Applied Soil Ecology 71 (2013) 7–14
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Arbuscular mycorrhizal fungi in the Brazilian Atlantic forest: A gradient of environmental restoration J.A. Bonfim a,∗ , R.L.F. Vasconcellos a , S.L. Stürmer b , E.J.B.N. Cardoso a a b
Soil Science Department, Soil Microbiology Lab, University of São Paulo, ESALQ, Pádua Dias Avenue, 11, CEP 13418-900 Piracicaba, SP, Brazil Departamento de Ciências Naturais, Universidade Regional de Blumenau, FURB, Caixa Postal 1507, CEP 89010-971 Blumenau, SC, Brazil
a r t i c l e
i n f o
Article history: Received 22 May 2012 Received in revised form 11 February 2013 Accepted 23 April 2013 Keywords: Spore number Species diversity Soil degradation
a b s t r a c t Arbuscular mycorrhizal fungi (AMF) are of great importance for the successful regeneration of degraded natural areas. The objective of this study was to examine how the time of environmental recuperation is affecting the occurrence and diversity of AMF species in riparian areas belonging to the Atlantic Forest biome in the State of São Paulo, Brazil. The study involved a native forest area (NT) and a gradient of environmental restoration: five (R05), ten (R10), and twenty (R20) years after reforestation. Soil samples were collected in the rainy (January) and dry season (June). Chemical, physical and microbiological analyses were performed including the amount of glomalin and quantification of AMF spores. The frequency of occurrence of genera and ecological indices, as richness (R), Shannon’s diversity (H) and Simpson’s dominance index (Is) were calculated. The largest spore number was found in R05 and the highest richness and diversity indices of AMF species in NT. Considering the two sampling periods and the four areas studied, we found 22 AMF species, and the genera Glomus and Acaulospora were the most frequent. A Canonical Discriminant Analysis showed that Glomus viscosum, Acaulospora scrobiculata, Acaulospora mellea and Scutellospora heterogama were the species that contributed the most to distinguishing the areas. Moisture, density and glomalin were positively correlated with the number of spores, however, soil nitrate showed a negative correlation. This work gives a better understanding of the interactions between AMF and forest soils and allows to know the distribution of AMF species according to environmental recovery time. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The Mata Atlantica biome (Atlantic Rain Forest) has suffered an intensive process of exploitation mainly due to deforestation followed by human activities like farming, grazing, logging and establishment of cities (Colombo and Joly, 2010). This process has generated a major environmental concern because it is responsible for soil erosion, siltation of water reservoirs, loss of soil nutrients, and reduction of biodiversity (Rodrigues et al., 2009). The revegetation process with native or exotic species in areas that had the original vegetation removed is a way to accelerate the natural regeneration (Zangaro et al., 2008). However, for a successful process of revegetation, it is important that seedlings absorb water and soil nutrients efficiently after transplanting to the field and are able to cope with environmental stresses (Carrenho et al., 2001; Aidar et al., 2004). In this context, the association of seedlings with arbuscular mycorrhizal fungi (AMF) is of paramount importance since the establishment of the arbuscular mycorrhizal associations
∗ Corresponding author. Tel.: +55 19 34172118; fax: +55 19 34172110. E-mail address:
[email protected] (J.A. Bonfim). 0929-1393/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsoil.2013.04.005
with plants provides several benefits for their hosts, such as greater plant vigor, greater tolerance to climatic stress, increased competitiveness due to a higher initial growth rate, disease resistance and survival in poor soils (Zangaro et al., 2008; Moreira et al., 2009). AMF also contribute to improve soil structure as their external mycelium is responsible for the exudation of a hydrophobic glycoprotein called glomalin, which acts as a cementing agent of soil particles, helping directly in the formation of stable aggregates, and also favoring the soil carbon stocks (Purin and Rillig, 2007; Rillig, 2004). In Brazil, several studies were carried out demonstrating the occurrence of AMF in the Mata Atlantica biome (Trufem and Viriato 1990; Carrenho et al., 2001; Moreira-Souza et al., 2003; Moreira et al., 2009). However, there are few works reporting the dynamics of the mycorrhizal association in distinct stages of natural secondary succession or man-induced revegetation. Generally, the diversity of AMF species, but not the number of spores, is lower in Atlantic Forest areas at initial stages of a succession than in more advanced or mature forest (Aidar et al., 2004; Stürmer et al., 2006; Zangaro et al., 2008). Moreover, Acaulospora and Glomus are predominant in the mycorrhizal fungal community associated with plants along the successional gradient, in the Mata Atlantica biome,
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Table 1 Description of selected areas for study, the Atlantic Forest ecosystem, State of Sao Paulo, Brazil. Areas
City
Geographic location
Size of area (ha)
Number of species used in revegetation
Soil type
NT R20 R10 R05
Campinas Iracemápolis Santa Bárbara d’Oeste Piracicaba
22◦ 50 13 S–46◦ 55 58 W 22◦ 35 S–47◦ 31 W 22◦ 49 06 S–47◦ 24 53 W 22◦ 42 02 S–47◦ 38 32 W
233 20 30 16
– 140 80 25
Acrisola Ferralsol Acrisol Nitisol
a
US Soil Taxonomy.
accounting for 83% of the total spores recovered in three stages of secondary succession in Santa Catarina state, South Brazil (Stürmer et al., 2006), 73% of the total spores in a chronosequence (Aidar et al., 2004) and 72% in revegetated areas of riparian vegetation (Carrenho et al., 2001), both in São Paulo state. Despite the studies mentioned above, there is no report on the dynamics of AMF species associated with plants after distinct periods of man-induced revegetation. This approach is important in order to understand if the revegetation process is also recovering the below ground microbial diversity. The objective of this paper was to determine the occurrence and diversity of AMF species in areas of riparian forest within the Mata Atlantica biome, representing a gradient of ecological restoration.
