Eugénio Mendes Ferreira*, Nuno Simões**, Isabel Videira e Castro* and Luís Carneiro*

*Senior Researchers

Instituto Nacional de Investigação Agrária/INRB, IP. Av. da República, Quinta do Marquês, 2780-159 OEIRAS

**Researcher

Instituto Nacional de Investigação Agrária/INRB, IP. Apartado 6, 7350-951 ELVAS

Abstract

Chemical, physical and biological soil parameters (OM, total N, pH, P₂O₅, K₂O, Mg, B, WHC, free–living nitrogen fixing and rhizobial microflora properties) and pastures yield were characterized and used to determine relationships in 40 locations covering different growth conditions of the "montado" ecosystem. Soil samples were collected in Spring. Reasonable soil fertility was found. The soils presented, in general, intermediate values for OM, K₂O, Mg, and Bo and low values for pH and P₂O₅. Populations of free-living nitrogen fixing bacteria were high, with an average of 4.2 x 10⁷ CFU g^-1 of soil, being nitrogenase activity highly variable, with an average of 34 nmoles of C₂H₄ g^-1 of soil h^-1. Rhizobial population associated to Trifolium subterraneum was high, with an average of 10⁶ bacteria g^-1 of soil, having adequate nitrogen fixing potential for the majority of the soils. Rhizobial population associated to Medicago polymorpha was low, with an average of 6.5 x 10⁴ bacteria g^-1 of soil, an insufficient value for a good nodulation, having low nitrogen fixing potential for the majority of the soils. Annual yield of natural pastures varied highly among locals, with an average of 3245 kg ha^-1, a usual value for this ecosystem. In general, soil properties were independent of the soil samples. The chemical parameters OM, P₂O₅, K₂O and total N were important factors to pasture yield. A great variability, even in soil samples collected within a short distance, was observed. A relationship between geological origin and productivity was found, being the granitic soils more productive in non-legume plants.

Relações Entre Parâmetros Seleccionados de Solo e Produção de Pastagens Naturais no Ecossistema Montado da Região do Mediterrâneo Através de Análise Multivariada

Sumário

Parâmetros químicos, físicos e biológicos do solo (MO, N total, pH, P₂O₅, K₂O, Mg, B, CR, e as propriedades da microflora fixadora de azoto de vida livre e dos rizóbios) e a produção de pastagens foram caracterizados e utilizados para determinar as relações em 40 locais abrangendo diferentes condições de crescimento dentro do ecossistema "montado". As amostras de solo foram colhidas na Primavera. Uma razoável fertilidade dos solos foi encontrada; apresentando em geral valores intermédios para a MO, K₂O, Mg e Bo e valores baixos para o pH e P₂O₅. As populações de bactérias fixadoras de azoto, de vida livre, foram elevadas, com uma média de 4.2 x 10⁷ UFC g^-1 de solo, com uma actividade da nitrogenase altamente variável e com uma média de 34 nmoles de C₂H₄ g^-1 de solo h^-1. A população rizobiana associada ao Trifolium subterraneum foi elevada, com uma média de 10⁶ bactérias por grama de solo, tendo um potencial de fixação de azoto adequado para a maioria dos solos. A população rizobiana associada à Medicago polymorpha foi baixa, com uma média de 6.5 x 10⁴ bactérias por grama de solo, um valor insuficiente para uma boa nodulação (no Outono), com um baixo potencial fixador de azoto para a maioria dos solos. A produtividade anual das pastagens naturais variou muito entre os locais, com uma média de 3245 kg ha^-1, um valor normal para este ecossistema. Em geral, as propriedades do solo foram independentes das amostras de solo. Os parâmetros químicos MO, P₂O₅, K₂O e N total foram factores importantes para a produção das pastagens. Uma grande variabilidade nas amostras de solo colhidas a uma curta distância foi observada. Uma relação entre a origem geológica e a produtividade foi também verificada, sendo os solos graníticos mais produtivos para as plantas não-leguminosas.

