Symbiosis (2010) 52:95101 DOI 10.

1007/s13199-010-0097-8

Differential effects of two species of arbuscular mycorrhiza on the growth and water relations of Spartium junceum and Anthyllis cytisoides
Marta Busquets & Cinta Calvet & Amelia Camprub & Victoria Estan

Received: 17 June 2010 / Accepted: 19 October 2010 / Published online: 15 November 2010 # Springer Science+Business Media B.V. 2010

Abstract Anthyllis cytisoides and Spartium junceum are two leguminous shrubs native of semiarid mediterranean areas, often used in revegetation strategies. Mycorrhization of both shrubs with Glomus intraradices BEG 72 enhanced both plants growth and water relations under drought stress. Root colonization achieved by Glomus mosseae was lower than the level achieved by G. intraradices in both plants studied, and the effects of the inoculation with G. mosseae BEG 116 were less positive than those observed for G. intraradices. Before the onset of the drought stress period the specific leaf weight (SLW) of S. junceum plants inoculated with G. mosseae was lower than the SLW of control and G. intraradices plants. At the end of the stress period, after 15 days of withholding water, the relative water content of S. junceum twigs was lower for G. mosseae inoculated plants and higher for G. intraradices inoculated plants, compared to control, non-inoculated plants. At the end of the recovery period, 15 days after the reestablishment of watering, there were no differences between inoculation treatments on the parameters related to the plants water status. Anthyllis cytisoides plants inoculated with G. intraradices had lower leaf osmotic potential, more leaves, and higher chlorophyll content (measured as SPAD values). Anthyllis cytisoides plants responded to drought defoliating, but defoliation was lower for the plants inoculated with G. intraradices. At the end of the drought, the leaf osmotic potential was lowest for G. intraradices plants as was the relative water
Submitted to the special issue The Potential of exploiting Mycorrhizal associations in semi arid regions. M. Busquets : C. Calvet : A. Camprub : V. Estan (*) IRTA, Carretera de Cabrils Km 2 08348 Cabrils, Barcelona, Spain e-mail: victoria.estaun@irta.es

content (RWC) whilst Glomus mosseae inoculated plants had the highest RWC, SLW and osmotic potential values. At the end of the recovery period, all plants recuperated the osmotic potential values measured at the pre-stress period. In our experiments, G. intraradices BEG 72 was found to be superior to G. mosseae BEG 116, this difference could be attributed to the origin of the fungus, native from a Mediterranean area, compared to G. mosseae (BEG116) isolated from the UK. Keywords Relative water content (RWC) . Specific leaf weight (SLW) . SPAD chlorophyll meter measurements . Osmotic potential . Drought . Glomus mosseae . Glomus intraradices

1 Introduction In semiarid environments the establishment of a plant cover is the most important step in the restoration of degraded areas, to avoid further degradation and desertification. In this aspect leguminous shrubs are important components of re-vegetation processes in the Mediterranean because they are well adapted to the prevailing edaphoclimatic conditions of the area. The Mediterranean Basins climate combines cool or cold and wet winters, and long, hot and dry summers. Summer drought is of variable duration, but frequent periods of drought can occur at any time of the year (Vallejo et al. 2006). The anthropogenic pressure over the land in the Mediterranean basin has caused the loss of the original vegetation (Puigdefbregas and Mendizabal 1998) with a higher risk of soil erosion and desertification. As a result of these conditions, soils are shallow and low in nutrients (Yaalon 1997), other characteristics include low organic matter content (Aranda