0.01 M. Ca2+ , Mg2+ and P was extracted with an ion exchange resin. P was quantified colorimetrically using ammonium molybdate, while Ca2+ and Mg2+ were determined by atomic absorption spectrophotometry. The K+ was extracted from soil by Mehlich 1 (HCl 0.05 M + H2 SO4 0.0125 M) and quantified by flame photometry. Al3+ was extracted with KCl 1 M and quantified by titration. Soil NO3 − -N was determined by distillation (Coelho et al., 1992). The total soil carbon (TC) was obtained by total combustion using an elemental analyzer LECO CN-2000. For soil density, microporosity and macroporosity 15 undisturbed soil samples were collected with volumetric rings 5 cm in diameter and 5 cm deep. Moisture was determined in deformed samples collected between 0 and 15 cm and the proportions of sand, silt and clay were determined by the pipette method (Embrapa, 1997) (Table 2).
2. Materials and methods 2.1. Study area
2.3. Microbial biomass carbon and soil basal respiration
The AMF communities were surveyed in a native forest area (NT) and three sites with increasing periods after revegetation: R05 (5 years), R10 (10 years), and R20 (20 years). All areas are riparian pertaining to the Mata Atlantica biome and recognized as fragments of the semideciduous forest located in the State of São Paulo, Brazil (Table 1). The climate of the areas is mesothermic humid subtropical (type Cwa), according to Köppen’s classification. The area is characterized by a seasonal climate: rainy summers between September and March, and dry winters between April and August. Average temperature of the coldest and warmest month is below 18 ◦ C and between 23 ◦ C and 24 ◦ C, respectively. The mean annual rainfall is 1100–1700 mm. The areas R05, R10 and R20 had been used for many years for sugar cane plantation, being plowed and fertilized regularly. The revegetation model of these areas was based on the concept of secondary succession and used regional native species belonging to distinct successional stages (pioneer, early and late secondary, and climax species). The main species used were: Myracrodruon urundeuva, Schinus terebinthifolius, Aspidosperma ramiflorum, Erythrina speciosa, Myroxylon peruiferum, Cedrela fissilis, Psidium cattleianum, Esenbeckia leiocarpa, Citharexylum myrianthum, and Luehea divaricata. It would not be possible here to cite the names of hundreds of different tropical plant species, but, in general, they had a similar flora in the environmental recovery areas. The soils also were somewhat similar in physical and chemical soil attributes, although belonging to different soil types: the native area and R10 were sandier and the areas R05 and R20 were more clayey (Table 1). In each area 30 plots (10 m × 10 m) were established, and 15 plots of them were randomly selected for soil sampling. In each plot three single soil samples were collected and pooled to form one compound sample. Soil samples were collected with an auger at a depth of 0–15 cm in January 2010 (rainy season and high temperatures) and June 2010 (dry season and mild temperatures).
The microbial biomass carbon (MBC) was estimated by the fumigation–extraction method (Vance et al., 1987). Microbial activity was determined by soil respiration (CO2 -C) from 50 g samples incubated for 20 days at 28 ◦ C. The CO2 released was captured in 50 mmol L−1 NaOH, precipitated with 0.5 mol L−1 BaCl2 ·2H2 O and quantified by titrating the remaining NaOH with 50 mmol L−1 HCl in the presence of phenolphthalein (Alef, 1995). The metabolic quotient (qCO2 ), representing the release rate of CO2 -C per unit microbial biomass C and per time unit, was calculated using the results of basal respiration and MBC.
2.2. Chemical and physical analyses The soil chemical analyses followed the methodology described by Raij et al. (2001). Soil pH was determined in a solution of CaCl2
2.4. AMF identification AMF spores were extracted from soil by wet sieving (Gerdemann and Nicolson, 1963), a 50 g soil aliquot from each sample, followed by sucrose centrifugation. Two nested sieves (0.42 mm and 0.053 mm) were used and the material retained in the smallest sieve was poured into plates containing concentric grooves and total number of spores was counted under a dissecting microscope at 40×. After counting, spores were separated into groups according to their morphology and mounted on slides with PVLG (polyvinyl alcohol–lactoglycerol) (Morton et al., 1993). Identification was made at the species level using an optical microscope (100–400× magnification), with the aid of the Schenck and Pérez (1990) manual, descriptions provided by the International Collection of Vesicular and Arbuscular Mycorrhizal Fungi (http://invam.caf.wvu.edu) and original species descriptions. The number of spores in 50 g dry soil was recorded for each species. The following diversity indices were calculated: species richness (R), evaluated by the number of species present in 50 g soil, Simp L = ni(n − 1)/N(N − 1) and son’s dominance index (Is), Is = 1 − L, Shannon’s diversity index (H), H = − (pi log pi), pi = ni/N, where for both formulas: ni = total spore numbers of each AMF species “i” and N = total number of spores. The relative spore abundance for each AMF genus (RSA), [RSA = (number of spores per genus/total number of AMF spores) × 100] was also calculated (Magurran, 2004).
