophic soils. Each patch with NaOH19. can contain several hundreds of individu- Colonization by AM. Roots of als. Morphologically, this plant shows Paepalanthus bromelioides and Bulbostylis convergence with the Bromeliaceae, hav- sp. were collected, and fixed in FAA solu- ing channelled leaves arranged in a rosette tion (5 mL of formaldehyde, 5 mL of acetic which accumulates water, probably ab- acid, 90 mL of ethyl alcohol) until samples sorbed by trichomes and other epidermal could be processed. The entire root system structures at the base of the leaves. A large was collected from three plants of each number of plants from P. bromelioides are species. associated with at least four genera of ter- Roots were stained and assessed for my- mites, which construct their nests around corrhizal infection as follows: Roots were the roots and stems. They use the plant for taken from the FAA solution, washed sev- shelter and food8. eral times in tap water, and bleached in 10% Bulbostylis Kunth (Cyperaceae; subfami- (w/v) KOH26 overnight and then heated to ly Cyperoideae) comprises approximately approximately 90 C in a water bath for 1 150 species with centers of distribution in h. Cooled root samples were washed and South America and Africa16. About 44 spe- stained with 0.05% trypan blue according cies have been described from Brazil. This to Phillips and Hayman26. Fine roots were genus of heliophilous plants is found in cut into 1 cm segments, and root fragments rocky fields or flooding soils27. (31 cm) were examined per sample for Soils. Rhizospheric soil samples of their AM status under a microscope (100x; the two co-occurring plant species were Olympus BH-2, Japan). If at least one root kept in plastic bags, labelled, sealed, and segment was found to contain fungal myc- transported to the IMA (Instituto Mineiro elia, arbuscules or vesicles, then the sample de Agronomia) Agriculture and Cattle was considered as an AM plant, recorded Chemical Laboratory (Brazil). Soils were as ‘‘+’’. Plants were recorded as non-my- /when neither arbuscules (’’ـ‘‘) air-dried and sieved with a 2 mm mesh. corrhizal Organic matter was determined by the vesicles, nor fungal mycelia were detected Walkley and Black35 method, while phos- in their root cortical cells. Quantification of phorus by the colorimetric method24. mycorrhizal colonization was carried out Potassium (K+), calcium (Ca2+), magne- according to McGonigle et al.18, and results sium (Mg2+), and sodium (Na+) were de- were expressed as percentage of colonized termined by atomic absorption spectrom- segments. These data were arcsin (x/100)½ etry (Spectrophotometer 6800, Analytical transformed. The data were subjected to Instruments Division Kyoto Japan, one-way ANOVA, and means were com- Shimadzu Corporation). pared by the Tukey test (P < 0.05). Soil texture was determined by the hy- The AM intensity was assessed by the drometer method6, and the pH of the soil method of Trouvelot et al.33, in which %M 32 was measured in H2O . Exchangeable K, indicates the intensity of mycorrhization

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according to an arbitrary scale of 1 to 5 (1 - calculate the Shannon-Weaver biodiver- trace of AM colonization; 5= >90% of the sity index (H), species richness (S), and root cortex colonized). Then %M is calcu- evenness (E), according to Magurran17. lated as the proportion of root centimetres Differences in AM diversity among plant colonized by AM, but weighted by the in- species were determined by ANOVA, and