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and Oyonarte 2005) and low levels of microbial activity (Bastida et al. 2007). Additionally in this region the irregular rain with high summer temperatures generate episodes of drought that hold back the spontaneous revegetation of many of these areas. It is well established that the reclamation of degraded ecosystems should use recognized succession trajectories to improve the degraded ecosystem structure and function (Walker and del Moral 2008). The use of known reference systems, such as the original vegetation exploiting the potential of native species will increase the ecosystem resilience, especially in relation to drought events and a gradual drift to hotter and drier climates (Pausas 2004). Shrubs are pioneer plants which help the survival of tree seedlings and therefore can speed up the spontaneous (or human-mediated) succession (Gmez-Aparicio et al. 2004). Evergreen sclerophyllous and drought deciduous shrubs are both key components of the Mediterranean vegetation of arid and semi-arid environments and both life strategies are considered adaptations to drought (Freitas and Billib 1997). In the Mediterranean region wild legume shrubs comprise a group of woody Faboideae (Genista, Spartium, Cytisus, Anthyllis and others), able to colonise poor and arid soils. Woody legumes are important species of the sparse shrub land that is often associated with the primary stages of succession of anthropogenically degraded lands (Lpez-Pintor et al. 2006). The establishment of the tripartite symbiosis with rhizobia and mycorrhizal fungi in the roots of these shrubs facilitates the plants nutrition and represents a net influx of nitrogen into the ecosystem (Cardinale et al. 2010). Two woody legumes were chosen for this study: an evergreen sclerophyllous shrub, Spartium junceum L., and a drought deciduous legume shrub Anthyllis cytisoides L., both common in the Spanish Mediterranean region and often used in rehabilitated arid Mediterranean areas. Spanish broom (S. junceum) is a perennial, evergreen legume shrub that can reach six to ten feet tall. It is the only species of the genera and is the most drought resistant of the brooms family. It is found in areas with full sun and limited water and it can grow in poor, rocky soils. A. cytisoides is found on limestone soils. In nature both A. cytisoides (LpezSnchez et al. 1992) and S. junceum (Maremmani et al. 2003) present the arbuscular mycorrhizal (AM) symbiosis. As microsymbiont propagules might be scarce in disturbed environments (Estan et al. 2008), inoculation of wild legume plantlets with selected fungi prior to their establishment in a degraded area should improve both plant growth and soil quality (Rillig and Mummey 2006). Mycorrhizal effects on plant water relations are not as consistent as those on P acquisition and host growth, however there is no doubt that AM fungi can modify host water relations (Aug 2001). Mycorrhiza have been shown to influence, among other water related parameters, the leaf

osmotic pressure and also the relative water content (RWC) (Subramanian et al. 1995; Subramanian and Charest 1999); these influences might be circumstance and also symbiont specific. The objective of this work was to evaluate the response of two type-specific woody legumes, an evergreen sclerophyllous shrub and a drought deciduous shrub to the inoculation with two different arbuscular mycorrhizal fungi (AMF) isolates. Both shrubs are commonly used in restoration strategies and the aim of the study was to assess how two different mycorrhizal fungi isolates might influence plant development and the plants response to a short drought period.

2 Materials and methods Spartium junceum seeds and Anthyllis cytisoides seeds were collected from the wild in the region of El Maresme (Northern Catalunya, Spain). After collection, vital seeds, those that were not attacked by insects, or looked damaged in any other way, were kept in the dark at 4C. Spartium junceum seeds were immersed in concentrated sulphuric acid for 60 min, rinsed with running water and immersed in water for 1 h before planting. Anthyllis cytisoides seeds were immersed in concentrated sulphuric acid for 24 h, rinsed with tap water and then left immersed in water for another hour before planting. Seeds of both plants were sown in sterilised sandy soil (1 h at 120C, repeated twice with a 24 h interval). Forty five days after the emergence, when plants had two true leaves they were transplanted to a pasteurized potting mix (soil: quartz sand : sphagnum peat) in 5 L pots. At transplant one third of the plants were inoculated with Glomus intraradices Schenck & Smith (BEG 72); Glomus mosseae Nicolson & Gerdemann; Gerdemann & Trappe (BEG 116) or non inoculated. The roots of plants left over on the seedling tray were stained to confirm the nonestablishment of the arbuscular mycorrhizal symbiosis before transplant and inoculation. Inocula for arbuscular mycorrhizal fungi were produced in leek (Allium porrum L.) pot plants grown in sterilized sand. The number of spores was assessed for each fungal inoculum before being used in the experiments. Spore numbers for each batch were counted in a 50 g of the mixed soil inoculum sample after wet sieving and decanting (Daniels and Skipper 1982) and shredding the roots in a blender to recover intraradical spores. The resulting concentration of mycorrhizal propagules was approximately 1,000 spores/10 g of soil inoculum and 80 sporocarps/10 g of soil inoculum for G. intraradices BEG 72 and G. mosseae BEG 116 respectively. The inoculation was done by placing 20 g of the soil inoculum directly under the plantlet roots.