J.A. Bonfim et al. / Applied Soil Ecology 71 (2013) 7–14
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Table 2 Chemical and physical soil characteristics in native area (NT) and reforested (R20, R10 and R05), Atlantic Forest ecosystems. January 2010, State of São Paulo, SP, Brazil. Characteristics
NT
Physical Density (g cm−3 ) Microporosity (m3 m−3 ) Macroporosity (m3 m−3 ) Moisture (%) Sand (g kg−1 ) Silt (g kg−1 ) Clay (g kg−1 )
R20
1.33 0.27 0.21 17 612 137 251
± ± ± ± ± ± ±
0.15 0.07 0.06 4 33 33 0.1
1.37 0.41 0.1 22 345 71 584
± ± ± ± ± ± ±
0.09 0.03 0.04 2 62 29 38
1.57 0.32 0.09 16 646 54 301
± ± ± ± ± ± ±
0.10 0.02 0.04 2 63 29 50
1.58 0.41 0.05 23 331 135 534
± ± ± ± ± ± ±
0.07 0.02 0.02 5 44 21 29
Chemical pH CaCl2 Available P (mmolc kg−1 ) K+ (mmolc kg−1 ) Ca2+ (mmolc kg−1 ) Mg2+ (mmolc kg−1 ) Al+3 (mmolc kg−1 ) H + Al (mmolc kg−1 ) NH4 + (g g−1 ) NO3 − (g g−1 ) TC (%) CEC (mmolc kg−1 ) SB (%)
4.7 13.1 117 36.5 10.9 5.2 54 8.0 18.4 3.9 218 164
± ± ± ± ± ± ± ± ± ± ± ±
0.5 3.73 34 24.7 3.9 2.2 20 7.0 6.8 1.0 38 52
4.5 4.5 40 17.9 8.8 2.2 34 5.6 9.7 2.4 100 67
± ± ± ± ± ± ± ± ± ± ± ±
0.5 1.2 13 4.7 2.8 0.8 3.7 4.2 3.6 0.5 11 11
4.7 22.9 195 22.7 7.5 4.5 80 7.6 5.9 2.3 305 226
± ± ± ± ± ± ± ± ± ± ± ±
0.2 10 39 15.4 4.8 3.3 25 5.0 2.8 0.5 42 49
4.9 16.3 43 39.7 10.5 1.8 61 3.3 2.7 1.9 154 94
± ± ± ± ± ± ± ± ± ± ± ±
0.3 8.12 17 12.9 3.0 0.3 10 2.5 1.9 0.2 20 22
2.5. Glomalin analysis The soil glomalin was quantified according to Wright and Upadhyaya (1998) and was called using the nomenclature proposed by Rillig (2004): total glomalin, named BRSP (Bradford related soil protein) and the easily removed glomalin EE-BRSP (Easily extractable BRSP). This method is quantifies the total soil protein while glomalin is its main component. The protein was quantified by the Bradford assay using bovine serum albumin as standard. To detect the presence of glomalin the reagent Coomassie Brilliant Blue G-250 was used, causing a color change (blue). Readings were performed in a spectrophotometer (595 nm), with the standard curve for bovine serum albumin (BSA).
2.6. Statistical analysis Spore count data were transformed to log(X + 1) and submitted to univariate statistical analysis, using ANOVA followed by LSD test (p < 0.05) to separate means. Statistical analysis was performed with SAS software. We calculated Spearman’s correlation coefficients between spore count data and soil chemical, physical and microbiological data based on replicate data. The multivariate canonical discriminating analysis (CDA) was applied to verify which ecological attribute contributed the most to distinguish between the ecosystems (NT, R20, R10 and R05) by the CANOCO version 4.0 program. Calculations were made for the homogenized canonical
R10
R05
coefficient (HCC), coefficient of correlation (r), and parallel discrimination rate coefficient (PDRC = r × HCC). The averages of the HCC were compared by LSD (p < 0.05).
