Occurrence of Arbuscular Mycorrhizal Fungi in the Rhizosphere of Native Populations of Cambui (Myrciaria Floribunda O. Berg) in the State of Alagoas, Brazil

Occurrence of Arbuscular Mycorrhizal Fungi in the Rhizosphere of Native Populations of Cambui (Myrciaria Floribunda O. Berg) in the State of Alagoas, Brazil

Keywords: Myrtaceae, Tropical fruit tree, restinga vegetation, coastal areas, Quartzose soil

Abstract
Cambui is a tropical fruit tree native from Brazil that grows in the sandy coastal soils (Quartzose soil) of Alagoas State. To assess the composition and the diversity of arbuscular mycorrhizae fungi (AMF) in native plants, soil samples were collected from the rhizosphere between 0 and 20 cm depth in four points around the shading line of plants. The plants were randomly chosen and marked with a GPS. The spores of AMs were extracted and identified taxonomically. The relative density, the frequency of each species and the Shannon-Wiener and Simpson indices were evaluated. The census data indicated a total of 287 spores/100 g soil (mean of four sampling). The species Acaulospora longula, Glomus clarum e G. etunicatum were identified. It was observed a greater number of spores of Glomus species. The number of AM fungi spores increased with the number of sampled plants, while indices of diversity, evenness and richness of Glomales species decreased in the rhizosphere of cambui. The reduction in the richness of AM fungal populations or their functional diversity has drastic consequences for the equilibrium of natural plant community structure. These results support the theory of reduction of AM fungal propagation under severe disturbance which affects the natural plant community structure, leading to the ecosystem instability. This is the first report of AM symbiosis occurrence from the root of Myrciaria floribunda in native restinga vegetation from Brazil.

INTRODUCTION
Cambui (Myrciaria floribunda O. Berg) is a tropical native fruit tree from Brazil that grows on low-fertility soils in restinga vegetation of Alagoas State. Due to increasing deforestation and urbanization of coastal areas this species has had its natural germplasm bank severely reduced (Cardoso Filho et al., 2008).

In Northeastern Brazil, an important pulp market has developed based on native tropical fruits, including cambui (Manica, 2000). Propagation of elite cambui trees is desirable to increase the productivity in orchards, but the use of seeds or vegetative methods is very laborious (Lorenzi, 2009). In addition, very few plants survive in the nursery and in the field, after transplanting, when the mortality may reach 60% (Manica, 2000).
The mycorrhizal association is a mutualistic symbioses which is formed between the roots of terrestrial plants families and soil fungi and amongst the mycorrhizal associations, the arbuscular mycorrhizal (AM) association is the most common one (Smith and Read, 1997).
These fossils indicate that Glomeromycota-like fungi may have played a critical role in facilitating the colonisation of land by plants (Redecker et al., 2000). The Glomeromycota is divided into four orders, eight families and ten genera (Schüßler et al., 2001). The genera which include most of the described species are Acaulospora, Gigaspora, Glomus and Scutellospora (Schüßler et al., 2001). The AM fungi are named by their formation of highly branched intracellular fungal structures or “arbuscules” which are the site of phosphate exchange between fungus and plant (Smith and Smith, 1997). Vesicles, which contain lipids and are carbon storage structures, are formed commonly in most genera of Glomeromycota, although this will depend on environmental conditions (Smith and Read, 1997). Gianinazzi- Pearson (1996) pointed out that these obligatory biotrophs, the AM fungi, have a very broad host range, which makes them definitely different from the biotrophic fungal plant pathogens as well as other root symbionts.

In the establishment of this symbiosis, plants are benefited by greater P absorption, while the fungi extract carbon from the plant for their development and sporulation. AMF and their interactions contribute to cycling, transport of nutrients and plant productivity (Eldor, 2007).
Inoculation with arbuscular mycorrhiza (AM) seems to be an interesting tool to select elite Myrciaria floribunda genotypes and to help in the domestication and improvement of recalcitrant species, given that has shown to be highly responsive do AM, mainly in low-fertility soils.
The present work aims at understanding the diversity of the arbuscular mycorrhizal fungi associated with Cambui in native restinga vegetation of Alagoas State in Brazil.

MATERIALS AND METHODS
Study site and Samplings
The diversity, abundance of community of AMF was studied in sites located in the city of Piaçabuçu (10o 24’ 20,6” S and 36o 26’ 4” W, 10 m above sea level) located in the State of Alagoas, Northeastern Brazil, with vegetation characterized by Forest “Perenifolia” of restinga (Marra, 1989). The annual rainfall is around of 1,876 mm. According to Köppen’s classification, the region’s climate is a warm and humid tropical, with a dry season in spring and summer, and a rainy season in the fall and winter, with rainfalls from April to July, with a mean temperature is about 32oC (Marra, 1989). The soils in the region are Quartzose sands (Entisols), very low-fertility soil, according to FAO (1994).