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All plants were also inoculated with Rhizobium strains that were isolated from corresponding wild plants. Bacteria were grown on a yeast manitol specific media. Plants were inoculated (1 ml plant-1) at a concentration of about 109 CFUml-1 after germination. At transplant all plants had nodules, and a second inoculum dose was added one month after transplant. No attempt was made to identify the Rhizobium strains. Plants were grown for 4 months under greenhouse conditions. Plant growth was monitored by measuring the following parameters: plant height, stem diameter, and for A. cytisoides the two maximum values of crown and basal diameters (cm), which are perpendicular to each other, were also measured to calculate the canopy volume. There were 40 plants per treatment. The 1st week of September, after 4 months growth, 10 plants of each species per treatment were harvested and biomass allocation and plant water relation parameters were determined (pre-stress measurements). Irrigation was withheld for 15 days (stress period) in all plants. At the end of this period 10 plants per treatment were harvested and plant water relations were determined (end of drought measurements). Then, all the plants were reirrigated to run-off, and subsequently, as needed for 15 days when the water relation parameters were measured again (end of recovery measurements). 2.1 Measurements Plants were separated into leaf blades, leaf sheaths+stem, and roots and plant parts were dried at 65C to constant weight. The area of leaf blades of A. cytisoides was determined using a digital leaf area meter (Delta T Ltd, Cambridge, UK). The area of the leaves of S. junceum plants, due to their morphology, was only measured to calculate the specific leave weight (SLW) in the drought experiment, using 10 leaves per treatment. The relative chlorophyll (a+b) content of the leaves was determined using a portable chlorophyllmeter (SPAD-502, Minolta, Japan). SPAD chlorophyll meter readings have been shown to have a direct linear relationship to extracted leaf chlorophyll (Yadava 1986) and are also related leaf nitrogen concentration (Bullock and Anderson 1998). The following allometric parameters were calculated: root:shoot ratio, leaf weight ratio (LWR; leaf dry weight per plant dry weight), shoot : plant ratio (SWR), root : plant ratio (RWR). Leaf water related parameters were calculated before withholding the water, at the end of the drought period and at the end of the recovery period. Leaf osmotic potential was measured for A. cytisoides on 10 leaves per treatment, the leaves were frozen in liquid nitrogen and osmotic potential (o) was measured after thawing the samples and extracting the sap, using a Wescor 5500 vapour pressure osmometer, the specific leaf

weight (SLW; leaf dry weight per leaf area) was calculated for both plant species. Pressure-volume curves were calculated for fully expanded leaves or in the case of S. junceum, due to the morphology of the plant, small twigs, taken from the middle of the plant. Leaves (in the case of S. junceum twigs) were excised pre-dawn, placed in plastic bags, and allowed to reach full turgor by dipping in distilled water for 24 h in darkness at 4C. The re-saturated leaves (twigs) were weighed using an analytical balance ( 0.1 mg precision), placed into the pressure chamber (lined with damp filter paper) and slowly pressurized (0.025 MPas-1) until the balance pressure was reached (when the leaf sap appears through the cut petiole (stem) protruding from the chamber). Once depressurized, the leaves (twigs) were repeatedly weighed and their balance pressures determined over the full range of the pressure gauge. Leaves were finally dried at 65C to determine their dry weights. The curves were drawn using a type II transformation (Tyree and Richter 1982) to calculate the RWC (relative water content). To evaluate arbuscular mycorrhizal root colonisation a root subsample from 5 plants per treatment was taken and after clearing and staining (Koske and Gemma 1989) analyzed using the grid intersect method (Giovanetti and Mosse 1980). The number of nodules was counted in five root systems for each plant species and treatment. The statistics design was an ANOVA with three treatment levels for the inoculation factor.