3. Results For both sampling periods the CO2 -C was higher in NT when compared to revegetated areas. MBC was higher in NT and R20 when compared to R10 and R05 (Table 3). The qCO2 was not different across areas in the rainy season while in the dry season qCO2 values for R20 were lower compared to the other areas (Table 3). The EE-BRSP was higher in NT in both rainy and dry seasons while BRSP was not different across areas in the dry season (Table 3). The mean number of spores in both periods, considering the four areas was obtained after counting the spores present in fifteen 50 g soil samples and calculating the mean value for each area (Fig. 1). These mean values were summed up for all areas and rendered a total mean of 453 spores. No differences for mean spore numbers were found between seasons in NT, R20 and R10. In R05, the number of spores averaged 154 spores per 50 g dry soil in the rainy season and was different from the dry season (83 spores). The number of spores was also much higher in the R05 area, differing from all other areas. A total of 22 AMF morphotypes were identified and 15 of them could be attributed to known species (Table 4). Six genera were found and the most representative genera were Glomus followed
Table 3 Basal respiration (CO2 -C), microbial biomass carbon (MBC), metabolic quotient (qCO2 ), easily extractable glomalin (EE-BRSP) and total glomalin (BRSP) in the native area (NT) and in the recovery areas (R20, R10 and R05) of the Atlantic Forest ecosystem, in January (rainy season) and June (dry season) 2010, State of São Paulo, Brazil. Variables
NT
R20
R10
R05
Rainy C-CO2 (g CO2 g dry soil−1 day−1 ) MBC (g C g soil−1 ) qCO2 (g C-CO2 /g CBM day−1 ) EE-BRSP (mg g dry soil−1 ) BRSP (mg g dry soil−1 )
101aAa 210aA 0.53aA 1.73aB 2.29aA
72bA 172aB 0.55aA 1.42bA 1.45abA
59bcA 109bA 0.58aA 1.13cB 1.26bB
47cA 104bB 0.61aA 1.33bcB 1.81abB
Dry C-CO2 (g CO2 g dry soil−1 day−1 ) MBC (g C g soil−1 ) qCO2 (g C-CO2 /g CBM day−1 ) EE-BRSP(mg g dry soil−1 ) BRSP (mg g dry soil−1 )
84aA 243aA 0.35abB 2.62aA 2.65abA
51bA 220aA 0.23bB 1.82bA 1.81bA
56bA 138bA 0.44aA 1.67bA 2.18abA
50bA 168bA 0.35abB 1.82bA 2.77aA
a
Small letters in lines compare areas and capital letters in columns compare the sampling periods by LSD test (p < 0.05).
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Table 4 Numbers of identified spores (50 g dry soil) in the native area (NT), and recuperation areas (R20, R10 and R05), of the Atlantic Forest ecosystem, in the rainy and dry season, State São Paulo, Brazil, 2010 (average of 15 repetitions). AMF diversity
Ambispora appendicula (Spain, Sieverd. e Schenck) Spain, Oehl & Sieverd Acaulospora mellea Spain & Schenck A. foveata Trappe & Janos, A. colossica Schultz, Bever & Morton A. scrobiculata Trappe A. spinosa Walker & Trappe A. lacunosa Morton Gigaspora decipiens Hall & Abbott Gi. rosea Nicolson & Schenck, Gi. albida Schenck & Sm Gigaspora sp1 Gigaspora sp2 Scutellospora heterogama (Nicolson e Gerd.) Walker & Sanders S. pellucida (Nicol. & Schenck) Walker e Sanders Glomus geosporum (Nicolson & Gerd.) Walker Glomus viscosum Nicolson Glomus sp1 Glomus sp2 Glomus sp3 Glomus sp4 Glomus sp5 Racocetra intraornata Goto, Maia & Oehl Total spores Ecological index Species richness Shannon’s diversity index Simpson’s dominance index a
Rainy
Dry
NT
R20
R10
0
0
3 0 2 7 1 2 2 0 0 2 0 1
R05
NT
R20
R10
R05
0
0
1
0
0
0
6 0 0 2 0 1 1 1 0 0 0 1
4 0 0 12 0 0 0 0 0 0 0 0
15 0 0 19 0 0 0 0 0 0 0 4
5 0 1 3 3 3 1 0 0 0 1 0
5 0 0 1 0 0 1 1 0 0 0 0
2 1 0 12 0 0 0 2 1 0 0 2
24 0 0 21 0 0 0 0 0 0 0 1
0 2 0 11 16 8 0 0 0
0 0 0 6 5 0 0 0 0
0 1 0 8 13 0 0 0 0
0 0 62 0 54 0 0 0 0
0 0 0 20 5 10 0 2 2
2 0 0 0 4 0 3 0 0
0 4 0 0 4 0 0 0 0
0 0 15 0 22 0 0 0 0
57
23
38
154
56
17
28
83
7.7aa 0.7a 0.2b
3.6c 0.4b 0.4a
3.4c 0.4b 0.4a
4.5b 0.5b 0.3ab
7.5a 0.7a 0.2c
3.8bc 0.4b 0.3b
3.0c 0.3c 0.5a
4.4b 0.5b 0.3b
Letters in lines compare the area in each season by LSD test (p < 0.05).
by Acaulospora and Gigaspora. Different species differed in each season at the same area. Different areas presented different richness indices. Some species, independent of the area, were found only in the dry season such as Ambispora appendicula, Acaulospora foveata, Gigaspora albida, Gigaspora sp.2, Scutellospora pellucida, Glomus sp.5, Glomus sp.4, Racocetra intraornata, probably because they only sporulate in cold and dry weather. Gigaspora sp.1 was detected only in the rainy season. As a matter of fact, it seems to be more common for AMF species to sporulate during the period in which plant growth is very slow, although the number of spores was greater in the rainy period, but with a lower number of species.
