Effect of saline soil parameters on endomycorrhizal colonisation of dominant halophytes in four Hungarian sites

Soil and root samples were collected from the rhizosphere of dominant halophytes (Artemisia santonicum, Aster tripolium, Festuca pseudovina, Lepidium crassifolium, Plantago maritima and Puccinellia limosa) at four locations with saline soils in Hungary. The correlationsbetween arbuscular mycorrhiza (AM) fungal colonisation parameters (% colonisation, % arbuscules) and soil physical, chemical and biological parameters were determined Endomycorrhiza colonisation was found to be negatively correlated with the electric conductivity of the soil paste, the salt-specific ion concentrations and the cation exchange capacity, showing the sensitivity of AM fungi at increasing salt concentrations, independently of the types of salt-specif ic anions. A positive correlation was detected between the mycorrhiza colonisation and the abundance of oligotroph bacteria known to be the less variable and more stable (k-strategist) group. This fact and the negative correlation found with the humus content underlines the importance of nutrient availability and the limitations of the symbiotic interactions in stressed saline or sodic soils. Additional key words: correlations, halophytes, microbial abundance, salt stress, soil-parameters.

microbes) are both influenced by the negative impacts of high salt concentrations (Hasegawa et al., 1986), independently of the identity of salt-specific anions (Füzy et al., 2008b).The interaction between higher plants (halophytes), the microsymbiont arbuscular mycorrhizal fungi (AMF) and beneficial plant-growth promoting rhizobacteria (PGPR) might affect the survival capacity of the plants in these stressed environments.There are several stress factors in these environments: high osmotic pressures, ion-toxicity, imbalanced ion concentrations, unfavourable soil structure and suboptimal soil pH, besides nutrient deficiency that also tends to occur in saline and sodic soils.The water regime of saline soils is imbalanced contributing to create an extreme environment for plant growth.
When water is present in saline soils, these swell, become plastic, and the water present becomes unavailable to plants.When the soil dries out, it hardens, becoming less porous, hindering the roots development and subsequently, the nutrient uptake of the plants.Fluctuations of the water content, including flooding episodes are also a stress-factor that can result in oxygen deficiency and/or enhanced CO 2 content in the plant-rhizosphere.
A relatively high mycorrhizal colonisation rate and AM fungi spore numbers have been found in saline soils (Landwehr et al., 2002) showing the importance of the symbiosis in these environments.In these saline soils, a specific mycorrhiza population has been found (Carvalho et al., 2001;Landwehr et al., 2002), with a low diversity, showing that salinity as a selection pressure acts not only on the plant-communities but also on the soil-microbial components.The aim of this work was to study the interrelations between the physical, chemical and biological properties of the saline or sodic soils and AMF root colonisation values in four sites in Hungary to assess the importance of the diverse environmental factors on the symbiosis.
Root and soil samples were collected from the rhizosphere of dominant halophytes Artemisia santonicum (L.), Aster tripolium (Jacq.),Festuca pseudovina (Hack.exWiesb.),Lepidium crassifolium (W.et K.), Plantago maritima (L.), Puccinellia limosa [(Schur.)Homberg.] at Spring (April 2001) from four sampling sites in Hortobágy-and Kiskunság National Parks, Hungary.The samples were collected at a depth of 5-15 cm, each sample was a mixture of 5 sub-samples from the rhizosphere soil of five individuals of each halophyte plant species studied.The soil physicochemical parameters measured were: soil-moisture, clay content, higroscopicity, electric conductivity, T-value, Na-content, pH, the salt-specific carbonatecontent and the humus content (Buzás, 1988(Buzás, , 1993)).Soil biological parameters studied were root colonization of AMF and abundance of some cultivable microbes (micromycetes, oligotrophs, heterotrophs).To assess mycorrhizal colonisation 1-2 g fresh fine roots were cleared by boiling in 15% KOH solution for 40 min, stained in aniline blue (30 min) and fixed in lactic acid (40%).Endomycorrhizal colonisation of the roots was determined by the method of Trouvelot et al. (1986).The abundance of hyphae, vesicles and arbuscules in the roots was evaluated; the intensity of mycorrhizal colonisation (M%) and the arbuscules richness (A%) were calculated for all the samples.Dilution series using 1g of the sampled soils were prepared and the appropriate soil suspensions were spread on three types of selective agar plates: nutrient agar for the heterotroph counts, 1/100 strenght nutrient agar for the k-strategist oligotroph counts (Horváth, 1980) and Martin agar for the micromycetes (Martin, 1950).Plates were incubated for 24-72 hours in 28°C and colony-forming units (CFU) were estimated.
Data sets were analysed by linear regression; and significant correlation ratios were determined.CFU data of countable microbes were log transformed; mycorrhizal colonisation values were arc-sin transformed before the appropriate variance analysis.Statistical evaluation was done for the whole range of rootsamples, collected and also for the most common halophyte (Plantago maritima L.) in order to exclude hostplant effects.
Significant correlations between the mycorrhizal colonisation, both as the infection intensity (M %) and also as the arbuscules richness (A %) and some soil physicochemical properties are shown in Figure 1.The upper part of the figure shows the correlations considering all the six plant species sampled; the lower part shows the result of the statistical analysis limited to one plant species, the known salt-tolerant host, Plantago maritima.Statistically significant negative correlations were found between the mycorrhizal colonisation intensity (M %) and/or the arbuscules richness (A %) and some of the measured soil physicochemical parameters.Most of the significant physicochemical parameters relate to the soils salt content, such as the electric conductivity (Fig. 2a), cation exchange capacity and cation content of the soil samples, more particularly the Na + content of the soil (Fig. 2b).Some of these correlations are significant only for P. maritima.A  negative correlation was detected between mycorrhizal colonisation and the soil humus content (H %) (Fig. 2d), whereas soil pH values were correlated positively with the root colonisation rates (M%) (Fig. 2c), and the arbuscules content (A %).