3 Results Glomus intraradices and G. mosseae stimulated plant growth of both A. cytisoides and S. junceum (Tables 1 and 2). Glomus intraradices produced a more positive response than G. mosseae in plant height, stem diameter and plant dry weight of S. junceum, (Table 1) although G. mosseae plants were better than control non inoculated plants for these parameters. When considering the allometric parameters measured, only the relation between the leaves and the plant dry weight was significantly different between control and inoculated plants. Control plants had a higher leaf weight ratio (LWR) than inoculated plants with either fungi. Anthyllis cytisoides (Table 2) plants inoculated with G. intraradices were taller, had a thicker stem diameter and a higher dry weight, than control and G. mosseae inoculated plants. Glomus mosseae plants were only better than control plants when considering plant height. The allometric parameters measured showed that non-inoculated A. cytisoides plants had a higher root/shoot weight ratio than inoculated plants. In both plant species, G. mosseae achieved lower levels of root colonisation, when compared to G. intraradices, although the amount of inocula used per plant was more than enough to establish the symbiosis. The

98 Table 1 Spartium junceum plant development and biomass partitioning after 4 months growth in greenhouse conditions Non-inoculated Plant height (cm) Stem diameter (mm) Plant dry weight (g) LWR (leaf weight ratio) SWR (shoot weight ratio) RWR (root weight ratio) Root/shoot weight ratio Rhizobium nodules (per gram plant roots) AM root colonisation (%) 23.43 c 1.87 c 0.443 b 0.165 a 0.624 0.264 0.409 1.5 b 0 G. mosseae 45.10 b 2.54 b 1.195 b 0.062 b 0.711 0.228 0.305 4.02 a 33 b

M. Busquets et al. G. intraradices 76.98 a 3.05 a 3.009 a 0.057 b 0.725 0.218 0.409 3.25 a 76 a

Data are means of 10 replicates (non destructive measures: plant height and stem diameter 40 replicates) Letters in the same row indicate differences at p0.05

number of Rhizobium nodules was significantly higher for S. junceum mycorrhizal plants while in A. cytisoides the differences observed were not significant. At the end of the pre-stress period the specific leaf weight (SLW) of S. junceum plants inoculated with G. mosseae (Table 3) was lower than the SLW of control and G. intraradices plants. At the end of the stress period, after 15 days of withholding water, the relative water content of S. junceum twigs was lowest for G. mosseae inoculated plants and highest for G. intraradices inoculated plants. At the end of the recovery period, 15 days after the reestablishment of watering, there were no differences between inoculation treatments on the parameters evaluating the plants water status (RWC and SLW). In the case of A. cytisoides the parameters measured (Table 4) showed a positive influence of G. intraradices on some of the plants water relations at the pre-stress period. Glomus intraradices inoculated plants had more leaves, and lower leaf osmotic potential. The SPAD values were higher for the mycorrhizal plants and G. intraradices plants had 3 times more leaves than noninoculated control and G. mosseae plants. At the end of the stress period, after 15 days of withholding water, the number of leaves was reduced in all treatments, however G. intraradices plants still had significantly more leaves than the other A. cytisoides plants, the leaf osmotic potential was
Table 2 Anthyllis cytisoides plant development and biomass partitioning after 4 months growth in greenhouse conditions

lowest for G. intraradices plants as was the relative water content (RWC). Glomus mosseae inoculated plants had the highest RWC, SLW and osmotic potential values. The SPAD values were significantly lower for both AMF inoculation treatments. At the end of the recovery period, despite a severe defoliation in all treatments, G. intraradices plants still had more leaves than the other treatments, and the SPAD values were recovered. All plants recuperated the osmotic potential values measured at the pre-stress period, although G. intraradices inoculated plants had a higher osmotic potential than G. mosseae plants. The values of SLW and RWC remained significantly lower for G. intraradices inoculated plants.

4 Discussion Perennial leguminous shrubs are in some Mediterranean marginal lands the most important vegetation, interspersed with bare areas or grass (Puigdefbregas and Mendizabal 1998). The expansion of the shrub land in agriculturally abandoned areas represents an intermediate state of old field successions (Haase et al. 1997). Due to climatic and also land use changes water is becoming a scarcer resource in many areas of the Mediterranean, particularly of Spain

Non-inoculated Plant height (cm) Stem diameter (mm) Canopy volumen (cm3) Plant dry weight (g) Leaf area (cm2) LWR (leaf weight ratio) SWR (shoot weight ratio) RWR (root weight ratio) Root/shoot weight ratio Rhizobium nodules (per gram plant roots) AM root colonisation (%) 18.46 b 1.73 b 17.18 c 8.803 b 18 ab 0.323 0.169 0.508 1.351 a 6.857 0