Samples of soil were taken from 4 points at 0-20 cm depth, at the crown projection area, and 1 m away from trunk (four points per tree). The plants were randomly chosen and marked with a GPS and four transects oriented toward the cardinal points from a center point. The soil samples were placed in plastic bags for later determination of number of spores and AMF species identification. Each sample point was analyzed separately (four points per tree × five trees = 20 sampling points for ordination analysis). The total number of spores in 100 g of dry soil was determined by wet sieving (Gerdemann and Nicolson, 1963), based on a 100 g-aliquot for each point studied, followed by centrifugation in sucrose (Jenkins, 1964). Cambuí root samples were also stored at temperature of 4oC for the estimation of mycorrhizal infection rates.

Root stained and colonization quantification
Individual roots were picked randomly from roots samples collected, and cut into pieces 1 cm length. For each sample examined, 200 root fragments were analyzed for the presence of AM fungi material after roots were stained. Prior to stained, roots were clarified in 5% (v/v) KOH for 1 h, followed by acidification with 1% HCl (v/v) overnight. Staining was with 0.05% Tripan Blue (w/v( for 20 min in lactoglycerol acid (Kormanik and McGraw, 1982). Root colonization was quantified using grid line intersect method (Giovannetti and Mosse, 1980), using a stereomicroscope (40 X magnification).

AMF identification
The spores were counted under a stereoscopic microscope with 40 X magnification, on plates containing concentric grooves. After the total number was obtained, the spores were separated into groups, according to their morphology and color. The AMF spore groups were transferred and mounted onto permanent slides, with polyvinyl alcohol and glycerol resin (PVLG) and PVLG + Melzer’s reagent (1:1, v/v) (Kormanik and McGraw, 1982). Identification was made at the species level under an stereomicroscope (100 to 400X magnification), with the aid of the Schenck and Pérez (1990) manual and descriptions provided by the International Collection of Vesicular and Arbuscular Mycorrhizal Fungi (INVAM) (invam.caf.wvu.edu) and the original species descriptions. Differences between spore recovery data were inspected using Tukeys’s honestly significant difference test (HSD) at 5% level.

Diversity of AM fungi community
The diversity of AM fungi in 5 locations was assessed based on diversity indices (Magurran, 1988): Simpson’s index: Simpson’s index: D’ = 1/(pi)2, and Shannon index: H’ = –(pi ln pi) where, pi is the proportion of individuals that species i contributes to the total. The evenness was expressed by: J’ = H’/ H’max, where H’max is the maximum value of diversity for the number of species present. Paired t-test was performed to assess the differences in the diversity between samples. To compare the species richness among the samples, rarefaction indices were calculated (Ludwing and Reynolds, 1988). The number of spores (NS) in 100 g dry soil was evaluated for each morphologically distinct AMF group.

Statistical analysis
The data for root colonization and spores total number were submitted to analyses of two-way ANOVA, using the R statistical software, according to Rizzo (2008) and means were compared by the LSD test (p < 0.05). The diversity indices of AM fungi data were calculated with the package VEGAN (Community Ecological Package), using the R statistical software, according to Bolker (2008). The relative spore frequency for each AMF species (RSF), [RSF = Number of soil samples with spores of a particular species (in n = 25) / total number of samples analyzed (n = 25) x 100] and Relative abundance, [RA= Number of spores of a particular species (in n = 25) / total number of spores (in n = 25) x 100] were also calculated for the studied ecosystem.

RESULTS DISCUSSION
AMF identification and diversity of AM fungi community
AMF spores isolated from the rhizosphere soil samples of plants of Cambuí understudy was belonging to three genera (Acaulospora, Glomus and Gigaspora) and these species Acaulospora longula, Glomus etunicatum and G. clarum were identified (Table 1). The indices of diversity, evenness and richness of Glomeraceae species decreased in the rhizosphere of cambui, while the sporulation of Acaulosporaceae increased (Table 2). The reduction in the richness of AM fungal populations or their functional diversity has drastic consequences for the equilibrium of natural plant community structure. These results support the theory of reduction of AM fungal propagation under severe disturbance which affects the natural plant community structure, leading to the ecosystem instability (Kernaghan, 2005).

According to Cordoba et al. (2001) there was a striking difference in the relative distribution along the soil gradient, and a uniform distribution (high evenness, Table 3) may explain the dominance of Acaulosporaceae. Acaulosporaceae species may become evenly distributed and prevalent by allocating C to a smaller number of large multiple-germinating spores (Table 2), each spore capable of encountering a multitude of roots compared with the colonization potential of the other AM fungi present. The Shannon’s index H’ is an indicator of AM fungal diversity, reflecting the complexity level of the communities in ecosystem studied. In addition, this estimator of species diversity is sensitive to changes in species abundance pattern and presents a low bias for small sample sizes (Moulillot and Leprêtre, 1999). This is the first report of AM symbiosis occurrence from the root of Myrciaria floribunda in native restinga vegetation from Brazil.

