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The role of arbuscular mycorrhizal fungi and cattle manure in the establishment of Tocoyena selloana Schum. in mined dune areas
European Journal of Soil Biology 46 (2010) 237e242
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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
The role of arbuscular mycorrhizal fungi and cattle manure in the establishment of Tocoyena selloana Schum. in mined dune areas Renata Gomes de Souza a, Bruno Tomio Goto a, Danielle Karla Alves da Silva a, Fábio Sérgio Barbosa da Silva c, Everardo V.S.B. Sampaio b, Leonor Costa Maia a, * a
Programa de Pós-Graduação em Biologia de Fungos, Departamento de Micologia, Universidade Federal de Pernambuco, Av. Prof. Nelson Chaves s/n, 50670-420 Recife, PE, Brazil Departamento de Energia Nuclear, Universidade Federal de Pernambuco, Av. Prof. Luis Freire 1000, 50740-540 Recife, PE, Brazil c Universidade de Pernambuco e Campus Petrolina (UPE), BR 203 Km 2, 56300-000 Petrolina, PE, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 November 2009 Received in revised form 19 February 2010 Accepted 9 April 2010 Available online 24 April 2010 Handling editor: Hermann Verhoef
In mined dune areas, revegetation with manured seedlings of native species is a common practice. Establishment of mycorrhized Tocoyena selloana seedlings in the mined coastal sand dunes of Northeast Brazil was tested. In greenhouse, seedlings were grown in substrates with 0, 5, 10, 15 or 20% cattle manure proportions and inoculated with Acaulospora longula, a mixture of native arbuscular mycorrhizal fungi (AMF) or uninoculated. The seedlings responded positively to the inoculation, but growth was limited in the absence of manure, independently of inoculation, and was higher with fertilizing doses 10%. The seedlings transplanted to the field were grown in a substrate with 16.5% manure and inoculated with A. longula or Gigaspora albida. After 13 months, 19 AMF species were identified in the rhizosphere and the inoculated plants were more colonized than those uninoculated. Plants associated to G. albida were taller and those associated to A. longula had a tendency of higher biomass than the uninoculated ones. Even though this tendency was not statistically significant, considering the effect on height and the low cost of inoculation it may be a feasible practice to maximize environment restoration. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Dunes AMF Mycorrhizal dependency Symbiotic efficiency
1. Introduction Soil microorganisms are involved in nutrient cycles and are important in revegetation processes. Therefore, management of the microbial community, with emphasis on the symbionts, can play a crucial role in the recovery of impacted ecosystems [1]. Among the main plant, fungi and soil interactions, the beneficial symbiotic associations between arbuscular mycorrhizal fungi (AMF) and roots of terrestrial plants are the most widespread in nature. This symbiosis is established with the bi-directional transfer of nutrients, favoring the development of plant communities, especially when soil fertility is a limiting factor, and becoming indispensable for the establishment of seedlings on infertile tropical soils [11]. AMF provide higher tolerance to water [30] and saline [39] stresses and to root pathogen attack [21]. The extra-radicular mycelial net also contributes to soil structure. The positive effects of the symbiosis for AMF and plants depend on intra- and interspecific competitivity of communities [37], on the functional and
* Corresponding author. Tel.: þ55 81 2126 8865; fax: þ55 81 2126 8482. E-mail address: [email protected] (L.C. Maia). 1164-5563/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2010.04.004
physiological compatibility among symbionts, and on the interactions among plants, soil and fungi [23]. In revegetation, other important aspects are the successional categories of plant species to be used [29], the soil physico-chemical properties [1], the use of chemical [28] or organic [15] fertilizers, and AMF inoculation with strains suitable for the edaphoclimatic conditions [7]. The benefits of introducing mycorrhized seedlings in the revegetation processes are associated with plant survival and growth, and incorporation of microorganisms and organic matter to the soil [5]. Addition of organic fertilizer to the substrate used for seedling production increases nutrient levels, improves microbiological and enzymatic activities and decreases soil density [8]. However, excess of organic fertilizer can hamper spore production, mycorrhizal colonization and, sometimes, plant response [24,36]. Although important, the use of mycorrhized plants in the recovery of degraded areas is incipient, due to difficulties in management of native species and limited knowledge of the plant responses to mycorrhizal colonization. Thus, the objective of this study was the evaluation, in greenhouse and field, of the effects of AMF inoculation and cattle manure addition on growth of a plant species used in the revegetation of coastal sand dunes impacted by mining activities.
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2. Materials and methods
The experiment was a completely randomized block design, in a 5 3 factorial arrangement, with four replications, consisting of the five manure proportions (0, 5, 10, 15 and 20%) and the three inoculation treatments (native AMF, A. longula and control). The inoculum of A. longula (UFPE 21) was obtained after multiplication in sandy-clay soil (pH 5.4 e 3 mg P dm3) with Panicum miliaceum L. (millet). The inoculum of native AMF was obtained from a composite soil sample taken from dunes revegetated 16, 12, 8 and 4 years ago and from an undisturbed dune. To identify the AMF present in this inoculums, spores were extracted [13,19], mounted on slides with PVLG (polyvinylic alcohol in lactoglycerol) and Melzer’s reagent þ PVLG (1:1) and identified after consulting Schenck & Pérez [33], the INVAM [18] (International Culture Collection of Arbuscular Mycorrhizal Fungi) homepage and publications with descriptions of new species. The following species were registered: Acaulospora scrobiculata Trappe, Ambispora appendicula (Spain, Sieverd. & N.C. Schenck) C. Walker, Glomus coremioides (Becker & Broome) D. Redecker & J.B. Morton, Glomus glomerulatum Sieverd., Paraglomus occultum (C. Walker) D. Redecker & J.B. Morton, Gigaspora aff. margarita. W.N. Becker & I.R. Hall, Scutellospora biornata Spain, & S. Toro, Scutellospora aff. fulgida Koske & C. Walker and Scutellospora heterogama (T.H. Nicolson & Gerd.) C. Walker & F.E. Sanders. The seedlings were kept in a greenhouse until harvest, 112 days after being transferred to the bags, when they reached the recommended height for field transplanting. During the experiment, soil moisture was adjusted daily to occupy 40% of the total soil pore space. The average temperature, in the greenhouse, varied from 22.1 C (March) to 38.6 C (September). At harvest, shoot and root fresh and oven-dried matter were determined. From each plant, 0.5 g of fresh roots were washed, cleared with KOH (10%), stained with Trypan blue [27] and preserved in glycerol 50% until analysis of mycorrhizal colonization. The percentage of colonization was calculated according to McGonigle et al. [22]. The dried plant material was ground, digested with a nitroperchloric solution, and extracts were analyzed for phosphorus (P), by colorimetry, and for potassium (K), by photometry. Nitrogen (N) was determined after sulfuric digestion by the Kjeldahl method [32]. Mycorrhizal effectiveness was estimated by the formula: ME ¼ 1 (b/a), where a ¼ average value of each variable (biomass, N and P contents) for each inoculated treatment and b ¼ average value of the variables for the control. The symbiosis was considered beneficial to the plants when ME >0 [16]. Increment rates of total biomass promoted by the inoculation were calculated as proposed by Weber et al. [38]: I% ¼ 100 [(X Y)/Y], where X ¼ average value of the inoculated treatment and Y ¼ average value of the control.
