Effectiveness of the Application of Arbuscular Mycorrhiza Fungi and Organic Amendments to Improve Soil Quality and Plant Performance Under Stress Conditions

J. soil sci. plant nutr. 10 (3): 354 – 372 (2010)

EFFECTIVENESS OF THE APPLICATION OF ARBUSCULAR MYCORRHIZA FUNGI AND ORGANIC AMENDMENTS TO IMPROVE SOIL QUALITY AND PLANT PERFORMANCE UNDER STRESS CONDITIONS

Almudena Medina1 and Rosario Azcón2*

1Netherlands Institute of Ecology (NIOO-KNAW), Department of Microbial Ecology, Boterhoeksestraat 48, 6666 GA Heteren, The Netherlands. 2*Department of Soil Microbiology and Symbiotic Systems, CSIC-Estación Experimental del Zaidín, Profesor Albareda nº 1, 18008 Granada, Spain. *Corresponding author: [email protected]

ABSTRACT

Plant growth is limited in arid and/or contaminated sites due to the adverse conditions coming from heavy metal (HM) contamination and/or water stress. Moreover, soils from these areas are generally characterised by poor soil structure, low water-holding capacity, lack of organic matter and nutrient deficiency. In order to carry out a successful re-afforestation, it is necessary to improve soil quality and the ability of plants species to resist this harsh environment. The symbiosis with arbuscular mycorrhizal (AM) fungi has been proposed as one of the mechanisms of plant heavy metal tolerance and water stress avoidance. On the other hand, addition of organic amendments to the soil can reverse degradation of soil properties. Agro-waste residues such as dry olive cake (DOC) and sugar beet waste (SB) supplemented with rock phosphate (RP) can be used as organic amendments after fermentation by Aspergillus niger. The application of A. niger- treated DOC and/or SB to semi-arid soils and/or HM-contaminated soils increased aggregate stability, soil enzymatic activities, water soluble C and water soluble carbohydrates as well as nutrient availability, especially P. AM inoculation, using adapted endophytes, was more efficient with respect to increasing plant nutrition and growth as well as plant tolerance to drought or HM-stress conditions. The combined treatments involving mycorrhiza fungi inoculation and addition of the amendments into the soil can be proposed as a successful revegetation strategy for plant performance in P-deficient soils under semiarid Mediterranean conditions. The beneficial effectiveness of this symbiosis with suitable AM fungi in A. niger-treated agro-waste residue-amended soil can also be regarded as a successful biotechnological tool for reclamation of HM-contaminated soils.

Keywords: Arbuscular mycorrhiza (AM) fungi, organic amendment, heavy metal (HM) contamination, water stress, drought, organic matter, rock phosphate, revegetation, bioremediation, Aspergillus niger, phosphate solubilization.

INTRODUCTION

Plant productivity can be limited seriously this respect, microbial inoculants can help in heavy metal and/or semi-arid sites. In plants to cope with adverse conditions and