Fig. 1. Number of spores of arbuscular mycorrhizal fungi (AMF) in the native (N) and the recovery areas (R20, R10 and R05), in the Atlantic Forest ecosystem, in the rainy (January) and dry (June) season of 2010, State of São Paulo, Brazil (n = 15). *Small letters compare seasons in the same areas and capital letters compare areas in the same season by LSD test (p < 0.05).
Species diversity (R) and Shannon’s diversity (H) were significantly higher in NT in both seasons when compared to other areas (Table 4). Simpson’s dominance index (Is) was higher in the rainy season for areas R05, R10 and R20. In the dry season, Is was higher in R10 and lowest in NT. Between the two seasons, the indices H and Is showed no differences, except for R10, assuming higher values of H in the rainy epoch and Is in the dry epoch. R was not different between seasons for all areas. In NT and R20, Glomus had the highest relative abundance at both sampling seasons, followed by Acaulospora (Fig. 2). The relative abundance of Glomus was larger in the rainy season for R10 and R05 (57% and 75%, respectively) when compared to the dry season (28% and 44%, respectively). In R10 and R05 the genus Acaulospora was dominant in terms of spore abundance in the dry season (Fig. 2). In the rainy season, when separating the areas by means of the two canonical axes, the first canonical discriminating function (CDF1) explained 65% and the second canonical discriminating function (CDF2) explained 33% of the separation of data with high correlation between the two canonical axes (r = 0.97, p < 0.0001) (Fig. 3). In the dry epoch CDF1 explained 52% and CDF2 explained 39%, also with high correlation between the two canonical axes (r = 0.85, p < 0.0001). Fig. 3 shows a very complete discrimination between the area NT and R05, the native and the least recovered areas, in both seasons. However, the intermediate areas R10 and R20 were grouped together, revealing several common aspects of the AMF distribution. The LSD test of means of the homogenized canonical coefficients (HCC) of the CDF1 and CDF2 for all species analyzed demonstrated significant differences among the areas (Table 5). In both seasons, in CDF1 the AMF species were responsible for discriminating the area NT from the other areas. In the rainy season the highest values of HCC were found for NT, followed by R20 and R10 and finally
J.A. Bonfim et al. / Applied Soil Ecology 71 (2013) 7–14
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Table 5 Analysis of variance of mean homogenized canonical coefficients (CCH) of the first and second canonical discriminant function (CDF1 and CDF2) for two seasons, the values refer to the AMF species in the native (NT) and recovery areas (R05, R10 and R20). Areas
Rainy
Dry
CDF1 NT R20 R10 R05 a
a
4.62a −1.86b −1.73bc −1.02c
CDF2
CDF1
CDF2
0.12b −1.75c 3.08a −1.46c
3.67a −0.55b −2.95c −0.55b
1.57b −2.25c 2.48a −1.80c
Letters in columns compare the areas by LSD test (p < 0.05).
by R05. In the dry season, higher values of HCC of the CDF1 were observed for NT, followed by R20 and R05 and finally by R10. The HCC of the CDF2 for both seasons indicated that the AMF species discriminated NT and R20 from R10 and R05, the highest values were observed in R10 followed by NT, R20 and R05. In the rainy season the highest values of PDRC (parallel discrimination rate coefficient) were observed for the species Glomus viscosum, Glomus spp. and Scutellospora heterogama in CDF1 and for Glomus spp., Acaulospora colossica, A. lacunosa, Gigaspora decipiens and Gigaspora sp. in CDF2 (Table 6). For the dry season, highest values of PDRC were found for G. viscosum, Acaulospora scrobiculata and Acaulospora mellea in CDF1 and G. viscosum, Glomus spp., Acaulospora lacunosa, Acaulospora spinosa and R. intraornata in CDF2 (Table 6). The correlation analysis showed that among the physical, chemical and microbiological analyses, in both seasons, the number of spores was influenced by moisture, soil density, BRSP and nitrate in the soil. Positive correlation was observed between number of spores and moisture (r = 0.52, p ≤ 0.0001), soil density (r = 0.443, p < 0.0004) and BRSP (r = 0.389, p < 0.002), whereas soil nitrate concentration (r = −0.40, p = 0.0017) showed a negative correlation with the number of spores.
Fig. 2. Relative abundance of genera based on the occurrence of AMF spores in environmental recovery areas (R05, R10 and R20) and one native area (NT): (A) rainy season and (B) dry season, São Paulo, Brazil, 2010.
4. Discussion Our data on microbial biomass carbon and basal respiration were higher in NT and in later stages of induced revegetation, suggesting that in these areas the input of plant debris is larger compared to R05 and R10, which influences concentrations of organic carbon in these areas (Mariani et al., 2006). Higher values of microbial biomass and respiration indicate a more intense microbial activity, resulting in soil quality improvement, high nutrient cycling, formation of soil stable aggregates and higher functional diversity. Such characteristics certainly improve the functionality and stability of the ecosystems (Nogueira et al., 2006). Moreover, comparable levels of MBC and basal respiration between R20 and NT indicate that the revegetation model applied to these areas is appropriate, since some attributes of the microbial community are approaching those of the native area 20 years after seedling establishment.