2.1. Study area The experiment was conducted in the Mataraca municipality, Paraiba State, Northeast Brazil (6 280 2000 e6 300 0000 S, 34 550 5000 e34 570 1000 W), where the predominant geomorphological formation is clay-sand sedimentary rocks, overlaid by fixed dunes up to 100 m high. The dune sand is rich in minerals of economic interest, such as ilmenite, rutile and zirconita. The vegetation on the dunes varies in physiognomy from an open herb layer to sclerophyllous forests. The climate is tropical rainy (Am type of Köppen), with a short dry period (OctobereJanuary) and 25 C annual average temperature and 1700 mm rainfall [26]. Mining activities, dune physical restoration and revegetation are done in the site since 1989, by the Millennium Inorganic Chemicals Mining, PB (henceforth abbreviated to Millennium). For mineral extraction, the vegetation is removed, the soil top 30 cm layer is scraped and piled up, the dune is disassembled and the sand transported to the extraction plant unit. After extraction, the waste material, mostly formed by quartz sand, is taken back to the site and used to reassemble the dune shape. After physical reassembling, the dune is covered again with the top soil and revegetation carried out with previously prepared seedlings. The most commonly used species are: cashew, Anacardium occidentale L. (Anacardiaceae); “peroba”, Tabebuia roseo-alba (Ridl.) Sandw. (Bignoniaceae); “jenipapo bravo”, Tocoyena selloana Schum. (Rubiaceae); “joazeiro”, Ziziphus joazeiro Mart. (Rhamnaceae) and “mutamba”, Guazuma ulmifolia Lam. (Sterculiaceae). Experiments with T. selloana were conducted in a greenhouse, from March to September 2005, and in the field, in a restored dune area, from July 2006 to September 2007. 2.2. Greenhouse experiment Tocoyena selloana seeds were collected in the mining area of the Guaju River, in Mataraca, and germinated in plastic cups (200 ml), containing dune soil (mostly quartz sand) collected in the mining area, mixed with 20% (v : v) of commercial cattle manure (pH ¼ 7.0; N ¼ 14.2 g kg1; C ¼ 173.4 g kg1). When four leaves developed the seedlings were transplanted to similar cups filled with a mixture of dune soil and cattle manure, in the proportions of 0, 5, 10, 15 and 20% manure by volume, and disinfected with BromexÒ (98% methyl bromide and 2% chloropicrin). At the time of transplanting, 20 seedlings were inoculated near the roots with a mixture of native AMF, obtained from composite samples of dune soil, 20 were inoculated with Acaulospora longula Spain & N.C. Schenck, and 20 were not inoculated and taken as a control treatment. The inoculated treatments received suspensions with 150 glomerospores per plant. After five days, substrate and seedlings were transferred to black plastic bags (2 kg), containing the same proportions of soil and manure. The substrates were analyzed (Table 1) by the Instituto Agronômico de Pernambuco (IPA).
2.3. Field experiment Tocoyena selloana seedlings about 25 cm tall, grown in a substrate containing approximately 16.5% of cattle manure (usual practice), were supplied by Millennium. In four of these seedlings,
Table 1 Physicalechemical characteristics of the substrates with increasing proportions of cattle manure used in the greenhouse experiment with Tocoyena selloana. Manure (%)
0 5 10 15 20
P
Ca
mg dm3
cmolc dm3
K
5 54 90 >90 >90
0.40 1.00 1.45 1.75 2.25
0.04 0.24 0.38 0.44 0.47
Na
0.08 0.09 0.10 0.11 0.11
Mg
0.20 0.70 0.65 1.10 0.90
Al
0.60 0.40 0.30 0.40 0.35
CEC
5.60 6.30 5.10 6.90 6.80
pH H2O
4.20 4.36 4.58 4.80 5.08
O.M.
Global density
g kg1
g cm3
10.2 13.5 16.5 16.8 17.5
e 1.38 1.31 1.23 1.18
Particle density
Porosity %
e 2.60 2.53 2.50 2.53
e 46.9 48.2 50.8 53.4
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mycorrhizal colonization and number of glomerospores were estimated and the AMF species present in the rhizosphere were identified. A composite sample of the substrate was analyzed (Table 2), revealing weak acidity, low level of P and medium levels of organic matter, cation exchange capacity and Ca and Mg. The experiment was a completely randomized block design, with three treatments and seven replications. The treatments corresponded to different inoculations. At planting time, 35 seedlings received soil inoculum with approximately 150 spores of A. longula (UFPE 21), 35 received inoculum of Gigaspora albida Schenck & G.S. Sm. (UFPE 01) and 35 were not inoculated (control). The inoculum of G. albida was multiplied in clay-sand soil (pH ¼ 5.4 e P ¼ 5 mg dm3), in association with P. miliaceum, while the inoculum of A. longula was the same used in the greenhouse experiment. Planting followed the procedures used by Millennium. Pits, 70 cm deep and 20 cm in diameter, were opened with a manual soil auger, along a 36 m line. The pits were 2 m from each other, with 2 m of distance between the rows. In each pit, the top 20 cm of soil layer was set aside for closing the holes, which were filled with five gallons of a mixture of sugar cane bagasse and cattle manure (pH H2O ¼ 7.3; N ¼ 1.4 g kg1; C ¼ 147.2 g kg1; P ¼ 0.4 g kg1; and K ¼ 1.5 g kg1). On the same day, before planting, ten soil samples of the area were collected for chemical analysis (Table 2) and counting of AMF spores. Weeds were removed and a foliar fertilization (VITAN e organomineral, A Class) was applied eight months after planting. Height and stem diameter were measured every month, until the sixth one; and every two months, from then to the end of the experiment, 13 months after planting. The average temperature varied from 25.9 C to 30.2 C (JulyeDecember 2006) and from 29.7 C to 27.6 C (JanuaryeSeptember 2007). The total of rainfall was 1245.8 mm in 2006 and 1885.4 mm in 2007. At harvest, two plants per plot were excavated, their shoots and roots separated and the material oven dried and weighed. Fine root samples were taken for mycorrhizal colonization assessment, as previously described, and colonization percentage was determined according to the gridline intercept method [14]. Rhizosphere soil samples (100 cm3) were used for spore extraction and counting, and for AMF species identification. The relative abundance of each AMF species was calculated dividing the spore number of a species by the number of spores of all species [4]. Mycorrhizal effectiveness [16], increment rate of total biomass enhanced by inoculation [38], and P and N concentrations in the biomass [32] were determined as described.