354 Interactive effect of AM fungi organic amendments, Medina y Azcón

arbuscular mycorrhizal (AM) fungi have lignocellulosic composition, has been an extraordinary importance since they shown to have a detrimental effect on increase nutrient acquisition by the plant plant growth (Vassilev et al., 1986; as well as resistance to biotic and abiotic Vassileva et al., 1998). The complete bio- stress (Barea and Jeffries, 1995; Barea et recycling of these agroindustrial wastes is al., 2002a, b, c, d). In fact, the symbiosis now accepted as an important element of with AM fungi has been proposed as one sustainable agriculture (Vassilev et al., of the mechanisms of heavy metal plant 2006). Such lignocellulosic materials can tolerance (Hildebrandt et al., 2007) and be used as organic amendments after water stress avoidance (Augé, 2004; Ruiz- biotransformation processes, (Vassilev et Lozano and Azcón, 1996; Ruíz-Lozano et al., 1997, 2006, 2007). al., 1995). Nonetheless, the mycorrhizal In addition, phosphorus is a limiting component may disappear or, at least, be nutrient for plant growth in degraded soils severely depleted in degraded soils, so it and the possibility of using rock may be necessary to reinforce or replace it phosphate as a fertilizer has received by appropriate inoculation (Requena et considerable interest in recent years al., 1996). (Barea et al., 2002d). Rock phosphates These arid and/ or contaminated soils (RP) are natural, inexpensive and easily are generally characterised by poor soil obtainable fertilizers, but their solubility structure, low water-holding capacity, is very low in non-acidic soils. organic matter lack and nutrient The use of fermentation processes for deficiency. Thus, the inoculation of RP solubilization by Aspergillus niger mycorrhizal fungi may not be enough to grown on DOC and SB agrowastes has assure the establishment of plant cover. been proposed (Vassilev et al., 1998, Therefore, in order to carry out successful 2002, 2006). During fermentation, at least reafforestation, it is necessary to improve three simultaneous activities occur: soil quality and the ability of the plant mineralization of the lignocellulosic species to resist these harsh environments. substrates, biosynthesis of organic acids In this respect, the application of organic and, as a consequence, a solubilisation of amendments to the soil, prior to the RP with a strict correlation between both inoculation of AM fungi, has been last processes (Vassilev et al., 1995). The recommended (Medina et al., 2004a, b). percent of substrate mineralization ranged The beneficial effects of organic from 16% in experiments with DOC to amendments include provision of plant 69% in experiments with SB (Vassilev et nutrients, increased humus content and al., 1998). thereby increased water–holding capacity, A series of microcosm experiments improved soil structure, and increased has been performed in greenhouse to microbiology activity (Caravaca et al., evaluate the effectiveness of the resulting 2002). fermented products. All amendments One attractive approach is the use of improved plant growth and P acquisition, agro-wastes as organic amendments. which were further enhanced by Large amounts of dry olive cake waste mycorrhizal and/or bacterial inoculations (DOC) is produced when oil is extracted (Rodríguez et al.,1999; Vassilev et al., from olive fruit. On the other hand, sugar 1998, 2006). Moreover, it has been beet waste (SB) is a lignocellulosic reported that mycorrhizal plants benefited residue produced during sugar processing, from P solubilized from RP by A. niger which is abundant and cheap. However, (Vassilev et al., 2002) by the use of such material, because of its isotopic 32P dilution technique.

355 J. soil sci. plant nutr. 10 (3): 354 – 372 (2010)

The aim of the present review paper is to influence on plant health and productivity summarize the achievements reached by (Jeffries et al., 2003; Requena et al., the application of these two organic 2001). On the other hand, the quality and amendments together with the inoculation productivity of degraded soils can be of beneficial soil microorganisms, such as improved by organic amendments arbuscular mycorrhiza fungi in improving (Roldán et al., 1996a, b, c). soil properties and plant performance in semiarid and/or heavy metal contaminated Influence of the application of organic sites. amendments and AMF on plant growth

The viability of applying microbially USE OF BIOTRANSFORMED treated amendments (DOC or SB) it has AGROWASTES AND AMF FOR been demonstrated in order to improve the RECLAMATION OF DESERTIFIED growth and (NPK) nutrient status of the AREAS different target shrub species, Dorycnium pentaphylum (Medina et al., 2004a, b), Stressed arid areas may be the result of Cistus albidus L. (Alguacil et al., 2003b), progressive degradation of vegetation Juniperus oxycedrus (Caravaca 2006c) cover (species diversity) and soil quality, and Quercus coccifera (Caravaca et al., both of which involve soil structure, 2005b). All these plant species belong to nutrient availability and microbial activity the natural succession in certain plant (Barea and Jeffries, 1995). communities of semiarid Mediterranean The establishment of a plant cover ecosystems in the southeast of Spain. based on autochthonous plant species They are well adapted to drought constitutes the most effective strategy for conditions and form symbiosis with reclaiming degraded lands in semiarid mycorrhizal fungi. Thus, they represent Mediterranean areas. Revegetation target species selected for revegetation of programmes based on planting drought the semiarid Mediterranean area. The tolerant, native shrub species would assist effectiveness of these amendments relies in the conservation of biodiversity and on the improvement of soil fertility. help to prevent the process of erosion and Mineralization of lignocellulosic desertification (Caravaca et al., 2003b). agrowastes by microbial processes and The vegetation cover helps avoid soil simultaneous solubilisation of inorganic losses, together with improving soil insoluble phosphates provides the plants physical properties. However, in these with an organic amendment rich in semiarid areas the low productivity and mineral nutrients, especially available P fertility of the soil and the severe water (when RP is applied in the fermentation deficits seriously limit plant growth. To process). It has been shown that D. carry out successful re-afforestation, it is pentaphylum plants grown in A. niger- necessary to improve soil quality and the treated SB or DOC amended soils had ability of the plant species to resist the higher (N, P, K) nutrient contents in their semiarid environment, (Alguacil et al., tissues than non-amended control plants 2003b, Caravaca et al., 2005a).The (Medina et al., 2004a, b). Caravaca et al. establishment of a mycorrhizal symbiosis (2004a) found a correlation between the can improve the performance of the increase in N, P, K content in shoots of D. shrubs. Mycorrhiza represent ecological pentaphylum grown in A. niger-treated SB key factors governing the cycles of major amended soil and the increase in total N, plant nutrients and have significant available P and extractable K content in