tensity of the colonization: %M= (95n5 + means were compared by the Tukey test

70n4 + 30n3 + 5n2 + n1) ÷ N, where n5, n4,... (P< 0.05). Spore morphology and differ- n1 indicate the number of root centimetres ential staining were observed. Photos were with an intensity 5, 4,…1, and N is the taken with a microscope using an Olympus number of fine root centimetres observed. BH-2 camera (Japan). AM distribution and diversity. Paepalanthus bromelioides and Bulbostylis sp. rhizospheric soil was collected for anal- RESULTS ysis of mycorrhizal spores. Spores were extracted from 100 g soil. AM spores were Basic properties of the rhizospheric soil recovered from soil samples, separated by were as follows: the pH 5.3 and organic wet sieving12, decanting and sucrose cen- matter content 2.72%. Sand >78% and clay trifugation34. Analysed data were expressed >7% were other properties. Base saturation as number of spores/100 grams of dry soil. was low, P content was low, and acidity Just healthy spores were counted. Each was moderated (Table 1). The texture of spore type was mounted in PVLG (poly- the fine soil showed high content of sand vinyl alcohol-lactic acid glycerol), and a (68%) and low content of clay (7%) (Table mixture of PVLG and Melzer’s reagent23 1), belonging to the sand textural class. for identification, as well as to obtain per- In general, the extent of root coloni- manent voucher specimens. Morphological zation varied from about 46% to 67%. properties and subcellular structures were Assessments of percentage AM root length observed under light microscopy at 100x for Paepalanthus bromelioides are shown magnification. Identification was based in Table 2. In relation to spore density, P. on spore colour, size, surface ornamen- bromelioides showed 45% higher density tation and wall structure, with reference of AM species than Bulbostylis sp. (Table to the descriptions provided by Schenk 2). The average AM spore density for both and Pérez30, the International Culture plant species was 77-139 per 100 g air- Collection of Arbuscular and Vesicular- dried soil, and the species richness was 3 Arbuscular Mycorrhizal Fungi (INVAM, to 4 AM species per soil sample. West Virginia, USA; www.invam.caf.wvu. Four taxa of AM fungi were distin- edu), and the original species descriptions. guished in the rooting zone soil samples, Spore numbers were square root trans- of which two were identified at the - spe formed and statistically analyzed. cies level and two at the genus level. Two The frequency of occurrence of each of the four taxa belonged to the genus species from AMF was computed with the Glomus, one to Acaulospora, and one to formula: Xi / X0 x 100, where Xi = the popu- Scutellospora. Glomus brohultii Sieverd. & lation density for an individual species and Herrera and Scutellospora biornata Spain,

X0= the total population. The frequency Sieverd. & S. Toro (Fig. 1 A-B) were the of occurrence of each species was used to most common species (Table 3). Three

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Glomus morphotypes were observed (Fig. observed. Oblong vesicles (11-34 x 50-75 1 C-E). Paraglomus and Gigaspora spe- µm) were often observed within Bulbostylis cies were not found in this study, whereas sp. root segments. A terminal vesicle is Gigaspora-like auxiliary cells with narrow shown in Fig. 1-I. Hyphae, which varied in projections were observed in P. bromelio- diameter (Fig. 1 G-I), often grew for some ides roots (Fig. 1 F). distance in parallel to each other, connected Typical AM structures such as vesicles, by “h” branching pattern, showing Arum- and intraradical hyphae were observed in type colonization. Low frequency of ar- the root of Bulbostylis sp. (Fig. 1 G-I). buscules were detected only in Bulbostylis Arbuscules were not present in all samples sp. In P. bromelioides only hyphae, hyphal coils or vesicles (59%) were observed in the roots and the AM type of colonization could not be determined. Table 1. Chemical analysis of the soil from The rhizosphere of P. bromelioides Paepalanthus bromelioides and Bulbostylis sp. showed higher AM genus richness than rooting-zone. Bulbostylis sp., whereas AM diversity was lower (Table 4). Soil property a Value

DISCUSSION pH (H2O) 1:1 5.3 Soil organic matter (%) 2.72 The low levels of Ca and Mg, and the lower C (%) 1.58 base saturation value observed are linked N (%) 0.14 to the rather low fertility of the site, be- C/N ratio 11.28 ing similar to a Brazilian foredune where -3 Available P (mg dm ) 1.3 Scutellospora species were found4. Two + 3 Available K (mg dm ) 66 fertility indices (OM, K+) were high in the 3+ –1 Exchangeable Al [cmol (+) kg soil] 1.02 rhizospheric soils of P. bromelioides and 2+ –1 Exchangeable Ca [cmol (+) kg ] 0.18 Bulbostylis sp. The organic matter content 2+ –1 Exchangeable Mg [cmol(+) kg ] 0.07 and the pH of the soil were similar to previ- –1 CEC [cmol (+) kg ] 3.68 ous reports from Polylepis woodlands with Base saturation (%) 11.17 sandy loam substrate, where Carex fuscula b Texture (%) (a perennial Cyperaceae) showed AM colo- Coarse sand 10.5 nization (about 6 to 25%)21. Fontenla et al.9 Fine sand 68.24 found the same species of Cyperaceae col- Clay 7.04 onized by AMF in the Patagonian steppe. Silt 14.22 Both plant species showed high rhizosphere spore densities in this study, and a Mean of two measures from one composite dominant hyphae and vesicle colonization. sample. Only three genera and four species of AMF b Particle size distribution: coarse sand 2-0.2 were found. AM diversity was found to be mm, fine sand 0.2-0.02 mm, silt 0.02-0.002 low, but similar to Puna ecosystem (a high- mm, and clay < 0.002 mm. land in Argentina and Peru), where Lugo CEC = Cation exchange capacity. et al.15 reported a low richness with the oc-