2.4. Statistical analysis The number of glomerospores was transformed into log (x þ 1) pffiffiffi and the colonization percentage to arcsin x. Analysis of variance
239
and average comparisons (Duncan test p 0.05) were performed using the Statistica 5.0 program [35]. 3. Results 3.1. Greenhouse The seedlings inoculated with A. longula and with the AMF mixture accumulated significantly more biomass than the uninoculated ones (Table 3), with increments of 30 and 39%, respectively. Average height showed the same trend but the differences (13 and 23%) were not statistically significant (data not shown). Manure absence limited seedlings growth and 10 and 15% manure dosages were more suitable for preparing the seedlings (Fig. 1). P and K concentrations varied little in the three inoculation treatments (Table 3). Thus, the contents were mainly influenced by the plant biomasses and were higher in the inoculated treatments. The N concentration was slightly lower in plants associated to A. longula but the increase in biomass compensated for that, and the contents were similar. At the highest manure proportion in the substrate (20%), no colonization occurred in both inoculated treatments (Fig. 2). At lower proportions, A. longula and the AMF mixture seemed to have different behaviors. While colonization by A. longula increased with higher manure proportions that of the AMF mixture was higher in the treatment without manure or at the lowest manure proportion. Therefore, inoculation with A. longula seems to be a better option for manure treated seedlings. No colonization occurred in the uninoculated treatment. 3.2. Field Soil samples from the top soil layer, collected after dune reassembly but before planting, contained 46 spores per 100 g of soil, with three AMF species: Acaulospora morrowiae, Glomus etunicatum and G. glomerulatum. The substrate of the seedlings before inoculation had a lower number of glomerospores (6 spores 100 g1 soil) and shared the first two species with the top soil but contained an additional species: A. scrobiculata. Mycorrhizal colonization was not confirmed in the root samples of the seedlings because no vesicles or arbuscules were observed and just a few intraradical hyphae were present. After 13 months, 13 AMF species, belonging to five genera, were identified in the rhizosphere of the uninoculated control plants (Table 4). Eleven “new” species were then present but G. etunicatum was not found. Most glomerospores were from A. longula (55% of the total density) and A. morrowiae (28%). The rhizosphere of plants inoculated with A. longula had almost the same number of species (12) but only seven in common with the control. As expected, glomerospores of A. longula were the most abundant but with
Table 2 Chemical characteristics of the substrate (SS) of Tocoyena selloana seedlings, of the soil collected in the area before planting (BP) and of the rhizosphere of non-inoculated plants (NI), of plants inoculated with Acaulospora longula (AL) or with Gigaspora albida (GA) and of the soil between the lines of planting (LP). Treatments
pH H2O
Beginning of the experiment SS 5.66 BP 5.12 End of the experiment (13 months) NI 5.40 AL 5.60 GA 5.40 LP 5.10
P
K
mg dm3
cmolc dm3
Ca
Na
Mg
16 2
0.02 0.02
1.60 0.50
0.02 0.02
1.00 0.50
6 7 7 2
0.05 0.05 0.05 0.01
0.70 0.65 0.75 0.40
0.05 0.04 0.05 0.03
0.35 0.30 0.40 0.25
Al
OM
CEC
g kg1
cmolc dm3
0.10 0.25
15.5 7.4
5.8 4.3
0.25 0.20 0.20 0.25
11.5 14.5 11.0 9.5
3.9 4.0 4.9 3.5
240
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Table 3 Effects of inoculation with a mixture of native AMF or Acaulospora longula on biomass production, and on concentrations and contents of N, P and K in seedlings of Tocoyena selloana grown in greenhouse. Content (mg plant1)
Concentration (mg g1)
Treatments
Biomass (g plant1)
N
P
K
N
P
K
Mixture A. longula Uninoculated
9.42a 8.85a 6.76b
106.5a 92.1ab 89.7b
45.6a 41.7a 32.4b
173.5a 165.2a 126.8b
27.0a 23.6b 30.1a
8.1a 7.7a 7.9a
29.9a 30.1a 31.3a
Values followed by the same letter in the column do not differ by the Duncan’s test (p 0.05).
a relative density (59%) not much greater than in the control. The rhizosphere of the G. albida inoculated plants had a lower number of species (8) and only five in common with the control. It is remarkable that also in this treatment glomerospores of A. longula were the most abundant (47%), while those of G. albida ranked fourth in abundance with only 10% of the total density. Soil samples taken in-between the planting lines, away from the rhizosphere influence, had only three AMF species which were different from those originally present in the soil top layer; the species with more glomerospores, from a low total of only 14 spores per 100 g of soil, was G. albida (79%). Most of the seedlings (>95%) survived the first 13 months in the field, in all treatments, and grew 40e50 cm taller than when transplanted. In the seedlings inoculated with G. albida or A. longula colonization was about 40%, contrasting with only 2% in the uninoculated seedlings (Table 5). The seedlings inoculated with G. albida were significantly higher (79 cm) than those of the control (67 cm), while the A. longula inoculated ones (75 cm) did not differ from both. Apart from these differences, no other plant measurement differed significantly among the treatments. Although not statistically significant (p > 0.05), plants inoculated with A. longula tended to accumulate more biomass, P and N contents than others treatments (Fig. 3), with biomass increments (A. longula ¼ 41%, G. albida ¼ 14%) of similar magnitude of those obtained in the greenhouse. 4. Discussion Positive effects of mycorrhization were more evident in the greenhouse than in the field, in spite of similar increments in biomass and P and N contents. Certainly, in the field, one or more environmental factors affected the plants to a larger degree than the inoculation treatments. Wind exposure seems to be one of these factors. In spite of the absence of a significant response, the increments due to the inoculation of A. longula relative to the
Fig. 1. Effects of AMF inoculation and proportion of cattle manure added to the substrate on the total biomass dry weight (BDW) of Tocoyena selloana seedlings (n ¼ 60). Values followed by the same letter, small in the line and capital between the lines, do not differ by the Duncan’s test (p 0.05), NI ¼ uninoculated; MIX ¼ mixture of native AMF; AL ¼ Acaulospora longula.
control, in aboveground biomass (0.29) and P (0.16) and N contents (0.31), indicate that this inoculation may be recommended, until more tests are done. The low cost and relatively simplicity of the inoculation procedure may compensate even a small increase in plant growth. Formation of mycorrhizal structures by AMF used for inoculation in the greenhouse experiment varied with manure proportions. Colonization by the native AMF was higher at the lowest proportion (5%), while that of A. longula was higher at 15% manure. These differences did not imply in different responses on increment of host biomass by these AMF. In the field, the low colonization of the uninoculated plants suggests that the native AMF were inhibited by the 16.5% proportion of manure added to the substrate. They are probably better adapted to soils with low organic matter contents, which are their usual condition [2]. A change in these conditions may affect spore germination and colonization [20]. Wang et al. [36] observed that high manure proportions decreased colonization, spore density and species richness and also altered the species composition of the AMF in soils cropped to wheat and maize. In the greenhouse, fertilized substrates had high P and K content (Table 1) probable enough to maintain high plant growth rates, but may not had enough N. N limitation probably made the difference between the treatments, inoculated plants having higher ability to absorb N. The N limitation, also verified in the field, indicates that there should be a supplementation of this nutrient. It would be interesting to test the addition of a substrate richer in N for seedlings preparation, such as cotton or castor bean seed cakes, or the addition of green manure or mineral fertilizer in the field. The evaluation of different isolates under the same conditions can assist in a more efficient AMF selection and, in general, soils with low fertility and/or low infective potential favor the activity of introduced fungi, if they are compatible with the plants and the environmental conditions [34]. In this case, the AMF community present in the rhizosphere of inoculated plants was more effective in promoting T. selloana height development in the field, with also
Fig. 2. Mycorrhizal colonization in Tocoyena selloana seedlings inoculated with a mixture of native AMF (MIX) or Acaulospora longula and grown in substrates with increasing proportions of cattle manure in greenhouse. Values followed by the same letter, small in the line and capital between the lines, do not differ by the Duncan’s test (p 0.05).