356 Interactive effect of AM fungi organic amendments, Medina y Azcón

this amended soil. The greatest increase (Medina et al., 2007) which can be related was found in available P, confirming the to an increase in nutrient uptake and plant solubilisation of RP by A. niger during the growth. fermentation of the agrowaste. Similar to One important factor in revegetation these results, Alguacil et al. (2008a, b) programmes is the improvement in the found an improvement in the available development of the root system. Roots nutrient supply for the plant in soil contribute to the establishment of plants, amended with A. niger-treated DOC. and consequently, to the protection of the The amendment added to the soil can soil against erosion. Medina et al. (2004a, be used as C and energy source for soil b; 2010b) found that D. pentaphyllum root microorganisms. This could lead to an biomass was increased by both enhancement of soil enzyme activities inoculation with AM fungi and and, as a result of this, to an increase in emendation of the soil with the fermented the availability of nutrients to plants (see DOC, and the highest values were section 2.4.2). Alguacil et al. (2008a, reached in plants receiving the dual 2003b) reported an increase in enzyme treatment. Moreover, it has been found activities in soil amended with A. niger- that shoot and root biomass were higher treated SB waste and A. niger-treated in mycorrhizal plants grown in A. niger- DOC, respectively. This increase was treated SB amended soil, and no statistical associated with a rise in macronutrients significant difference was recorded in (NPK) in this amended soil. these plants grown under water stress Moreover, these amendments have or non-stress conditions (unpublished been shown to be highly efficient in data). association with arbuscular mycorrhiza. Alguacil et al. (2008a), showed a positive Influence of the application of organic interaction between the amendment A. amendments and AMF on plant niger-treated DOC and G. mosseae in biochemical parameters related to terms of plant growth. Similarly, drought tolerance Caravaca et al. (2005b, c; 2006c), reported that the combined treatments, Desertified areas are characterized by a involving mycorrhizal inoculation with G. severe climate with little and irregular intraradices and the addition of fermented precipitation and frequent drought. DOC increased the growth of J. Therefore, the main objective in oxycedrus to a higher extent than each restoration purposes is the improvement treatment applied separately. Medina et of plant drought tolerance. The osmolytes al. (2004a, b) found that in D. proline and total sugars are considered as pentaphylum N and P adquisition was indexes of drought avoidance and higher in mycorrhizal plants grown in A. antioxidative plant defence response niger-treated SB or DOC amended soil. (Ruiz-Lozano et al., 1995). Medina et al. While organic amendments can improve (2010a) compared the response of P nutrient supply, inoculation with fertilized Trifolium repens to that of mycorrhizal fungi can enhance plant mycorrhizal A. niger-treated DOC nutrient uptake. Furthermore, it has amended ones under water stress been found that the amendments conditions. Interestingly mycorrhizal increased nodule number Medina et al. plants grown in the amended soil reached (2004b), percentage of micorrhizal the highest proline and sugar contents and root colonization (Medina et al., were the least-damaged (in terms of plant 2004a), and hyphal length in soil growth) by drought.