Palavras-chave: Ecossistema montado; produção de pastagem; parâmetros do solo; agricultura sustentável

Relations de  Paramètres Sélectionnés des Sols et la Production des Pâturages Naturels Chez l'Écosystème «Montado» de la Région Méditerranéenne en Utilisant une Analyse Multivariée

Résumé

Paramètreschimiques, physiques et biologiques des sols (MO, N total, pH, P₂O₅, K₂O, Mg, B et propriétés de la microflore des microorganismes fixateurs de azote, vivant en vie libre, des Rhizobium) et la production des pâturages ont été caractérisés et utilisés pour déterminer les relations des 40 situations couvrant différentes conditions de croissance parmi l'écosystème "montado". Les terres ont été récoltées et analysées au Printemps. La fertilité des sols a été raisonnable, présentant en général, des valeurs intermédiaires pour la MO, K₂O, Mg, et Bo et des valeurs faibles pour pH et P₂O₅. Les populations de bactéries fixatrices de azote, non symbiotiques, ont été élevées, avec une moyenne de 4.2 x 10⁷ de colonies g^-1 de sol, la nitrogénase était très variable, avec une moyenne de 34 nmoles de C₂H₄ g^-1 de sol h^-1. La population des rhizobia associés au Trifolium subterraneum était élevée, avec une moyenne de 10⁶ bactéries g^-1 de sol, ayant un potentiel adéquat de fixation d'azote pour la majorité des sols. La  population des rhizobia associés à Medicago polymorpha était faible avec une moyenne de ]]>  6.5 x 10⁴ bactéries g^-1 de sol, une valeur insuffisante (à l'Automne) pour une bonne nodulation, ayant un faible potentiel de fixation pour la majorité des sols. La production annuelle des pâturages naturels a varié beaucoup selon les situations, avec une moyenne de 3245 kg ha^-1, une valeur normale pour cet écosystème. En général, les propriétés du sol sont indépendantes des situations. Les paramètres chimiques MO, P₂O₅, K₂O et N total ont été des facteurs importants pour la production des pâturages. Une grande variabilité, même dans les situations de sols prélevés à une courte distance, a été observée. Une relation entre l'origine géologique et la productivité a été trouvée,  les sols granitiques étant plus productifs pour les plantes non-légumineuses.

Mots clés: Écosystème montado; production des pâturages; paramètres du sol; agriculture soutenable

Introduction 

The "montado" is an agroforestry and pasture system of the regions of Southern of Iberian Peninsula, Portugal and Spain where it is called "dehesa", associated to extensive and sustainable livestock production under scattered oak trees, Quercus suber and Quercus ilex. It is the most extended agroforestry system in Europe, with more than 3 million hectares. This system has been known through centuries for its multiple use and renewable resources, mainly silvo-pastoral and cereal cropping (JOFFRE et al., 1988). When well established it can assume different functions for the ecosystem, such as optimisation of available energy through biomass production, soil preservation, nutrients circulation, water conservation and bioregulation of climate or microclimate stability (Trujillo and Mata, 2001).

In Portugal, this special ecosystem supports a sustainable extensive agriculture, where the natural pastures and the introduced legume based pastures play an important role, supporting the direct grazing by cows and sheep. Natural pastures are in their majority poor and managed with a low animal-stocking rate due the low soil fertility, associated to a diverse, but few productive natural flora.

Soil chemical and physical properties are usually associated to soil productivity providing information that can be used by land managers to make management decisions. In this rain-fed ecosystem, the nitrogen fixation by free-living bacteria and legume symbioses is a major process of providing nitrogen to the soils, being legumes an important component of a strategy for increasing productivity and sustainability of farming systems. The amount and effectiveness of background rhizobial populations are fundamental parameters for evaluation the need for legume seeds inoculation, procedure that can be determinant for improving pastures yield and quality. 

Our objectives were to assess some soil properties, to determine relationships among chemical, physical and biological soil parameters, sampling local, soil origin and natural pastures yield under Quercus suber and Quercus ilex trees on the Southern of Portugal. These results will allow the identification of parameters that can be used for development of practices for increasing soil quality and crop production.