G. mosseae 23.12 a 1.85 ab 32.51 b 8.976 b 16 b 0.381 0.177 0.442 0.868 b 3.571 14 b

G. intraradices 24.74 a 1.98 a 55.52 a 10.488 a 22 a 0.389 0.198 0.413 0.741 b 5.429 43 a

Data are means of 10 replicates (non destructive measures: plant height and stem diameter 40 replicates) Leaf area data are leaf mean values for 10 plants. Letters in the same row indicate differences at p0.05

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Table 3 Spartium junceum water relation parameters. Measures were done before withholding the water, at the end of the stress period and at the end of the recovery period Relative water content (%) (RWC) Control G. mosseae G. intraradices Control G. mosseae G. intraradices Control G. mosseae G. intraradices 58.34 66.36 64.79 48.43 b 31.96 c 52.65 a 78.15 86.57 81.57 Specific leaf weight (SLW) 9.21 a 7.76 b 10.51 a 8.44 9.61 7.04 8.16 7.73 8.65 Chlorophyll (SPAD) 50.55 50.21 52.93 54.48 52.62 53.53 47.29 45.25 45.11

Pre-stressed

End of drought

End of recovery

Data are means of 10 replicates Letters in the same subsection indicate differences at p0.05

(Archer et al. 2002). These shrub lands effectively increase ground cover and favor the rain water infiltration decreasing the rainfall run off, with the net effect of reducing soil erosion. Perennial leguminous shrubs have been shown to be mycorrhizal (Maremmani et al. 2003), and to respond positively to AM inoculation when assessing plant growth. Our results confirm the findings of Requena et al. (1996) in A. cytisoides, where the inoculation with G. intraradices was better at increasing plant development than the inoculation with G. mosseae. Spartium junceum has also been shown to benefit from mycorrhizal inoculation, (Quatrini et al. 2002) in a greenhouse experiment and in a landfill restoration project. Quatrini et al. (2002) studied plants inoculated with AMF and Rhizobia in contrast to plants that were inoculated with neither Rhizobium nor AMF. In our study all plants were inoculated with Rhizobium, and we found that mycorrhiza inoculation increased the number of nodules in S. junceum roots. When assessing the effect of mycorrhizal

inoculation on plant growth we found two different levels of response, G. mosseae increased plant growth compared to control, however G. intraradices was better than G. mosseae. The sequence: control G. mosseae G. intraradices was maintained in all the parameters measured, showing a specific functional response of the symbiosis between S. junceum and G. intraradices. The AM fungus G. intraradices BEG 72 was isolated from an area cultivated with citrus trees in the south of Catalunya (Camprub and Calvet 1996) where drought episodes are common, therefore this isolate might be particularly suited to establish the symbiosis with plants that are native of similar edaphoclimatic areas, such as S. junceum and A. cytisoides. Requena et al. 1997 also found that an indigenous fungus Glomus coronatum was better than a culture collection G. intraradices at increasing A. cytisoides growth. Inoculation with Glomus versiforme increased the number of leaves in tangerine (Citrus tangerine) under well watered and also under stressed conditions (Wu

Table 4 Anthyllis cytisoides water relations parameters. Measures were done before withholding the water, at the end of the stress period, and the end of the recovery period Relative water content (%) (RWC) Control G. mosseae G. intraradices Control G. mosseae G. intraradices Control G. mosseae G. intraradices 78.87 75.87 75.45 35.32 39.74 32.36 77.92 78.44 74.64 Specific leaf weight (SLW) 8.92 9.41 9.36 8.29 9.17 8.68 8.42 8.22 5.94 b a a b a ab a a b Osmotic potential (MPa) -1.48 -1.60 -1.81 -4.80 -4.49 -5.22 -1.78 -1.96 -1.68 a a b b a c ab b a Chlorophyll (SPAD) 50.10 60.10 59.33 53.64 47.68 47.10 35.37 45.02 54.17 b a a a b b c b a N Leaves 12.52 b 18.89 b 43.29 a 8.4 b 14.70 b 30.10 a 8.00 b 7.50 b 16.60 a

Pre-stressed

End of drought

End of recovery

ab a b ab a b

Data are means of 10 replicates Letters in the same subsection indicate differences at p0.05