R.G. de Souza et al. / European Journal of Soil Biology 46 (2010) 237e242 Table 4 Relative abundance (%) of AMF species in soil collected between the lines of planting (LP) or in the rhizosphere of Tocoyena selloana plants inoculated with Acaulospora longula (AL), Gigaspora albida (GA) or non-inoculated (NI), 13 months after seedling transplant to the field. Species of AMF
LP
AL
GA
NI
Acaulospora foveata Trappe & Janos A. longula Spain & N.C. Schenck A. morrowiae Spain & N.C. Schenck A. scrobiculata Trappe A. spinosa C. Walker & Trappe A. tuberculata Janos & Trappe Ambispora appendicula (Spain, Sieverd. & N.C. Schenck) C. Walker Gigaspora aff. albida N.C. Schenck & G.S. Sm. Glomus aff. fasciculatum (Thaxt.) Gerd. & Trappe emend. C. Walker & Koske G. sinuosum (Gerd. & Bakshi) R.T. Almeida & N.C. Schenck G. glomerulatum Sieverd. Scutellospora aurigloba (I.R. Hall) C. Walker & F.E. Sanders S. biornata Spain, Sieverd. & S. Toro S. calospora (T.H. Nicolson & Gerd.) C. Walker & F.E. Sanders S. aff. fulgida Koske & C. Walker S. gregaria (N.C. Schenck & T.H. Nicolson) C. Walker & F.E. Sanders S. pellucida (T.H. Nicolson & N.C. Schenck) C. Walker & F.E. Sanders S. rubra Stürmer & J.B. Morton S. aff. verrucosa (Koske & C. Walker) C. Walker & F.E. Sanders
e e e e e e 7.1
1.5 58.5 3.1 1.5 e e e
e 47.2 e 17.3 e 9.1 11.5
e 55.0 28.0 2.3 2.3 2.3 1.1
78.7 e
7.7 e
10.3 e
1.1 2.3
e
1.5
e
e
e e
12.4 3.1
e 1.1
1.1 e
e e
e 1.5
1.1 e
e 1.1
e e
4.6 e
2.3 e
e 1.1
e
3.1
e
e
1.5 e
e e
14.3 e
Total number of species
3 3
Number of glomerospores (50 cm soil)
14b
12 69.1a
8 91.4a
1.1 1.1 13 52.5a
Values followed by the same letter do not differ by the Duncan’s test (p 0.05).
a tendency of larger biomass and nutrient contents. Sharma et al. [31] observed that mycorrhizal plants were superior in height, weight and nutrient concentrations than uninoculated ones, but the extent of the responses varied with the inoculum used. It should be also considered that some mycorrhizal benefits can only be effective with periods longer than the 13 months of the experiment. Inoculation at planting time and field conditions associated with the host plant provided an increase of A. longula sporulation, whereas glomerospores of this species were not observed in the seedlings substrate or in soil samples from the area, before planting. Oehl et al. [25] found A. longula spores in organic systems. Members of Acaulosporaceae, especially A. longula [3] are favored by low pH, and that of the study site was between 5.1 and 5.6. G. albida was not the most abundant where it was inoculated; may be because its proliferation was hampered by the manure addition. Cordoba et al. [10] related an experiment in which Gigaspora spp. decreased in fertilized plots in comparison to non-fertilized ones. They also stated that low numbers of Gigasporaceae spores may
Table 5 Effects of inoculation with Acaulospora longula or Gigaspora albida on shoot (SDW) and root dry weight (RDW) and on the mycorrhizal colonization of Tocoyena selloana plants, after 13 months in the field. Treatments
Uninoculated A. longula G. albida
Height
Diameter
SDW
RDW
Colonization
cm
cm
g plant1
g plant1
%
67.4b 74.7ab 78.7a
1.6a 1.8a 1.8a
94.0a 140.0a 113.1a
69.3a 90.3a 74.5a
2.5b 39.8a 40.3a
Values followed by the same letter in the column do not differ by the Duncan’s test (p 0.05).
0.40
241
Biomas N
0.30
P
0.20 0.10 0.00
A. longula
G. albida
Fig. 3. Effectiveness of the symbiosis in biomass production and N and P contents of Tocoyena selloana plants inoculated with Acaulospora longula or Gigaspora albida, after 13 months in field.
result from its strategy of producing few but large multiplegerminating spores, each one capable of encountering a multitude of roots. The high abundance of A. longula in the rhizosphere of all treatments confirms the greenhouse results and shows that it thrives well in soils with about 15% of manure, although more research is needed to confirm these results. The highest relative abundance of A. longula and A. morrowiae spores suggests that these species are better adapted to the first recovery stage of the studied area. Higher sporulation may be associated with high competitive capacity [9], better adaptation to the environment [6] and a certain degree of specificity between the symbionts. The substrates for seedlings growth favored the introduction of AMF species in the field, as also observed by Caproni et al. [5], and may have contributed to the AMF spread and establishment. The increased number of taxa in the treatments, thirteen months after planting, in relation to the initial number, also may have occurred because of wind dispersion [12] and a reduction in dominance of some species [17]. Alteration in the rhizosphere AMF community may have resulted from dispersal mechanisms or due to the ability of the introduced species to compete with those that were in place or were established after seedling planting in the field. The proportions 15% of manure used in the substrates promoted seedling growth increases in the greenhouse but inhibited colonization by native AMF, both inoculated in the greenhouse and under natural conditions in the field. A. longula thrived well at this manure proportion, dominating the spore production and achieving high colonization percentages. Its contribution to the seedlings growth in the field was small (29% increase in biomass) but considering the low cost of inoculation it may be a feasible practice and an alternative to maximize environment restoration. Acknowledgements To the Millennium Inorganic Chemicals Mining, a Cristal Company, to the Instituto Agronômico de Pernambuco (IPA) for performing the soil analyses, to CAPES for providing a PhD scholarship to B.T. Goto and to CNPq for a PhD scholarship to R.G. Souza and research grants to E.V.S.B. Sampaio and L.C. Maia. References [1] R. Azcón, J.M. Barea, Mycorrhizal dependency of a representative plant species in mediterranean shrublands (Lavandula spica L.) as a key factor to its use for revegetation strategies in desertification-threatened areas. Appl. Soil Ecol. 7 (1997) 83e92.