Materials and methods

The study was carried out on a range of long term dryland natural pastures, at the "montado" ecosystem (Alentejo-Portugal). This ecosystem covers an area superior to 10⁶ ha. The climate is characterized by dry hot summers and wet cool winters with an average annual temperature ranging from 14 to 17ºC. Rainfall is very variable, with an average annual precipitation ranging from 500 to 700 mm (450-600 during the agricultural year of experiment, October-September), falling the majority during the cool season (October-April). Soils, mainly derived from granite and schist, are mostly poor. Lightly hilly lands are the dominant landscape, with average altitudes ranging from 200 to 250 m.

Field plants sampling and pasture yield

In Autumn (October), 13 farms (locals) in different edaphic and climatic environmental conditions were selected for this study. At each farm, outside canopy shade, three exclusion cages (1 x 1m) were placed at upper, mid and lower slope positions, trying to embrace different plant growth conditions. On one farm, due their larger area and different soils, four cages were placed. Dung patches visually affected none of the cages places.

Two cuttings per season were undertaken, one in the end of February (winter) and the other in the end of May (Spring). The shoot material was handcut with shears to a height of 2 cm. The plants were separated into legumes (leg) and non-legumes (n-leg), dried at 70ºC and weight for total yield (TY).

Soil sampling and processing

 In May, forty soil samples were collected on 13 farms (40 exclusion cages) covering different growth conditions. Sampling was carried out in spring, when soil biological activity in Mediterranean environmental conditions is higher (GARCIA et al., 1997). Five soil cores from the upper 10 cm of soil, were collected aseptically from each exclusion cage, bulked, and stored in sealed plastic bags. Samples were transported to the laboratory under cooled conditions where they were thoroughly mixed, passed through a 2 mm sieve to remove stones and large pieces of organic matter and stored in the refrigerator (6ºC) before physical, chemical and microbial analysis. 

Chemical and physical analysis of the soil samples: organic matter (OM), total N, pH (H₂O), extractable P (P₂O₅), K (K₂O), Mg and B, and water holding capacity (WHC) were done according to the usual procedures of the Analytical Service.

Biological properties

Free-living nitrogen fixing microflora properties

Cultivable nitrogen-fixing bacteria were enumerated by plate counting from colony forming units (CFU) growing on a N-free medium (POCHON and TCHAN, 1948; PARKINSON et al., 1971), containing actidione at 20 mg l^-1. The results (Free-liv) were expressed as log of number of colony forming unities (CFU) per gram of dried soil.

Potential nitrogen fixation by free-living microflora was evaluated indirectly by acetylene reduction assay (HARDY et al., 1973), a sensitive method for assaying nitrogenase activity, using the gas chromatography technique. Two seedlings of Lolium multiflorum were grown in glass bottles containing 15 g of soil adjusted to 75% WHC in a controlled environmental growth room (18-20ºC, 12 h day) during 6 weeks. For measurement of acetylene reduction activity (ARA), acetylene was injected at 10% of the bottle volume, after removal of the same volume of air, being the soils exposed to C₂H₂ for 1 h at 20ºC. The results (ARA Free-liv) were expressed as nanomoles (nmol) of C₂H₄ per gram of soil and per hour.

Rhizobial microflora properties

Rhizobial microflora associated to Trifolium subterraneum (Rhizobium leguminosarum bv. trifolii) and Medicago polymorpha (Sinorhizobium meliloti) were estimated by the most probable number (MPN) method (VINCENT, 1970) using a ten-fold dilution series with the test plants Trifolium subterraneum cv. Clare and Medicago polymorpha cv. 66, growing in Jensen's agar medium (VINCENT, 1970). The results were expressed as log of number of rhizobia bacteria (Rh trifolii for clover and Rh meliloti for annual medic) per gram of dried soil (Table 1). The plants were grown for 8 weeks in a controlled environmental growth room (18-20ºC, 12 h day) and harvested. The roots were examined for nodulation and the dry matter from the shoots was measured after oven drying at 70ºC. The "whole-soil inoculation technique" (BROCKWELL et al., 1988; QUIGLEY et al., 1997) was used to assess the nitrogen fixing potential of the soil rhizobial populations. The results obtained from dry matter production of the nodulated plants (Trifolium subterraneum - DM Trifolium and Medicago polymorpha - DM Medicago), from the soil dilutions 10^-1 and 10^-2, allowed to differentiate the rhizobial resident populations.