357 J. soil sci. plant nutr. 10 (3): 354 – 372 (2010)

Water stress was less compensated in P- favours the establishment or viability of a fertilized than in A. niger-treated DOC stable plant cover (Alguacil et al., 2008a, amended plants. The mechanisms b). Soil structure has a prevailing role in underlining these results may be related to soil infiltration and biogeochemical the coordination effects achieved by the processes. Therefore, improved soil improved physical characteristics of structure means increased water retention, composted soil and the ability of these nutrient uptake, drainage, aeration and fungi to acquire water (Porcel et al., 2006; root growth. Ruíz-Lozano and Azcón, 1996). Nitrate reductase (NR) has been Influence of the application of organic proposed as an index for assaying the amendments and AMF on symbiotic effectiveness of AM fungi-host plant parameters combinations for mitigation of water- deficit stress (Caravaca et al., 2003a, Medina et al. (2004a, b; 2010b) found 2005c). This enzyme, responsible for that in the degraded soil from semiarid nitrate assimilation, is highly sensitive to areas, natural rhizobial and arbuscular metabolic and physiological plant status mycorrhizal populations were very low and it is induced by high nitrate supply and inefficient in promoting the growth of (Kandlbinder et al., 2000). A synergistic legume D. pentaphylum. The inoculation effect between the fermented DOC with authoctonous AM fungi was required amendment and AM fungi in increasing to get colonized plants. Moreover, the NR activity in roots of J. Oxycedrus and addition of A.niger-treated SB and or D. pentaphylum has been reported A.niger-treated DOC to the soil enhanced (Caravaca et al., 2006a, c, Caravaca et al., the formation of such symbiotic structures 2004b). The higher nutrient supply arising (Medina et al., 2004b). from the fermented amendment can Similarly, Caravaca et al. (2005a), explain the increase in NR activity reported no natural ectomycorrhizal recorder in those experiments. Moreover, colonization in non-inoculated C.albidus nitrate is the preferential N source of AM and Q. coccifera one year after being fungi associated with plants grown in planted in the field. neutral to alkaline soils (Azcón et al., Caravaca et al. (2005a) found the 2001). The reduction process is highly highest levels of Scleroderma verrucosum energy-demanding and hence frequently root colonization in inoculated seedlings limited by P availability. Some authors of C.albidus and Q. coccifera grown in have indicated that the increase in NR of soil amended with SB waste, rock mycorrhizal plants with respect to non- phosphate and A. niger added directly into mycorrhizal ones can be related to the the planting hole. On the contrary, phosphate requirements of this enzyme Alguacil et al. (2003b) found no (Ruíz-Lozano and Azcón, 1996). In these significant difference in Pisolithus experiments, the combining effect from tinctorius root colonization between the NR activation through nitrate addition C.albidus grown in A.niger-treated via organic amendment with improved P amended or in non amended soil. These uptake through mycorrhizal fungi enhance contrasting results could be due to the the N assimilation of the plant. different ectomycorrhizal strains used for Moreover, the benefits of organic the experiments. amendments are also due to the D. pentaphylum is a woody legume improvement of the physical very useful for revegetation of semiarid characteristics of the soil, which in turn sites because of its ability to develop

360358 Interactive effect of AM fungi organic amendments, Medina y Azcón

symbiotic associations with both rhizobial products on a number of soil properties bacteria and arbuscular mycorrhizal fungi. such as soil structure, soil enzyme activity Medina et al. (2004a, b) reported that the and soil microbial community have been percentage of AM root colonization in demonstrated. AM-inoculated D. pentaphylum was increased by the amendments A. niger- Physical and physico-chemical soil treated DOC and SB. Moreover, these parameters plants reached the highest nodule formation. The important role of AM As we explained in section 2.1, the fungi in increasing nodule number is a effectiveness of fermented agrowaste with well-documented subject (Barea et al., respect to stimulation of plant growth 2002c). Besides, the addition of the could be due to an improvement in the mineralized amendments represent a available nutrient supply in soil. During source of C and nutrients that provide the process of A. niger fermentation, the energy sources for the growth and RP solubilizes increasing the level of bio- metabolic activity of soil heterotrophic available P in the amendment. Thus, the bacteria such as Rhizobium. use of the fermented improves potentially Organic matter has been shown to limiting soil nutrients, such as N and P. increase mycorrhizal fungal growth (Joner However, the benefits of organic and Jakobsen, 1995a, b). Medina et al. amendments are also due to the (2004a, b) reported an increase on the improvement of the physical soil number and biodiversity of AM fungi and characteristics. Soil structure largely spores populations in soils amended with determines soil quality and fertility, A.niger-treated DOC and SB. Moreover, which, in turn, favours the establishment A. niger-treated SB has been shown to and viability of a stable plant cover increase hyphal length density and (Caravaca et al., 2002). Structural soil activity of AM fungi grown in the hyphal stability of a semiarid soil has been shown compartment (HC) of compartmentalized to be significantly improved by the pots (Medina et al., 2007). Changes in addition of A. niger-SB waste, on soil microbial community structure average, by about 79% compared with induced by the amendments has been non-amended soils (Alguacil et al., 2003a, suggested to be involved in those findings b). Moreover, these authors found a (Medina et al., 2007). In addition, the reduction in bulk density of that soil when chemical composition of the amendments the amendment was added. The organic has been shown to have a strong influence materials are less dense than the mineral on AM hyphal growth (Medina et al., fraction of soils. In consequence, their unpublished data; Ravnskov et al., 1999). application reduces soil bulk density and On the contrary Medina et al., 2007 leads to an increase in soil porosity reported no AM fungal growth in the HC improving its structural stability (Pagliai soil amended with the non-fermented SB. et al., 1981). Aggregate stability is an important Influence of the application of organic criterion of a healthy, managed ecosystem amendments and AMF on soil (Miller et al., 1992). The mechanisms properties involved in aggregate stabilization are based on the enmeshment of soil particles During the course of the studies with A. by hyphae and roots, and on the exudation niger-treated agrowastes, the multiple of polysaccharides (Bearden and Petersen, beneficial effects of the biotechnological 2000). According to Roldán et al. (1994)