Figure 1 shows the correlation data between the AM fungi and the three groups of cultivable microorganisms (micromycetes, heterotrophs and oligotrophs), assessed with selective plates from the rhizosphere of the studied halophytes.Among these cultivable microbes the oligotroph bacteria showed the most stable count; there were less than one order of magnitude variability among the sampling sites and sub samples.The variability in the counts of the heterotroph bacteria and the microscopic fungi from different host-plants rhizosphere was three orders of magnitude, meaning more than 1000-times difference in the counts.A positive correlation was found between the AMF colonisation values (M %, A %) and the abundance of oligotroph bacteria in the rhizosphere.
The soil physico-chemical parameters of saline and sodic soils have a defined effect on the studied endomycorrhizal colonisation.Soil physical parameters, have been shown to influence soil biological parameters (Al-Karaki, 2000;Hildebrandt et al., 2000;Carvalho et al., 2001).Seasonal samplings done at the same saline locations (Füzy et al., 2006(Füzy et al., , 2008a) ) showed that soil water content have an effect on the soil biota and on the mycorrhizal colonisation.Parameters describing the quality and quantity of salts in the soil are strongly correlated with mycorrhizal colonisation.These results are in agreement with other studies, which showed a negative impact of high salt concentrations on the symbiosis formation (Juniper and Abbott, 1993;Aguilera et al., 1998;Al-Karaki, 2000), the spore germination (Juniper and Abbott, 1993), and on hypha elongation (McMillen et al., 1998).The negative effects of continuous salt stress might also select a more salt tolerant mycorrhizal population.Dominance (at about 80% abundance) of Glomus geosporum species was found by Landwehr et al. (2002) in salt-affected sites in Germany and in Hungary, irrespectively of the type of saltspecific anions in the sampled soil.
Mycorrhizal colonisation in saline and sodic areas has been found higher than average values found in other non stressed areas, showing an increase of mycorrhiza dependent plants in areas under this environmental stress (Hildebrandt et al., 2000).Some AM fungi have shown to be able to tolerate a specif ic environmental stress (Regvar et al., 2003;Ruiz-Lozano, 2003;Biró et al., 2005) and might be capable to confer this tolerance to their non-adapted host-plants, as shown by Hildebrandt et al. (1999) on heavy metal contaminated sites.Besides salinity other factors linked to specific characteristics of the soil can influence the establishment and development of the symbiosis.In our study the alkalinity of the soil ranged from 7.5 to 10, the increase of pH was found to correlate positively with AMF colonisation.Sidhu and Behl (1996) showed that alkaline soils are favourable for mycorrhizal colonisation and for extraradical hyphal growth.The plant available P content of soils depends highly on the soil pH.As mycorrhizal symbiosis play an important role in phosphorus uptake, soil parameters involved in the P cycle can form a complex system with the biological parameters determining the ecophysiology of the hostplants.In saline conditions the need for a better nutrient supply and thus a stronger mycorrhizal colonization increases.The negative effect of the humus content (H %) of soil on the mycorrhizal colonisation also suggests the important role of the symbiosis in the nutrient uptake, specially in nutrient deficient soils (Marschner, 1998).When nutrient availability increases mycorrhiza dependency decreases, as found in this study.
There is a positive correlation between the mycorrhizal colonisation and the oligotrophic bacterial counts in the rhizosphere soil of halophytes.The abundance, ratio and dynamism of the so-called «k-strategist» oligotroph bacteria might be an important factor of soil quality and a biological indicator, as it was shown by Karlen et al. (1997) and Biró et al. (2002).The oligotrophs require a low-nutrient availability media, have a slow reproduction capacity, and are able to survive more efficiently in unfavourable (stressed) environmental (soil) conditions.This group of bacteria form a cluster of stable microbes, increasing the k-strategist ratio of the soil ecosystems.These k-strategist microbes, correlate positively with the mycorrhizal fungi, and might interact with the symbiosis acting either as mycorrhiza helper bacteria or have a plant growth promoting effect.
Adaptation to extreme environmental conditions is a complex phenomenon, specially when considering plant-microbe interactions.The plant's physiology and the rhizosphere microbiota are both capable of adaptative changes, interacting in multifactorial stressed environments (Schwarz and Gale, 1984).This interaction results in a complex, more plastic, system, which can tolerate saline habitats more efficiently.The tight relations between rhizobiological parameters and environmental stress factors can be the basis for the development good indicators for soil quality (O'Neill et al., 1986;Visser and Parkinson, 1992;Kling and Jakobsen, 1998).

S146A.Figure 1 .Figure 2 .
Figure1.Correlation matrix between the main mycorrhizal colonisation values (infection intensity, arbusculum richness) and some other soil properties, including the physical-chemical and other biological parameters.Upper part of the figure is the cumulative results of all the six plant species tested (Artemisia santonicum, Aster tripolium, Festuca pseudovina, Lepidium crassifolium, Plantago maritima and Puccinellia limosa); lower part shows the data of Plantago maritima only.0: no correlation, +/-: significant correlations at p = 0.95 level, ++/--: significant correlations at p = 0.99 level.