Fig. 3. Relationship between the first and second canonical discriminating functions (CDF1 and CDF2) by means of the homogenized canonical coefficients (HCC), regarding the values of the analyzed AMF species in environmental recuperation areas (R05, R10 and R20) and one native area (NT), (A) rainy season and (B) dry season, São Paulo, Brazil.
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Table 6 Values of the canonical correlation coefficient (r), homogenized canonical coefficients (CCH) and parallel discrimination rate coefficient (PDRC) within the first and second canonical discriminating function (CDF1 and CDF2), regarding the values of the species studied in areas: native (NT) and recovery area (R05, R10 and R20) in January (rainy season) and June (dry season), 2010, Sao Paulo, Brazil. Species identified only at the genus level were pooled together to facilitate understanding of the analysis. AMF species
CDF1
CDF2
r
CCH
PDRC
r
CCH
PDRC
Rainy Glomus geosporum G. viscosum Glomus spp. Acaulospora mellea A. colossica A. spinosa A. scrobiculata A. lacunosa Gigaspora decipiens G. rosea Gigaspora spp. Scutellospora heterogama
−0.07 0.84 0.58 0.16 0.04 −0.16 0.22 −0.36 −0.15 −0.14 −0.22 0.48
−0.08 0.55 0.37 0.21 −0.07 −0.03 0.16 −0.13 −0.15 −0.04 −0.08 0.28
0 0.47 0.22 0.03 −0 0 0.03 0.05 0.02 0 0.02 0.13
−0.07 0.84 0.58 0.16 0.04 −0.16 0.22 −0.36 −0.15 −0.14 −0.22 0.48
−0.08 0.55 0.37 0.21 −0.07 −0.03 0.16 −0.13 −0.15 −0.04 −0.08 0.28
0.08 0 0.14 0 0.11 0.02 −0 0.12 0.21 0 0.15 0.02
Dry Glomus viscosum Glomus spp. Acaulospora mellea A. foveata A. colossica A. spinosa A. scrobiculata A. lacunosa Gigaspora decipiens G. rosea G. albida Gigaspora spp. Scutellospora heterogama S. pellucida Ambispora appendicula Racocetra intraornata
0.66 0.04 0.47 −0.2 −0.02 −0.52 0.46 −0.92 0.35 −0 0.03 0.28 0.03 −0.09 −0.12 −0.61
0.60 −0.11 0.28 −0 −0.12 −0.17 0.28 −0.22 −0.16 −0.01 −0.01 −0.13 0.06 −0.04 −0.15 −0.15
0.39 −0 0.13 0 0 0.09 0.13 0.20 −0.05 1.3E−05 −0 −0.04 0 0 0.02 0.09
0.36 0.68 0.30 −0.10 0.05 0.76 0.36 0.63 −0.39 −0.07 −0.15 −0.10 −0.22 −0.24 0.30 0.68
0.34 0.42 0.19 −0.05 0.13 0.19 0.10 0.24 0.13 −0.13 −0.13 0.13 −0.06 −0.24 0.17 0.17
0.12 0.29 0.06 0 0 0.15 0.04 0.15 −0.05 0.01 0.02 −0.01 0.01 0.06 0.05 0.12
The higher amount of total glomalin in NT compared to other areas is possibly related with the accumulation of this protein over time. Glomalin in soil has a high stability and its residence time is calculated as up to 42 years until complete mineralization (Rillig, 2004). In this study, R05, R10 and R20 had been agricultural areas and the lower amounts of BRSP and EE-BRSP glomalin compared to NT indicates that even after 20 years under recuperation has not been enough for a maximum accumulation of this protein in soil. Glomalin concentration is considered one of the most sensitive biological attributes of soil management practices (Bedini et al., 2007), and our data suggest that glomalin can be used to assess soil quality evolution during revegetation processes. Interesting is that the NT had the highest AMF species diversity, which may have contributed to increase the levels of this protein in the soil (Purin and Rillig, 2007). Large amounts of glomalin in NT may have contributed to higher levels of carbon in the soil in this area, since the glomalin, through the formation of soil aggregates, plays an important role in carbon storage in soil (physical protection of C within the aggregates, protecting it from microbial degradation) (Rillig, 2004). The positive correlation between glomalin and AMF spore numbers (r = 0.389, p < 0.002) corroborates the hypothesis that AMF spores are the main propagules responsible for the production of this glycoprotein, with high amounts of glomalin mainly in the walls of spores (Rillig, 2004). We observed a large variation in total number of AMF spores produced between areas and no correlation was found between sporulation and the time of ecological restoration. Indeed, high spore numbers were found in R05, the area undergoing regeneration most recently. Some studies show a high sporulation in soils with cultivation, when compared with natural vegetation (Moreira et al., 2006). This can explain the higher number of spores found in R05, since this area was covered by an agricultural crop only five
years ago. Possibly, in the already established forest, in a well balanced climax, the fungi can spend more energy for mycelial growth than for the production of spores (Stürmer and Siqueira, 2011). The AMF community of all areas was dominated by Glomus and Acaulospora. This pattern is similar to that observed in other surveys in the Mata Atlantica biome in the state of São Paulo (Carrenho et al., 2001; Aidar et al., 2004; Moreira-Souza et al., 2003; Moreira et al., 2009), in Santa Catarina (Stürmer et al., 2006), and in Rio Grande do Sul (Zandavalli et al., 2008). Similar results were also obtained in tropical forest in China (Zhao et al., 2001) and in the ˜ et al., 2007). Thus, these two Colombian Amazon (Pena-Venegas genera seem to be more common and more adapted to different