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Table 2. Arbuscular mycorrhizal (AM) fungal status in roots, as well as AM spore species density and richness in the rooting zone soils of Paepalanthus bromelioides and Bulbostylis sp. in the dry period (May, 2006).

Host Length NS Coils A V Intensity CT Density S

Paepalanthus bromelioides 67.7a nd + - 59.6 0.6 nd 139 4 Bulbostylis sp. 46.6 + - + 33.3 0.6 Arum 77 3 a Values represent the mean of three samples. Relative development of structures shown as: + = Always present; – = Not detected; nd= Not determined; Length= AM root length (%); NS= Intercellular non-septate hyphae; Coils= Hyphal coils; A= Arbuscules; V= Vesicles; Intensity= Intensity of AM colonization (%); CT= AM colonization type; Density= Spore density per 100 g air-dried soil; S= Species richness. currence of the same Scutellospora species The presence of AM in the Cyperaceae and Glomus dominance. (sedges) studied was noteworthy because it A high spore density of AM species is frequently considered a non-mycorrhizal found in this work (77 to 139 spores 100 family. Likewise, Lovera and Cuenca14 re- g-1 soil) may be related to the plant age or ported Cyperaceae species showing high to environmental conditions31. The density levels of colonization (45%) and the pres- of Glomus was high in this study, and com- ence of arbuscules, structures that confirm parable to the density (121 spores 100 g-1) AM functionality. Moreover, Miller et al.22 of this genus in Puna15. found AM to occur in 16 out of the 23 stud-

Table 3. Arbuscular mycorrhizal (AM) spore Table 4. Arbuscular mycorrhizal (AM) diversity diversity in Paepalanthus bromelioides and in Paepalanthus bromelioides and Bulbostylis Bulbostylis sp. sp. rhizospheres at Serra do Cipó, Brazil.

AM fungal species P. bromelioides Bulbostylis Host plant species Indicator P. bromelioides Bulbostylis Gigasporaceae

Scutellospora biornata 4 3 a Acaulosporaceae Genus richness 3 3 b Acaulospora sp. 4 5 Diversity 0.36 0.43 Glomeraceae Evennessc 0.18 0.27 Glomus spp.a 131 69 a AM fungal genus richness. b Shannon diversity a Total spore number of Glomus. index. c Shannon evenness index.

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B C

A E a

G D F

V V

H I

Fig. 1. Spores and arbuscular mycorrhizal (AM) colonization of species studied. A-B: Scutellospora biornata spores showing the germination shield in Paepalanthus bromelioides (A) and Bulbostylis sp. (B) rhizospheric soils. C-E. Glomus spp. in polyvinyl alcohol-lactic acid glycerol (PVLG) showing the attachment of the subtending hypha at the spore base. F: External auxiliary cell in P. bromelioides root. G: Bulbostylis sp. root showing hyphae of different sizes, often growing in parallel to each other, connected by distinct “h” branching pattern (h), and arbuscules (a). H: Intraradical hyphae bearing vesicles (V) in Bulbostylis sp. I: Intracellular terminal vesicle (V) in Bulbostylis sp. Bars for A, C, D, E, F= 50 µm; for B= 10µm; and for G-I= 25 µm. ied Carex species (mean colonization of Plant species studied that belong to fam- 5-20%), and Cuenca et al.5 observed a 10% ily Cyperaceae, which usually do not form AM colonization in Bulbostylis capillaris. mycorrhizae, appear to be well-colonized