Root colonization and spore density
With regard to root colonization, all Cambuí roots segments analyzed were colonized by AM fungi, with the occurrence of hyphae, vesicles, arbuscules, and auxiliary cells, both internally and/or in association with the roots at all sampling sites (Figure 1). The colonization of AM fungi was greatest (50%) at MF05PA, while it was least (12%) at MF03PA (Figure 1). It was observed a greater number of spores of Glomus species, according to the relative spore frequency (RSF), accounting for 77% of all spores recovered at all sampling sites (Table 2). In contrast, Acaulosporaceae spores, accounted for only 22% of recovered spores (Table 2).

According to (Black and Tinker, 1979) the rapid growth rate of roots may result in a low colonization rate, which explains low spore reproduction as related for Ammophila arenaria in British sand dunes. On the other hand, the level of AM fungal association depends on root morphology, metabolism and rate of plant growth (Guadarrama et al., 1999). Certain stress conditions as well as low-fertility soils, as observed in sampling sites located in the city of Piaçabuçu, might elicit a stimulus for greater spore production (Zangaro et al., 2007). Although root colonization and sporulation seem to be correlated, at least in some cases, with plant phenology and physiology (Escudero and Mendonza, 2005), it has also been shown that the community of AM fungi may determine host plant community’s association and production (Van der Heijden et al., 1998). On the other hand, Bever (2002) demonstrated that each endophyte may be multiplied quite differently on different host plants, and that there may be positive as well as negative feedback occurring between specific host plants and their endophytes.

CONCLUSIONS
The species Acaulospora longula, Glomus clarum e G. etunicatum were identified in the rhizosphere of native populations of Cambui (Myrciaria floribunda).
The number of AM fungi spores increased with the number of sampled plants, while indices of diversity, evenness and richness of Glomales species decreased in the rhizosphere of cambui.

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Tables
Table 1. The relative spore frequency (RSF) for each AMF species and relative abundance (RA) of AM fungal spores in the rhizosphere native populations of Cambui (Myrciaria floribunda O. Berg) in the State of Alagoas, Brazil.
TAXON RSF (%)* RA (%)**
Acaulospora longula Spain & Schenck 16.2 5.37
Acaulospora sp.a 6 0.54
Glomus etunicatum Becker & Gerdemann 42.2 32.35
Glomus clarum Nicolson & Schenck 25 17.9
Gloums sp.a 10 1.35
Gigaspora sp.a 14 4.46
*Number of soil samples with spores of a particular species (in n = 25) / total number of samples analyzed (n = 25) x 100. **Number of spores of a particular species (in n = 25) / total number of spores (in n = 25) x 100.
a)Non-identified species

Table 2. Species richness, diversity and evenness of AM fungi associated with native populations of Cambui (Myrciaria floribunda O. Berg) in the State of Alagoas , Brazil.

Diversity index

TAXON Species richness Simpson (D’) Shannon (H’) Shannon evenness (J’)
Acaulospora longula Spain & Schenck 19 0.891 3.307 0.893
Glomus etunicatum Becker & Gerdemann 7 0.811 1.044 0.606
Glomus clarum Nicolson & Schenck 2 0.460 1.813 0.463
Acaulospora sp.a 12 0.602 2.178 0.869
Gloums sp.a 5 0.773 1.105 0.540
Gigaspora sp.a 3 0.448 1.095 0.691
a)Non-identified species

Table 3. Species richness, diversity and evenness of AM fungi associated with native populations of Cambui (Myrciaria floribunda O. Berg) in the State of Alagoas , Brazil

Diversity index
GPS coordinates of sampling sites Species richness Simpson
(D’) Shannon
(H’) Shannon evenness (J’)
22º 03.0’S/23º37.6’Wa 6 0.807 2.347 0.793
22o02.8’S/23o36.5’Wb 7 0.691 2.544 0.606
21o32.9’S/24o25.2’Wc 2 0.476 1.863 0.663
21o27.7’S/24o19.0’Wd 3 0.620 1.378 0.669
21o28.8’S/24o20.7’We 5 0.473 1.105 0.640
a,b,c. and d) Code of location ( aMF01PA, bMF02PA, cMF03PA, dMF04PA and eMF05PA)

Further details are as follows:
Abstract Category: T03-Crop physiology
Provisional day and time of presentation: Aug 25 2010 / 3:30PM
Abstract Number: 5476
Abstract title: Occurrence of Arbuscular Mycorrhizae Fungi in the Rhizosphere of Native Populations of Cambuí (Myrciaria Floribunda O. Berg) in the State of Alagoas, Brazil
Author: Santos Dias, E.; Cardoso Filho Alves J.; Lemos Eduardo Pinto E.; Campos Silva R.; Araújo Rocha R.; Rezende Paula L.; Lima Salvador T.