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Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
The role of arbuscular mycorrhizal fungi and cattle manure in the establishment of Tocoyena selloana Schum. in mined dune areas Renata Gomes de Souza a, Bruno Tomio Goto a, Danielle Karla Alves da Silva a, Fábio Sérgio Barbosa da Silva c, Everardo V.S.B. Sampaio b, Leonor Costa Maia a, * a
Programa de Pós-Graduação em Biologia de Fungos, Departamento de Micologia, Universidade Federal de Pernambuco, Av. Prof. Nelson Chaves s/n, 50670-420 Recife, PE, Brazil Departamento de Energia Nuclear, Universidade Federal de Pernambuco, Av. Prof. Luis Freire 1000, 50740-540 Recife, PE, Brazil c Universidade de Pernambuco e Campus Petrolina (UPE), BR 203 Km 2, 56300-000 Petrolina, PE, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 November 2009 Received in revised form 19 February 2010 Accepted 9 April 2010 Available online 24 April 2010 Handling editor: Hermann Verhoef
In mined dune areas, revegetation with manured seedlings of native species is a common practice. Establishment of mycorrhized Tocoyena selloana seedlings in the mined coastal sand dunes of Northeast Brazil was tested. In greenhouse, seedlings were grown in substrates with 0, 5, 10, 15 or 20% cattle manure proportions and inoculated with Acaulospora longula, a mixture of native arbuscular mycorrhizal fungi (AMF) or uninoculated. The seedlings responded positively to the inoculation, but growth was limited in the absence of manure, independently of inoculation, and was higher with fertilizing doses 10%. The seedlings transplanted to the field were grown in a substrate with 16.5% manure and inoculated with A. longula or Gigaspora albida. After 13 months, 19 AMF species were identified in the rhizosphere and the inoculated plants were more colonized than those uninoculated. Plants associated to G. albida were taller and those associated to A. longula had a tendency of higher biomass than the uninoculated ones. Even though this tendency was not statistically significant, considering the effect on height and the low cost of inoculation it may be a feasible practice to maximize environment restoration. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Dunes AMF Mycorrhizal dependency Symbiotic efficiency
1. Introduction Soil microorganisms are involved in nutrient cycles and are important in revegetation processes. Therefore, management of the microbial community, with emphasis on the symbionts, can play a crucial role in the recovery of impacted ecosystems [1]. Among the main plant, fungi and soil interactions, the beneficial symbiotic associations between arbuscular mycorrhizal fungi (AMF) and roots of terrestrial plants are the most widespread in nature. This symbiosis is established with the bi-directional transfer of nutrients, favoring the development of plant communities, especially when soil fertility is a limiting factor, and becoming indispensable for the establishment of seedlings on infertile tropical soils [11]. AMF provide higher tolerance to water [30] and saline [39] stresses and to root pathogen attack [21]. The extra-radicular mycelial net also contributes to soil structure. The positive effects of the symbiosis for AMF and plants depend on intra- and interspecific competitivity of communities [37], on the functional and
* Corresponding author. Tel.: þ55 81 2126 8865; fax: þ55 81 2126 8482. E-mail address: [email protected] (L.C. Maia). 1164-5563/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2010.04.004
physiological compatibility among symbionts, and on the interactions among plants, soil and fungi [23]. In revegetation, other important aspects are the successional categories of plant species to be used [29], the soil physico-chemical properties [1], the use of chemical [28] or organic [15] fertilizers, and AMF inoculation with strains suitable for the edaphoclimatic conditions [7]. The benefits of introducing mycorrhized seedlings in the revegetation processes are associated with plant survival and growth, and incorporation of microorganisms and organic matter to the soil [5]. Addition of organic fertilizer to the substrate used for seedling production increases nutrient levels, improves microbiological and enzymatic activities and decreases soil density [8]. However, excess of organic fertilizer can hamper spore production, mycorrhizal colonization and, sometimes, plant response [24,36]. Although important, the use of mycorrhized plants in the recovery of degraded areas is incipient, due to difficulties in management of native species and limited knowledge of the plant responses to mycorrhizal colonization. Thus, the objective of this study was the evaluation, in greenhouse and field, of the effects of AMF inoculation and cattle manure addition on growth of a plant species used in the revegetation of coastal sand dunes impacted by mining activities.
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2. Materials and methods
The experiment was a completely randomized block design, in a 5 3 factorial arrangement, with four replications, consisting of the five manure proportions (0, 5, 10, 15 and 20%) and the three inoculation treatments (native AMF, A. longula and control). The inoculum of A. longula (UFPE 21) was obtained after multiplication in sandy-clay soil (pH 5.4 e 3 mg P dm3) with Panicum miliaceum L. (millet). The inoculum of native AMF was obtained from a composite soil sample taken from dunes revegetated 16, 12, 8 and 4 years ago and from an undisturbed dune. To identify the AMF present in this inoculums, spores were extracted [13,19], mounted on slides with PVLG (polyvinylic alcohol in lactoglycerol) and Melzer’s reagent þ PVLG (1:1) and identified after consulting Schenck & Pérez [33], the INVAM [18] (International Culture Collection of Arbuscular Mycorrhizal Fungi) homepage and publications with descriptions of new species. The following species were registered: Acaulospora scrobiculata Trappe, Ambispora appendicula (Spain, Sieverd. & N.C. Schenck) C. Walker, Glomus coremioides (Becker & Broome) D. Redecker & J.B. Morton, Glomus glomerulatum Sieverd., Paraglomus occultum (C. Walker) D. Redecker & J.B. Morton, Gigaspora aff. margarita. W.N. Becker & I.R. Hall, Scutellospora biornata Spain, & S. Toro, Scutellospora aff. fulgida Koske & C. Walker and Scutellospora heterogama (T.H. Nicolson & Gerd.) C. Walker & F.E. Sanders. The seedlings were kept in a greenhouse until harvest, 112 days after being transferred to the bags, when they reached the recommended height for field transplanting. During the experiment, soil moisture was adjusted daily to occupy 40% of the total soil pore space. The average temperature, in the greenhouse, varied from 22.1 C (March) to 38.6 C (September). At harvest, shoot and root fresh and oven-dried matter were determined. From each plant, 0.5 g of fresh roots were washed, cleared with KOH (10%), stained with Trypan blue [27] and preserved in glycerol 50% until analysis of mycorrhizal colonization. The percentage of colonization was calculated according to McGonigle et al. [22]. The dried plant material was ground, digested with a nitroperchloric solution, and extracts were analyzed for phosphorus (P), by colorimetry, and for potassium (K), by photometry. Nitrogen (N) was determined after sulfuric digestion by the Kjeldahl method [32]. Mycorrhizal effectiveness was estimated by the formula: ME ¼ 1 (b/a), where a ¼ average value of each variable (biomass, N and P contents) for each inoculated treatment and b ¼ average value of the variables for the control. The symbiosis was considered beneficial to the plants when ME >0 [16]. Increment rates of total biomass promoted by the inoculation were calculated as proposed by Weber et al. [38]: I% ¼ 100 [(X Y)/Y], where X ¼ average value of the inoculated treatment and Y ¼ average value of the control.