soil characteristics in this study, as well as in different ecosystems. Low numbers of Ambispora, Gigaspora, Racocetra and Scutellospora were also detected in this study. Records from the literature indicate that Acaulospora species are favored by low pH values whereas the occurrence of some of the Glomus species is favored in soils with pH values between 6.0 and 8.0 (Stürmer et al., 2006). Our results do not support this pattern, as Glomus accounted for 32% of the total number of species recovered and soil pH ranged from 4.4 to 4.9. Although the pH of the soil under study is within the range that favors Gigaspora and Scutellospora (Stürmer et al., 2006; Zandavalli et al., 2008), they were less abundant in all areas. However, we cannot rule out the possibility that these genera are present predominantly as external mycelium. In fact, analyses of AMF communities based solely on spore morphology may not be very accurate, as molecular data evidenced that the identified AMF species represent only a fraction of the total AMF community diversity (Helgason and Fitter, 2009). Moreira et al. (2006) reported on the presence of a large amount of auxiliary cells in the roots, what indicates an abundant occurrence of Gigaspora, Scutellospora and/or Racocetra. Other soil
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attributes, as texture may contribute to a predominance of Glomus and Acaulospora over Gigaspora and Scutellospora that may be better adapted to sandy soils (Lekberg et al., 2007). The high number of spores of G. viscosum in R05 perhaps indicates that this species is better adapted to areas under human influence and soils with higher density and moisture. The species G. macrocarpum and Claroideoglomus etunicatum, commonly found in surveys of the Atlantic Forest biome in São Paulo (Carrenho et al., 2001; Aidar et al., 2004; Moreira et al., 2009), were not found in this study. It is possible that these species were present, but could not be readily identified; some Glomus spores easily loose important structures for their identification as the subtending hypha and evanescent layers. Conversely, species such as A. foveata and A. scrobiculata that predominated in other studies in the Mata Atlantica biome were also common in this work. A wide distribution of A. mellea and A. scrobiculata (occurring in all areas and seasons) indicates that these species can tolerate large variation of soil conditions, climate and host plant phenologies. A compilation of Glomeromycota species occurring in Brazilian soils shows that these species are usually found with high frequency (Stürmer and Siqueira, 2011). Only in R05 there were significant differences in the number of spores between summer and winter, with the largest number occurring in the rainy season. The seasonal effect on the dynamics of AMF is not well understood, many divergent results are found in the literature (Trufem and Viriato, 1990; Aidar et al., 2004; Moreira et al., 2006). These differences are associated with the characteristics of each environment, and vary according to the AMF species present and also due to the genetic tendency for a species to sporulate more than another in a determinate period of the year (Entry et al., 2002; Moreira et al., 2009). In this study the sporulation of some AMF species was season dependent. Some species were found to sporulate only in the rainy season and many others sporulated exclusively in the dry season. This result evidences the need of sampling in distinct seasons to recover most of the AMF species, especially in regions were only two seasons are found. Only in R05 the species did not change with the time of sampling. Possibly, since this area is theoretically the most disturbed one, due to recent agriculture, many AMF species had been lost and it may be that only those species that survived human intervention in this ecosystem still prevail. Higher indices of species diversity (H) and species richness (R) were observed in NT, when comparing with areas under recovery, and AMF species richness was correlated with the gradient of environmental restoration in both seasons. This fact has been confirmed by others, since often areas under anthropogenic influence have greater numbers of spores and lower species diversity (Moreira-Souza et al., 2003). The higher plant diversity in NT may have contributed to a greater diversity of AMF in this area, since different AMF species can possibly be associated with many host plants, which results in differential sporulation (Carrenho et al., 2001; Boeger et al., 2005). Although high plant diversity does not necessarily reflect a high AMF diversity (Stürmer et al., 2006). Other features in NT, such as absence of disturbance (Kernaghan, 2005), and more intense microbial activity, which provides more efficient nutrient cycling, may also have contributed to a greater diversity of AMF. The CDA analysis confirms large differences between AMF communities and ecosystems in different seasons. Positive values of the PDRC indicate an effect of separation among areas, where the species with the highest values were those that contributed most for the separation. This indicates that among all AMF species found G. viscosum, Glomus spp. (Glomus sp.1, sp.2, sp.3, sp.4 and sp.5), S. heterogama, A. scrobiculata, A. mellea, A. spinosa, A. lacunosa, R. intraornata, G. decipiens and Gigaspora spp. (Gigaspora sp.1 and sp.2) were the most important in separating areas. This separation is related to the characteristics of each environment, as well as to