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and Xia 2006); in our experiment A. cytisoides inoculated with G. intraradices had more leaves in all measurements, although the plants responded to drought defoliating, confirming previous results (Haase et al. 2000; Archer et al. 2002) that describe the shrub as drought deciduous. At the end of the drought episode, control plants retained more chlorophyll, and mycorrhizal plants were more yellow, the situation was reversed at the end of the recovery period when control plants had the least chlorophyll, and plants inoculated with G. intraradices had almost pre-stress measurements of SPAD. In Rosmarinus officinalis subjected to drought the chlorophyll content of leaves was reduced by up to 85% during the drought with an increase in the de-epoxidation state of the xanthophyll cycle which conferred protection against irreversible damage to the photosystem under a severe drought stress, enabling the recovery of the plants after the autumn rains (Munn-Bosch and Alegre 2009). In our experiment the early onset of the xantophyll cycle shown by the G. intraradices plants might explain the quick recovery of the chlorophyll once the irrigation is re-established. Lower leaf osmotic potentials have been associated to plants adapted to growing on xeric sites (Gebre et al. 1998; Liu et al. 2003), A. cytisoides shows low water potential values, even when well watered and the symbiosis lowers the values measured at the pre-stress phase and also at the end of the stress period. The low relative water content (RWC) measures and the relatively high specific leaf weight (SLW) show that A. cytisoides possess typical xeric characteristics such as thick leaves, also enhanced by the establishment of the symbiosis. It has been shown for Fragaria virginiana (ONeill 1983) that osmotic adjustments are dependent on leaf age, with the highest capacity for adjustment in the intermediate age leaves; old leaves senesce and promote the osmotic adjustment of the rest. In our experiment, at the end of the recovery period the SLW of plants inoculated with G. intraradices had a higher SLW than the other treatments, inoculated with G. mosseae and non mycorrhizal control, and their osmotic potential was higher than the values observed at the pre-stress period for the same treatment. This could be the result of the senescence of older leaves followed by the severe defoliation observed during the drought stress. Most of the leaves that remained in the G. intraradices plants might be young, explaining the values both of SLW, of osmotic pressure and of SPAD observed. The data found for A. cytisoides show that the plant has a double system of drought tolerance, it defoliates thus avoiding drought but also lowers the osmotic potential of the leaves thus increasing its intrinsic drought resistance. At the pre-stress measurements S. junceum water relation parameters were not significantly changed by the symbiosis, except for the specific leaf weight (SLW) that was higher for G. intraradices plants, whilst A. cytisoides SLW was higher for plants inoculated with both fungi assayed. Similar results were found for Erythrina variegata

(Manoharan et al. 2010) confirming that mycorrhization can favour the adaptation to xeric environments. At the end of the drought period the relative water content of plants inoculated with G. intraradices was higher that the RWC of control and of G. mosseae plants. Soya bean plants were also shown to have an increased RWC due to mycorrhization (Aliasgharzad et al. 2006) at all soil moisture levels. In that case both fungi assayed, G. mosseae and Glomus etunicatum gave similar results; in our experiment G. intraradices was significantly better than G. mosseae at improving the plant response to drought stress. At the end of the recovery period, water relation parameters in S. junceum were not significantly different among treatments. Both plants, indigenous legumes from semi arid Mediterranean shrub lands, are often used in revegetation strategies (Requena et al. 2001; Preti and Giadrossich 2009). The use of mycorrhiza inoculated plants can enhance plant growth and also improve the response to drought, thus increasing the possibilities of survival and growth of these plants in an arid environment. The selection of the fungal inoculum is decisive as not all the AM fungi elicit the same response. In these experiments, G. intraradices BEG 72 was found to be superior to G. mosseae BEG 116, this difference could be attributed to the origin of the fungus, a native isolate from a Mediterranean area, compared to G. mosseae (BEG 116) isolated from the UK. These results confirm that the use of mycorrhizal inoculation should be included as a standard practice in revegetation programs (Estan et al. 2008), specially in degraded semi arid soils where the AMF might be in low numbers and unable to establish an effective symbiosis.
Acknowledgements This work has been partly funded by the Spanish Ministry of Science (MICINN) grants CGL2006-05648/ BOS and INIA RTA2007-00039.

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