Statistical analyses

The data were analysed using multivariate analysis: Principal component analysis (PCA), using NTSYS-pc version 2.1 (ROHLF, 2000) and discriminant analysis (DA) using NCSS software package (HINTZE, 2001).

Analyses were conducted on the complete dataset for the 40 soil samples characterized by 16 properties (variables) (Table 1). The principal component analysis allows us to classify the soil samples and to obtain different similarity groups and to find relationships among variables. The discriminant analysis allows us to validate the existence of geographical or geological patterns of soil samples distribution.

Table 1 - Soil chemical, physical and biological parameters and pasture yield (legumes and non-legumes)

Results and discussion

Pasture yield

Annual yield of natural pastures varied highly among locals (Table 1). The shoot material ranged from 0 to 1257 kg ha^-1 with an average of 404 kg ha^-1 for legumes (TY leg) and from 255 to 6255 kg ha^-1 with an average of 2842 kg ha^-1 for non-legumes (TY n-leg). Total yield ranged from 287 to 6866 kg ha^-1 with an average of 3245 kg ha^-1.

In this study we found pasture yields that confirm those results, being our results slightly superior, possibly as a consequence of a regular annual rainfall (450-600 mm).

Chemical and physical properties

Values for the soil variables presented in Table 1 showed a wide range either among the sampling locals or the samples from the same local. According to the recommended levels for crops fertilisation in Portugal (Laboratório Químico Agrícola Rebelo da Silva 2000) the soils presented in general, intermediate values for OM, K₂O, Mg, and B  and  low  values for pH and P₂O₅.

In spite of the acceptable soil fertility in the "montado" ecosystem, it can be easily increased using a phosphorus and calcium based fertilizer, as for example the calcium superphosphate (18% P₂O₅).

Biological properties

Free-living nitrogen fixing microflora properties

Free-living nitrogen-fixing microbial populations in the natural ecosystem are crucial for the ecosystem maintenance and productivity due to the special role of these organisms in the global biogeochemical cycle of nitrogen (KAVADIA et al., 2007). It is known that free-living nitrogen-fixing populations in soil are affected by environmental, physical and chemical conditions, including the organic components excreted by plant roots, as well as by interactions with other microbial populations.

ARA was highly variable among the soil samples (Table 1), ranging from 0 to 170 nmoles of C₂H₄ g^-1 of soil h^-1_, with an average of 34 nmoles of C₂H₄ g^-1 of soil h^-1_, being detected higher rates of ARA in schistose soils.

CHOTE et al., 2002, working on 3 and 19 years old natural fallows, found a potential N₂ fixation of 6.4 and 110 nmoles of C₂H₄ g^-1 of soil day^-1, respectively.

RODRÍGUEZ-ZARAGOZA et al., 2008, in soils under perennial shrubs in a desert in Mexico, found rates of ARA between 20.1 and 29 nmoles of C₂H₄ g^-1 of soil day^-1.

Working with soil cores (6 cm diam x 15 cm length) containing grasses, obtainned from 25 sites in Texas, USA, MORRIS et al., 1985, found ARA rates ranging from 0 to 7.6 mmol of C₂H₄core^-1 day^-1. Also in Texas, WRIGHT and WEAVER, 1981, had found ARA rates ranging from 0 to 5.5 mmol of C₂H₄ core^-1 day^-1, in root-soil cores (8 cm diam x 15 cm length) collected from forage grasses.

Nitrogenase activity associated with several grass species was measured by THOMPSON et al., 1984, in soil cores (12.5 cm diam x 15 cm length) at 67 sites in New South Wales (Australia) finding a maximum of 246 nmol of C₂H₄ core^-1day^-1. Also in Australia, in soil-plant cores (12 cm diam x 16.5 cm length), WEIER, 1980, obtained media values for ARA ranging from 5.3 to 17.2 mmol of C₂H₄ core^-1 day^-1, for three grass species in pasture soil.