Mi c o l . Ap l . In t ., 21(1), 2009, p p . 1-10 Ar b u s c u l a r m y c o r r h i z a i n r u p e s t r i a n l a n d s c a p e 9 with AM and to possess arbuscules in this reported from rupestrian soils. Considering rupestrian fields. the predominance of AM in these species, Paepalanthus showed the greatest per- further studies must consider seasonal sam- cent values of AM colonization, as it was plings for a longer period of time, as well also found by Aristizábal et al.2 (66.53%), as greenhouse inoculation trials. although they did not find vesicles in the roots. The small and round vesicles found in root segments suggest colonization by ACKNOWLEDGEMENTS Glomeraceae, in accord with the abun- dant presence of Glomus sp. in the stud- The author is grateful to FAPEMIG (Processo 311/07) for granting Post-doctoral scholarships, and to Prof. Dr J. E. ied rhizosphere. However, the presence of C. Figueira for material collection. arbuscules is yet to be ascertained. While the absence of arbuscules (a structure of nutrient interchange, which have one or two weeks of life period), could suggest a LITERATURE CITED non-functional association, the presence of 1. Anwar, Q. M. K. and M. Jalaluddin. 1994. Significance vesicles is also a significant evidence of the of VAM in weeds of wheat. Mycorrhiza News presence of AM in the roots. In this work, 5: 9-11. it is noteworthy that the intensity of AM 2. Aristizábal, C., E. L. Rivera and D. P Janos. 2004. Arbuscular mycorrhizal fungi colonize de- colonization did not differ between the two composing leaves of Myrica parvifolia, M. plant species. The presence of auxiliary pubescens and Paepalanthus sp. Mycorrhiza cells with narrow projections indicated 14: 221-228. the presence of Gigaspora sp., although 3. Cardoso, I. M. and T. W. Kuyper. 2006. Mycorrhizas and tropical soil fertility. Agriculture, Ecosys- spores of this genus were not found in the tems & Environment 116: 72-84. rhizospheric soils. 4. Cordoba, A. S., M. M. de Mendonça, S. L. Stürmer According to the results on P. bromelio- and P. T. Rygiewicz. 2001. Diversity of ar- ides and Bulbostylis sp., it can be conclud- buscular mycorrhizal fungi along a sand dune stabilization gradient: a case study at Praia da ed that: 1) The presence of AM suggested Joaquina, Ilha de Santa Catarina, south Brazil. that P. bromelioides and Bulbostylis sp. Mycoscience 42: 379-387. are arbuscular-mycorrhiza dependent; 2) 5. Cuenca, G., Z. De Andrade and G. Escalante. 1998. Arbuscular mycorrhizae in the rehabilitation The dominance of Glomus sp. suggested of fragile degraded tropical lands. Biology a potential native AM inoculum for both and Fertility of Soils 26: 107-111. plant species in poor soils, and species 6. Day, P. R. 1965. Particle fractionation and particle-size identification is recommended; and 3) The analysis. Pp. 545-567. In: Methods of Soils Analysis. Ed. C. A. Black. Part 1. Agronomy presence of Scutellospora species, as well 9. American Society of Agronomy, Madison. as auxiliary cells-like Gigaspora sp., es- 7. Dickson, S. 2004. The Arum-Paris continuum of my- pecially in P. bromelioides, indicated that corrhizal symbioses. New Phytologist 163: these fungal genera could be used as in- 187-200. 8. Figueira, J. E. C. and P. Vasconcellos Neto. 1992. oculants, due to the different colonization Paepalanthus, cupins e aranhas. Ciência strategy of the Gigasporaceae in relation Hoje, Eco-Brasil (Special edition, pp. 89-93). to the Glomeraceae. Instituto Ciência Hoje, Rio de Janiero. 9. Fontenla, S., J. Puntieri and J. A. Ocampo. 2001. Myc- This study contributed to the mycodi- orrhizal associations in the Patagonian steppe, versity analysis in the north of the Minas Argentina. Plant and Soil 233: 13-29. Gerais region, as plant species studied are 10. Freitas, R. O. 1951. Ensaio sobre o relêvo tectônico

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