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anthropogenic disturbances that impose various restrictions on the survival of certain species. In the Araucaria angustifolia ecosystems, other species, such as Scutellospora sp.1, Acaulospora sp.1, G. decipiens and S. pellucida, were those that contributed most to separating the native and reforested areas (Moreira et al., 2009). The results of this study contribute to the knowledge of the distribution of AMF species in soils of native rainforest and in the process of environmental recovery. Furthermore, they permit to learn which AMF species contributed the most to distinguish between forest ecosystems and which are more adapted to the environment with higher anthropogenic influences. This work leads to a better understanding of AMF in tropical forests that can be used for the management and recovery of threatened ecosystems, such as the Brazilian Atlantic Forest. Acknowledgments JAB and RLFV thank Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) (process number 135756/20090) and “Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP)” (process number 09/07506-1) for the respective research fellowships. SLS would like to thank CNPq for a Research Assistantship (Process 302667/2009-1). EJBNC acknowledges a Research Grant from CNPq. Special thanks are due to Denise Mescolotti and Luis Fernando Baldesin for technical assistance. References Aidar, M.P.M., Carrenho, R., Joly, C.A., 2004. Aspects of arbuscular mycorrhizal fungi in an Atlantic Forest chronosequence in Parque Estadual Turístico do Alto Ribeira (PETAR), SP. Biota Neotrop. 4, 1–15. Alef, K., 1995. Soil respiration. In: Alef, K., Nannipieri, P. (Eds.), Methods in applied soil microbiology. Academic Press, London, pp. 214–219. Bedini, S., Avio, L., Agrese, E., Giovannetti, M., 2007. Effects of long-term land use on arbuscular mycorrhizal fungi and glomalin-related soil protein. Agric. Ecos. Environ. 120, 463–466. Boeger, M.R.T., Wisniewski, C., Reissmann, C.B., 2005. Nutrientes foliares de espécies arbóreas de três estádios sucessionais de Floresta Ombrófila Densa no Sul do Brasil. Acta Bot. Bras. 19, 167–181. Carrenho, R., Trufem, S.F.B., Bononi, V.L.R., 2001. Fungos micorrízicos arbusculares em rizosferas de três espécies de fitobiontes instaladas em área de mata ciliar revegetada. Acta Bot. Bras. 15, 115–124. Coelho, N.M.M., Andrade, J.C., Cantarella, H., 1992. Determinac¸ão de amônio e nitrato em solos por injec¸ão em fluxo, pelo método difusão-condutividade. Rev. Bras. Ci. Solo 16, 325–329. Colombo, A.F., Joly, C.A., 2010. Brazilian Atlantic Forest lato sensu: the most ancient Brazilian forest, and a biodiversity hotspot, is highly threatened by climate change. Braz. J. Biol. 70, 697–708. Embrapa, 1997. Manual de métodos de análise de solo, 2nd ed. Embrapa, Rio de Janeiro. Entry, J.A., Rygiewiez, P.T., Watrud, L.S., Donnelly, P.K., 2002. Influence of adverse soil conditions on the formation and function of arbuscular mycorrhizas. Adv. Environ. Res. 7, 123–138. Gerdemann, J.W., Nicolson, T.H., 1963. Spores of mycorrhizal endegone species extracted from soil by sieving and decanting. Trans. Br. Mycol. Soc. 46, 235–246. Helgason, T., Fitter, A.H., 2009. Natural selection and the evolutionary ecology of the arbuscular mycorrhizal fungi (Phylum Glomeromycota). J. Exp. Bot. 60, 2465–2480. Kernaghan, G., 2005. Mycorrhizal diversity: cause and effect? Pedobiologia 49, 511–520. Lekberg, Y.R.T., Koide, R., Rohr, J.R., Aldrich-Wolfe, L., Morton, J.B., 2007. Role of niche restrictions and dispersal in the composition of arbuscular mycorrhizal fungal communities. J. Ecol. 5, 95–105. Magurran, A.E., 2004. Measuring Biological Diversity. Blackwell Science, Oxford. Mariani, L., Changa, S.X., Kabzems, R., 2006. Effects of tree harvesting, forest floor removal, and compaction on soil microbial biomass, microbial respiration, and N availability in a boreal aspen forest in British Columbia. Soil Biol. Biochem. 38, 1734–1744. Moreira, M., Baretta, D., Tsai, S.M., Cardoso, E.J.B.N., 2006. Spore density and root colonization by arbuscular mycorrhizal fungi in preserved or disturbed Araucaria angustifolia (Bert.) O. Ktze. ecosystems. Sci. Agric. 63, 380–385. Moreira, M., Baretta, D., Tsai, S.M., Cardoso, E.J.B.N., 2009. Arbuscular mycorrhizal fungal communities in native and in replanted Araucaria forest. Sci. Agric. 66, 677–684. Moreira-Souza, M., Trufem, S.F.B., Gomes-da-Costa, S.M., Cardoso, E.J.B.N., 2003. Arbuscular mycorrizal fungi associated with Araucaria angustifolia (Bert.) O. Ktze. Mycorrhiza 13, 211–215.
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