Correlations between populations of free-living nitrogen fixing microflora (in field conditions) and ARA (in laboratory controlled conditions) are poor and clear explanations cannot be given for the relatively low rates of ARA, but high amounts of CFU. This discrepancy can be a result of various factors including the plate count method used (it determines only the cultivable bacteria), the different plant rhizospheres and the environmental soil conditions used for counting the populations of free–living nitrogen fixing microflora and for ARA evaluation. However, in general terms, larger populations of free-living nitrogen fixers suggest considerable nitrogen fixation (XIE and STEINBERGER, 2002). Microflora around the roots is largely dependent of organic nutrients from the roots exudates and the factors controlling exudation also control nitrogenase (DOMMERGUES et al., (1973). Different plant species induce different environment (favourable or adverse) for nitrogenase activity in the rhizospheres. Also the plant growth stages and the physical, biological and chemical soil characteristics can influence the nitrogenase activity.

Rhizobial microflora properties

Subterranean clovers and annual medics play a major role in supplying nitrogen to dryland pastures on the Southern of Portugal, place where these legume plants occur naturally. The numbers of the nodulating indigenous rhizobial populations and their symbiotic effectiveness with the hosts are fundamental in satisfying the nitrogen requirements of the legume plants, but a considerable variation of the number of rhizobia in soils and a ample range of symbiotic effectiveness can be found. Agricultural soils are often constrained in their ability to sustain productive farming systems due to factors associated to low fertility and water stress, that can have a negative impact on the legume-Rhizobiumsymbiotic relationship.

Results from this study, presented in Table 1, showed that, in spring, the indigenous rhizobial populations associated to Trifolium subterraneum, were in general high, ranging from 0 (only 1 soil sample) to 1.15 x 10⁷ rhizobia bacteria per gram of soil, with an average of 10⁶. In a general way, the size of the rhizobial populations (5 x 10⁴ in autumn) are enough for a good nodulation of the subterranean clovers and the introduction of new and more efficient rhizobial strains can be difficult and often unsuccessful (BROCKWELL, 1981; HERRIDGE et al., 2002; SLATTERY and PEARCE, 2002). The nitrogen fixing potential of the rhizobial populations associated to clover was adequate for the majority of the soils, originating a dry mater production similar to the inoculated control. In these conditions, the introduction of new clover seeds implies their inoculation with a large number of bacteria, superior to 10⁵ per seed, using competitive and efficient rhizobial strains.

The rhizobial populations associated to Medicago polymorpha (annual medic), even in spring, were low, ranging from 4 x 10 to 10⁶ (only 1 soil sample) rhizobia bacteria per gram of soil, with an average of 6.5 x 10⁴, (1.3 x 10³, in autumn) being in their majority inferior to 10³, an insufficient value for a good nodulation (BROCKWELL, 1981; HERRIDGE et al., 2002; SLATTERY and PEARCE, 2002). The nitrogen fixing potential of the rhizobial populations associated to annual medics was low for the majority of the soils, originating a dry matter production about 50% of the inoculated control. The introduction of annual medics in these pastures requires the inoculation of the seeds with efficient rhizobial strains.

Statistical analyses

Principal component analysis

A principal component analysis based on 16 variables (physical, chemical, biological and pasture yield) from 40 soils, sampled on 13 farms was made (Figure 1).

Figure 1 - Projections of the 40 soil samples onto the plane defined by the first and the second principal axes with minimum spanning tree superimposed. The ellipses enclose the soil samples with similar properties

Table 2 - Correlation between the original properties and principal components

Figure 2 - Projections of the 16 soil properties used to characterize the 40 soil samples onto the plane defined by the first and the second principal axes

The PC1 separated the soil samples with higher values for the variables: Total N, pH, K₂O, Mg, B, WHC, Free-liv, ARA Free-liv, Rh meliloti and DM Medicago, on right side of Figure 1, from the soil samples with lower values for the same variables. The PC2 separated the soil samples with higher values for the variables OM, P₂O₅, and TY (leg and non-leg), on upper side of Figure 1, from the soil samples with lower values for the same variables. The PC3 (not shown) separated the soil samples with higher values for the variable DM Trifolium from the soil samples with lower values for this variable (Table 2).