2.1. Study area The experiment was conducted in the Mataraca municipality, Paraiba State, Northeast Brazil (6 280 2000 e6 300 0000 S, 34 550 5000 e34 570 1000 W), where the predominant geomorphological formation is clay-sand sedimentary rocks, overlaid by fixed dunes up to 100 m high. The dune sand is rich in minerals of economic interest, such as ilmenite, rutile and zirconita. The vegetation on the dunes varies in physiognomy from an open herb layer to sclerophyllous forests. The climate is tropical rainy (Am type of Köppen), with a short dry period (OctobereJanuary) and 25 C annual average temperature and 1700 mm rainfall [26]. Mining activities, dune physical restoration and revegetation are done in the site since 1989, by the Millennium Inorganic Chemicals Mining, PB (henceforth abbreviated to Millennium). For mineral extraction, the vegetation is removed, the soil top 30 cm layer is scraped and piled up, the dune is disassembled and the sand transported to the extraction plant unit. After extraction, the waste material, mostly formed by quartz sand, is taken back to the site and used to reassemble the dune shape. After physical reassembling, the dune is covered again with the top soil and revegetation carried out with previously prepared seedlings. The most commonly used species are: cashew, Anacardium occidentale L. (Anacardiaceae); “peroba”, Tabebuia roseo-alba (Ridl.) Sandw. (Bignoniaceae); “jenipapo bravo”, Tocoyena selloana Schum. (Rubiaceae); “joazeiro”, Ziziphus joazeiro Mart. (Rhamnaceae) and “mutamba”, Guazuma ulmifolia Lam. (Sterculiaceae). Experiments with T. selloana were conducted in a greenhouse, from March to September 2005, and in the field, in a restored dune area, from July 2006 to September 2007. 2.2. Greenhouse experiment Tocoyena selloana seeds were collected in the mining area of the Guaju River, in Mataraca, and germinated in plastic cups (200 ml), containing dune soil (mostly quartz sand) collected in the mining area, mixed with 20% (v : v) of commercial cattle manure (pH ¼ 7.0; N ¼ 14.2 g kg1; C ¼ 173.4 g kg1). When four leaves developed the seedlings were transplanted to similar cups filled with a mixture of dune soil and cattle manure, in the proportions of 0, 5, 10, 15 and 20% manure by volume, and disinfected with BromexÒ (98% methyl bromide and 2% chloropicrin). At the time of transplanting, 20 seedlings were inoculated near the roots with a mixture of native AMF, obtained from composite samples of dune soil, 20 were inoculated with Acaulospora longula Spain & N.C. Schenck, and 20 were not inoculated and taken as a control treatment. The inoculated treatments received suspensions with 150 glomerospores per plant. After five days, substrate and seedlings were transferred to black plastic bags (2 kg), containing the same proportions of soil and manure. The substrates were analyzed (Table 1) by the Instituto Agronômico de Pernambuco (IPA).
2.3. Field experiment Tocoyena selloana seedlings about 25 cm tall, grown in a substrate containing approximately 16.5% of cattle manure (usual practice), were supplied by Millennium. In four of these seedlings,
Table 1 Physicalechemical characteristics of the substrates with increasing proportions of cattle manure used in the greenhouse experiment with Tocoyena selloana. Manure (%)
0 5 10 15 20
P
Ca
mg dm3
cmolc dm3
K
5 54 90 >90 >90
0.40 1.00 1.45 1.75 2.25
0.04 0.24 0.38 0.44 0.47
Na
0.08 0.09 0.10 0.11 0.11
Mg
0.20 0.70 0.65 1.10 0.90
Al
0.60 0.40 0.30 0.40 0.35
CEC
5.60 6.30 5.10 6.90 6.80
pH H2O
4.20 4.36 4.58 4.80 5.08
O.M.
Global density
g kg1
g cm3
10.2 13.5 16.5 16.8 17.5
e 1.38 1.31 1.23 1.18
Particle density
Porosity %
e 2.60 2.53 2.50 2.53
e 46.9 48.2 50.8 53.4
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mycorrhizal colonization and number of glomerospores were estimated and the AMF species present in the rhizosphere were identified. A composite sample of the substrate was analyzed (Table 2), revealing weak acidity, low level of P and medium levels of organic matter, cation exchange capacity and Ca and Mg. The experiment was a completely randomized block design, with three treatments and seven replications. The treatments corresponded to different inoculations. At planting time, 35 seedlings received soil inoculum with approximately 150 spores of A. longula (UFPE 21), 35 received inoculum of Gigaspora albida Schenck & G.S. Sm. (UFPE 01) and 35 were not inoculated (control). The inoculum of G. albida was multiplied in clay-sand soil (pH ¼ 5.4 e P ¼ 5 mg dm3), in association with P. miliaceum, while the inoculum of A. longula was the same used in the greenhouse experiment. Planting followed the procedures used by Millennium. Pits, 70 cm deep and 20 cm in diameter, were opened with a manual soil auger, along a 36 m line. The pits were 2 m from each other, with 2 m of distance between the rows. In each pit, the top 20 cm of soil layer was set aside for closing the holes, which were filled with five gallons of a mixture of sugar cane bagasse and cattle manure (pH H2O ¼ 7.3; N ¼ 1.4 g kg1; C ¼ 147.2 g kg1; P ¼ 0.4 g kg1; and K ¼ 1.5 g kg1). On the same day, before planting, ten soil samples of the area were collected for chemical analysis (Table 2) and counting of AMF spores. Weeds were removed and a foliar fertilization (VITAN e organomineral, A Class) was applied eight months after planting. Height and stem diameter were measured every month, until the sixth one; and every two months, from then to the end of the experiment, 13 months after planting. The average temperature varied from 25.9 C to 30.2 C (JulyeDecember 2006) and from 29.7 C to 27.6 C (JanuaryeSeptember 2007). The total of rainfall was 1245.8 mm in 2006 and 1885.4 mm in 2007. At harvest, two plants per plot were excavated, their shoots and roots separated and the material oven dried and weighed. Fine root samples were taken for mycorrhizal colonization assessment, as previously described, and colonization percentage was determined according to the gridline intercept method [14]. Rhizosphere soil samples (100 cm3) were used for spore extraction and counting, and for AMF species identification. The relative abundance of each AMF species was calculated dividing the spore number of a species by the number of spores of all species [4]. Mycorrhizal effectiveness [16], increment rate of total biomass enhanced by inoculation [38], and P and N concentrations in the biomass [32] were determined as described.