359 J. soil sci. plant nutr. 10 (3): 354 – 372 (2010)

the binding effect of roots and hyphae is important mechanism affecting available long-lived, while that of polysaccharides water in soil is aggregate stabilization. is transient because they are decomposed This mechanism could explain the rapidly by microbes. enhanced development of A. niger-treated Medina et al., 2004b found a DOC amended mycorrhizal plants under significant increase of aggregate stability drought-stress conditions (Medina et al., in the rhizosphere of D. pentaphylum by 2010b). the addition of A. niger-treated SB and DOC. This increase was correlated with Soil biological and biochemical an increase in water-soluble C and water parameters –soluble carbohydrates. The water-soluble organic matter fraction consists of a It is widely accepted that soil enzyme heterogeneous mixture of components of activities are highly sensitive biochemical varying molecular weight, such as mono- parameters indicating perturbations and polysaccharides, polyphenols, caused by soil treatments (Naseby and proteins and low molecular weight Lynch, 1997). In the degraded soil organic acids. Many authors suggest that belonging to semiarid sites, the activity of the production of polysaccharides is microbiota is low because of the lack of responsible for improvement aggregates suitable organic substrates (Medina et al., stability since they act as cementing 2004b). Nevertheless, the addition to the agents (Jastrow et al., 1998). soil of the organic amendments, A. niger- Moreover, this fraction can be used as treated SB and/or DOC, significantly carbon and energy sources by soil increased soil microbial activity as microorganisms, such as bacteria and reflected by an increase in dehydrogenase fungi, which are mainly responsible for activity (Medina et al., 2004b). the formation of aggregates larger than Measurement of soil hydrolases 0.2 mm (Roldán et al., 1994). In fact, an provides an early indication of changes in increase in microbial activity has been soil fertility, since they are involved in the reported by the addition of these mineralization of important nutrient amendments (Alguacil et al., 2003a, b; elements such as N, P, and C. Thus, the Medina et al., 2004a, b). Microorganisms increase of enzymatic activities in soil is participate mechanically (union by involved in an increase in the nutrient hyphae) or by the excretion of availability to plants, which, in turn, have polysaccharides into the medium. On the a positive influence on soil fertility other hand, A. niger-treated SB has been (García et al., 1997). Many researchers shown to increase root development and have found that soil hydrolase activities stimulate hyphal growth (Medina et al., are enhanced by the addition of organic 2007, 2010b). Both roots and associated materials (Garcia et al., 1999) and can hyphae may form a three-dimensional remain active in an extracellular soil network that enmeshes fine particles of environment. Alguacil et al. (2003b) soil into aggregates. In addition, the found that urease, protease-BAA, acid organic C released by roots promotes a phosphatase and glucosidase activities dense microbial community in the were higher in soil amended with the immediate environment of the root. fermented SB residue than in the non- Moreover, it is known that glomalin, amended soil. Similarly, Medina et al. produced by AM fungi, acts as an (2004a, b), reported an increase in all insoluble glue to stabilise aggregates these enzymatic activities when soil was (Wright and Anderson, 2000). An amended with A. niger-treated DOC. The