In Figure 1, we can verify the existence of 4 groups of soil samples and 2 outliers (gr 31, a very poor soil with low values for almost all variables and gr 34 a rich soil for almost all variables). The group A includes the soil samples: sch 8, sch 15, sch 28, gr 19, with higher values for the variables P₂O₅, OM, K₂O total N and TY leg and non-leg. The group B includes the soil samples: sch 26, and sch 27 with higher values for almost all biological variables and some chemical and physical variables (B, WHC, Mg and pH). The group C includes the soil samples: sch 1, sch 2, sch 3, sch 5, sch 7, sch 10, sch 12, sch 13, sch 14, sch 25, gr 17, and gr 18, with intermediate values for almost all biological variables and for pH, Mg, B and WHC. The group D includes the remaining soil samples with lower values for biological variables. The obtained groups of similarity join soil samples with similar geological origin.

In Figure 2, we can verify the importance of each variable in the spatial distribution of soil samples (Figure 1) and to observe that some chemical variables, mainly OM, P₂O_5, K₂O and total N seem to be related to legume and non-legume pasture yield.

Discriminant analysis

The discriminant analysis used to verify the existence of a geographical pattern of soil samples distribution, showed accuracy of 91.9% in the separation of the different sampling locals (Figure 3).

Figure 3 - Discriminant analysis of the soil samples geographical origin with the properties vectors superimposed

The first discriminant function accounted for 58.1% of the variance, discriminating the soil samples from different locals, being Nisa on the upper left side and Serpa and Portalegre on the down right side of the figure. It represents the contrast of the variables B, Mg, pH, Rh meliloti, DM Medicago and ARA Free-liv, with high levels at Serpa and Portalegre and low levels at Nisa. The second discriminant function accounted for 12.7% of the variance and allowed the individualization of the remaining sampling locals, representing the contrast between the two variables TY n-leg and P₂O₅ against K₂O, Total N and OM, being the soil samples positioned according to the values of these variables. These results can be used as a complement of the PCA providing information on which variables are best adequate for discriminating the sampling locals.

The soil samples from each local were considered as uncorrelated soil samples. This methodology seems to be adequate, since the soil samples dispersion (Figure 3) in each local shows a general variability.

When the discriminant analysis was used to group the soil samples according to their geological origin (Figure 4), it was verified a good separation (89% accuracy) between the 2 origins, except for 2 misclassified soil samples, 1 schistose soil (sch 11) that is classified as granitic soil and 1 granitic soil (gr 18) that is classified as schistose soil. The soil sch 6 can be classified either as schistose (48%) or as granitic (52%). The soil samples sch 26 and sch 27 are a different group as already seen in PCA. The granite-derived soils, in general, have higher values for TY n-leg, being TY leg not important to discriminate the soils from different geological origin.

Correlations of soil physical, chemical and biological properties with yield are often low due to the interactions with weather, topography, and other biotic or abiotic factors. Associated to these constraints, the soil properties evaluated differ among the authors; the obtained results are frequently discrepant and cannot be applied without critical adjustments. Studies related to soil properties and herbaceous vegetation yield in the Mediterranean area are scarce. REZAEI et al., 2006, studying the relationships between soil chemical and physical properties and plant growth on the mountainous rangeland of northern Iran, a Mediterranean area, obtained results indicating that plant variables were more sensitive to soil physical properties than to soil chemical properties. Our results are different, showing that chemical properties are key factors to pasture yield.

Conclusions

Soils chemical and physical properties in the "montado" ecosystem are acceptable having low values for pH and P₂O₅. Properties related to number and quality of free-living nitrogen fixing bacteria and rhizobial populations associated to subclovers are adequate, contrasting with properties of rhizobial populations associated to annual medics, that are insufficient for a good nodulation, having a low nitrogen fixing potential.

With the principal component analysis it were obtained groups of similarity, being the schistose soils dominant in the groups A, B and C and the granitic soils dominant in the group D. The groups are dispersed over all the study area, suggesting that soil properties are independent of the soil samples and that some chemical properties are important factors to pasture yield.

 The geographical distribution of the soil samples, when using the discriminant analysis, showed a large variability even in soil samples collected within a short distance. A good separation of the soil samples according to their geological origin was verified, being the granitic soils more productive in non-legume pasture.

Acknowledgements

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Entregue para publicação em Setembro de 2010

Aceite para publicação em Outubro de 2010