2.4. Statistical analysis The number of glomerospores was transformed into log (x þ 1) pffiffiffi and the colonization percentage to arcsin x. Analysis of variance
239
and average comparisons (Duncan test p 0.05) were performed using the Statistica 5.0 program [35]. 3. Results 3.1. Greenhouse The seedlings inoculated with A. longula and with the AMF mixture accumulated significantly more biomass than the uninoculated ones (Table 3), with increments of 30 and 39%, respectively. Average height showed the same trend but the differences (13 and 23%) were not statistically significant (data not shown). Manure absence limited seedlings growth and 10 and 15% manure dosages were more suitable for preparing the seedlings (Fig. 1). P and K concentrations varied little in the three inoculation treatments (Table 3). Thus, the contents were mainly influenced by the plant biomasses and were higher in the inoculated treatments. The N concentration was slightly lower in plants associated to A. longula but the increase in biomass compensated for that, and the contents were similar. At the highest manure proportion in the substrate (20%), no colonization occurred in both inoculated treatments (Fig. 2). At lower proportions, A. longula and the AMF mixture seemed to have different behaviors. While colonization by A. longula increased with higher manure proportions that of the AMF mixture was higher in the treatment without manure or at the lowest manure proportion. Therefore, inoculation with A. longula seems to be a better option for manure treated seedlings. No colonization occurred in the uninoculated treatment. 3.2. Field Soil samples from the top soil layer, collected after dune reassembly but before planting, contained 46 spores per 100 g of soil, with three AMF species: Acaulospora morrowiae, Glomus etunicatum and G. glomerulatum. The substrate of the seedlings before inoculation had a lower number of glomerospores (6 spores 100 g1 soil) and shared the first two species with the top soil but contained an additional species: A. scrobiculata. Mycorrhizal colonization was not confirmed in the root samples of the seedlings because no vesicles or arbuscules were observed and just a few intraradical hyphae were present. After 13 months, 13 AMF species, belonging to five genera, were identified in the rhizosphere of the uninoculated control plants (Table 4). Eleven “new” species were then present but G. etunicatum was not found. Most glomerospores were from A. longula (55% of the total density) and A. morrowiae (28%). The rhizosphere of plants inoculated with A. longula had almost the same number of species (12) but only seven in common with the control. As expected, glomerospores of A. longula were the most abundant but with
Table 2 Chemical characteristics of the substrate (SS) of Tocoyena selloana seedlings, of the soil collected in the area before planting (BP) and of the rhizosphere of non-inoculated plants (NI), of plants inoculated with Acaulospora longula (AL) or with Gigaspora albida (GA) and of the soil between the lines of planting (LP). Treatments
pH H2O
Beginning of the experiment SS 5.66 BP 5.12 End of the experiment (13 months) NI 5.40 AL 5.60 GA 5.40 LP 5.10
P
K
mg dm3
cmolc dm3
Ca
Na
Mg
16 2
0.02 0.02
1.60 0.50
0.02 0.02
1.00 0.50
6 7 7 2
0.05 0.05 0.05 0.01
0.70 0.65 0.75 0.40
0.05 0.04 0.05 0.03
0.35 0.30 0.40 0.25
Al
OM
CEC
g kg1
cmolc dm3
0.10 0.25
15.5 7.4
5.8 4.3
0.25 0.20 0.20 0.25
11.5 14.5 11.0 9.5
3.9 4.0 4.9 3.5
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Table 3 Effects of inoculation with a mixture of native AMF or Acaulospora longula on biomass production, and on concentrations and contents of N, P and K in seedlings of Tocoyena selloana grown in greenhouse. Content (mg plant1)
Concentration (mg g1)
Treatments
Biomass (g plant1)
N
P
K
N
P
K
Mixture A. longula Uninoculated
9.42a 8.85a 6.76b
106.5a 92.1ab 89.7b
45.6a 41.7a 32.4b
173.5a 165.2a 126.8b
27.0a 23.6b 30.1a
8.1a 7.7a 7.9a
29.9a 30.1a 31.3a
Values followed by the same letter in the column do not differ by the Duncan’s test (p 0.05).
a relative density (59%) not much greater than in the control. The rhizosphere of the G. albida inoculated plants had a lower number of species (8) and only five in common with the control. It is remarkable that also in this treatment glomerospores of A. longula were the most abundant (47%), while those of G. albida ranked fourth in abundance with only 10% of the total density. Soil samples taken in-between the planting lines, away from the rhizosphere influence, had only three AMF species which were different from those originally present in the soil top layer; the species with more glomerospores, from a low total of only 14 spores per 100 g of soil, was G. albida (79%). Most of the seedlings (>95%) survived the first 13 months in the field, in all treatments, and grew 40e50 cm taller than when transplanted. In the seedlings inoculated with G. albida or A. longula colonization was about 40%, contrasting with only 2% in the uninoculated seedlings (Table 5). The seedlings inoculated with G. albida were significantly higher (79 cm) than those of the control (67 cm), while the A. longula inoculated ones (75 cm) did not differ from both. Apart from these differences, no other plant measurement differed significantly among the treatments. Although not statistically significant (p > 0.05), plants inoculated with A. longula tended to accumulate more biomass, P and N contents than others treatments (Fig. 3), with biomass increments (A. longula ¼ 41%, G. albida ¼ 14%) of similar magnitude of those obtained in the greenhouse. 4. Discussion Positive effects of mycorrhization were more evident in the greenhouse than in the field, in spite of similar increments in biomass and P and N contents. Certainly, in the field, one or more environmental factors affected the plants to a larger degree than the inoculation treatments. Wind exposure seems to be one of these factors. In spite of the absence of a significant response, the increments due to the inoculation of A. longula relative to the
Fig. 1. Effects of AMF inoculation and proportion of cattle manure added to the substrate on the total biomass dry weight (BDW) of Tocoyena selloana seedlings (n ¼ 60). Values followed by the same letter, small in the line and capital between the lines, do not differ by the Duncan’s test (p 0.05), NI ¼ uninoculated; MIX ¼ mixture of native AMF; AL ¼ Acaulospora longula.
control, in aboveground biomass (0.29) and P (0.16) and N contents (0.31), indicate that this inoculation may be recommended, until more tests are done. The low cost and relatively simplicity of the inoculation procedure may compensate even a small increase in plant growth. Formation of mycorrhizal structures by AMF used for inoculation in the greenhouse experiment varied with manure proportions. Colonization by the native AMF was higher at the lowest proportion (5%), while that of A. longula was higher at 15% manure. These differences did not imply in different responses on increment of host biomass by these AMF. In the field, the low colonization of the uninoculated plants suggests that the native AMF were inhibited by the 16.5% proportion of manure added to the substrate. They are probably better adapted to soils with low organic matter contents, which are their usual condition [2]. A change in these conditions may affect spore germination and colonization [20]. Wang et al. [36] observed that high manure proportions decreased colonization, spore density and species richness and also altered the species composition of the AMF in soils cropped to wheat and maize. In the greenhouse, fertilized substrates had high P and K content (Table 1) probable enough to maintain high plant growth rates, but may not had enough N. N limitation probably made the difference between the treatments, inoculated plants having higher ability to absorb N. The N limitation, also verified in the field, indicates that there should be a supplementation of this nutrient. It would be interesting to test the addition of a substrate richer in N for seedlings preparation, such as cotton or castor bean seed cakes, or the addition of green manure or mineral fertilizer in the field. The evaluation of different isolates under the same conditions can assist in a more efficient AMF selection and, in general, soils with low fertility and/or low infective potential favor the activity of introduced fungi, if they are compatible with the plants and the environmental conditions [34]. In this case, the AMF community present in the rhizosphere of inoculated plants was more effective in promoting T. selloana height development in the field, with also
Fig. 2. Mycorrhizal colonization in Tocoyena selloana seedlings inoculated with a mixture of native AMF (MIX) or Acaulospora longula and grown in substrates with increasing proportions of cattle manure in greenhouse. Values followed by the same letter, small in the line and capital between the lines, do not differ by the Duncan’s test (p 0.05).
R.G. de Souza et al. / European Journal of Soil Biology 46 (2010) 237e242 Table 4 Relative abundance (%) of AMF species in soil collected between the lines of planting (LP) or in the rhizosphere of Tocoyena selloana plants inoculated with Acaulospora longula (AL), Gigaspora albida (GA) or non-inoculated (NI), 13 months after seedling transplant to the field. Species of AMF
LP
AL
GA
NI
Acaulospora foveata Trappe & Janos A. longula Spain & N.C. Schenck A. morrowiae Spain & N.C. Schenck A. scrobiculata Trappe A. spinosa C. Walker & Trappe A. tuberculata Janos & Trappe Ambispora appendicula (Spain, Sieverd. & N.C. Schenck) C. Walker Gigaspora aff. albida N.C. Schenck & G.S. Sm. Glomus aff. fasciculatum (Thaxt.) Gerd. & Trappe emend. C. Walker & Koske G. sinuosum (Gerd. & Bakshi) R.T. Almeida & N.C. Schenck G. glomerulatum Sieverd. Scutellospora aurigloba (I.R. Hall) C. Walker & F.E. Sanders S. biornata Spain, Sieverd. & S. Toro S. calospora (T.H. Nicolson & Gerd.) C. Walker & F.E. Sanders S. aff. fulgida Koske & C. Walker S. gregaria (N.C. Schenck & T.H. Nicolson) C. Walker & F.E. Sanders S. pellucida (T.H. Nicolson & N.C. Schenck) C. Walker & F.E. Sanders S. rubra Stürmer & J.B. Morton S. aff. verrucosa (Koske & C. Walker) C. Walker & F.E. Sanders
e e e e e e 7.1
1.5 58.5 3.1 1.5 e e e
e 47.2 e 17.3 e 9.1 11.5
e 55.0 28.0 2.3 2.3 2.3 1.1
78.7 e
7.7 e
10.3 e
1.1 2.3
e
1.5
e
e
e e
12.4 3.1
e 1.1
1.1 e
e e
e 1.5
1.1 e
e 1.1
e e
4.6 e
2.3 e
e 1.1
e
3.1
e
e
1.5 e
e e
14.3 e
Total number of species
3 3
Number of glomerospores (50 cm soil)
14b
12 69.1a
8 91.4a
1.1 1.1 13 52.5a
Values followed by the same letter do not differ by the Duncan’s test (p 0.05).
a tendency of larger biomass and nutrient contents. Sharma et al. [31] observed that mycorrhizal plants were superior in height, weight and nutrient concentrations than uninoculated ones, but the extent of the responses varied with the inoculum used. It should be also considered that some mycorrhizal benefits can only be effective with periods longer than the 13 months of the experiment. Inoculation at planting time and field conditions associated with the host plant provided an increase of A. longula sporulation, whereas glomerospores of this species were not observed in the seedlings substrate or in soil samples from the area, before planting. Oehl et al. [25] found A. longula spores in organic systems. Members of Acaulosporaceae, especially A. longula [3] are favored by low pH, and that of the study site was between 5.1 and 5.6. G. albida was not the most abundant where it was inoculated; may be because its proliferation was hampered by the manure addition. Cordoba et al. [10] related an experiment in which Gigaspora spp. decreased in fertilized plots in comparison to non-fertilized ones. They also stated that low numbers of Gigasporaceae spores may
Table 5 Effects of inoculation with Acaulospora longula or Gigaspora albida on shoot (SDW) and root dry weight (RDW) and on the mycorrhizal colonization of Tocoyena selloana plants, after 13 months in the field. Treatments
Uninoculated A. longula G. albida
Height
Diameter
SDW
RDW
Colonization
cm
cm
g plant1
g plant1
%
67.4b 74.7ab 78.7a
1.6a 1.8a 1.8a
94.0a 140.0a 113.1a
69.3a 90.3a 74.5a
2.5b 39.8a 40.3a
Values followed by the same letter in the column do not differ by the Duncan’s test (p 0.05).
0.40
241
Biomas N
0.30
P
0.20 0.10 0.00
A. longula
G. albida
Fig. 3. Effectiveness of the symbiosis in biomass production and N and P contents of Tocoyena selloana plants inoculated with Acaulospora longula or Gigaspora albida, after 13 months in field.
result from its strategy of producing few but large multiplegerminating spores, each one capable of encountering a multitude of roots. The high abundance of A. longula in the rhizosphere of all treatments confirms the greenhouse results and shows that it thrives well in soils with about 15% of manure, although more research is needed to confirm these results. The highest relative abundance of A. longula and A. morrowiae spores suggests that these species are better adapted to the first recovery stage of the studied area. Higher sporulation may be associated with high competitive capacity [9], better adaptation to the environment [6] and a certain degree of specificity between the symbionts. The substrates for seedlings growth favored the introduction of AMF species in the field, as also observed by Caproni et al. [5], and may have contributed to the AMF spread and establishment. The increased number of taxa in the treatments, thirteen months after planting, in relation to the initial number, also may have occurred because of wind dispersion [12] and a reduction in dominance of some species [17]. Alteration in the rhizosphere AMF community may have resulted from dispersal mechanisms or due to the ability of the introduced species to compete with those that were in place or were established after seedling planting in the field. The proportions 15% of manure used in the substrates promoted seedling growth increases in the greenhouse but inhibited colonization by native AMF, both inoculated in the greenhouse and under natural conditions in the field. A. longula thrived well at this manure proportion, dominating the spore production and achieving high colonization percentages. Its contribution to the seedlings growth in the field was small (29% increase in biomass) but considering the low cost of inoculation it may be a feasible practice and an alternative to maximize environment restoration. Acknowledgements To the Millennium Inorganic Chemicals Mining, a Cristal Company, to the Instituto Agronômico de Pernambuco (IPA) for performing the soil analyses, to CAPES for providing a PhD scholarship to B.T. Goto and to CNPq for a PhD scholarship to R.G. Souza and research grants to E.V.S.B. Sampaio and L.C. Maia. References [1] R. Azcón, J.M. Barea, Mycorrhizal dependency of a representative plant species in mediterranean shrublands (Lavandula spica L.) as a key factor to its use for revegetation strategies in desertification-threatened areas. Appl. Soil Ecol. 7 (1997) 83e92.
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