360 Interactive effect of AM fungi organic amendments, Medina y Azcón

increases observed in enzyme activities strategic for reclaiming contaminated may be mainly related to reactivation of areas. In polluted zones, the decreases in the rhizosphere microbial population as a the characteristic biodiversity and the consequence of the addition of the degradation of the natural ecosystem have fermented product in combination or not been proved. with mycorrhiza, which indicates a In general, vegetation cover decreases rehabilitation of the degraded soil the danger of HM dispersal by water and (Medina et al., 2004a; Naseby and Lynch, wind erosion and, in this context, the 1997) establishment of plant species is recommended for reclamation of HM- contaminated soils (Jeffries et al., 2003). BIOTRANSFORMED AGROWASTE The application of AM fungus and A. AND AM USE FOR REMEDIATION niger-treated SB amendment can be OF HEAVY METALS CONTA- regarded as a successful biotechnological MINATED SOILS tool for decontaminating a polluted soil, for the recovery of polluted soils and as Heavy metals (HM) have caused serious an important strategy for bioremediation environmental problems and may enter purposes. Soil microorganisms may play the ecosystem through mining, an important role in nutrient cycling in atmosphere deposition and agrochemicals soils amended with organic materials and as pesticides, fertilizer and anthropogenic their activity can also alleviate the metal activities (Liu et al., 1997). Soil toxicity in the environment. contamination by heavy metals is an Measurements of this microbial important problem in industrialized areas, activity in a treated and/or inoculated soil and HMs can cause detrimental effects on provide information about the effect of ecosystems in soil. High concentrations of AM symbiosis, treated agrowaste or both, metals, such as zinc, nickel, mercury, on some biochemical values in the cadmium, or copper, cause environmental contaminated rhizosphere soil (Medina et pollution because they have a strong al., 2006; 2010b) persistence. Bioremediation has been defined as The use of plants for bioremediation the use of microorganisms for the purpose is based on their ability to treatment of soil pollution (Leyval et al., tolerate HMs. 2002) and can be applied in association Nevertheless, plants are more with different strategies of dependent on microbial activity and phytoremediation. microorganisms are able to enhance their Phytoremediation, the use of plants in metabolic activity to combat stress in order to remove toxic metals from soil, is polluted areas (Killham and Firestone, emerging as a potential strategy for cost- 1983; Moreno-Ortego et al., 1999). effective and environmentally friendly Depending on the severity of the remediation of contaminated soils (Glass, environmental damage, the diversity and 2000). activity of the soil biota are increased to a The deterioration of biological certain threshold value preceding the final properties of metal contaminated soils is function loss. in part due to their progressive decrease in There is a duality in plant tolerance to organic matter content (Turnau et al., pollutants and its response to pollutant 2002). Thus, the application of stress, but the establishment of plant appropriate levels and kinds of organic species is considered as an effective amendments may be a valid choice to

361 J. soil sci. plant nutr. 10 (3): 354 – 372 (2010)

improve soil characteristics (Martens and Influence of the application of organic Frankenberger, 1992a, b). amendments and AMF on Cu polluted For phytoremediation purpose we soils need a better understanding of the interactions between plant and soil Copper is an essential element controlling microorganisms, particularly beneficial diverse biochemical and regulatory events microbes as arbuscular mycorrhizal (AM) for plant and fungal metabolism, but it fungi and plant growth promoting can be toxic to plant and AM fungi at rhizobacteria (PGPR) (Vivas et al., 2003a, high concentration by interfering b, c, d; Whitfield et al., 2004a, b). respiratory processes and protein Many rhizosphere colonizing bacteria synthesis. The extraradical mycelium of produce typical metabolites, such as AM colonized plants may operate binding siderophores, biosurfactants or organic processes and have an important role in acids that stimulate plant growth (Glick, plant tolerance to Cu. The extraradical 1995) and also may reduce metal mycelium may exclude selectively toxic availability in the medium. Until now, elements by intracellular precipitation y mycorrhizal fungi and bacteria have been extracellular glycoprotein or chitin- found in HM contaminated soils which containing cell walls (Zhou, 1999). are an indication on AM fungal and Metal tolerant plant species bacterial tolerance. (metallophytes) can grow on metal HMs exert a negative influence on polluted soils (Whiting et al., 2002), and soil’s microorganisms (Biró et al., 2007; microorganisms as AMF resistant to Khan, 2005) which are considered as an copper would form the dominant index to test soil pollution, because HMs populations (Bååth, 1989; Ferrol et al., have a deleterious influence on cell 2009). functioning and consequently on soil In a Cu contaminated soil with and microbial community (Azcón et al., without treated agro-wastes, the 2009b). alleviating effect of the inoculation of an Root exudates have a variety of roles autochthonous Cu-adapted arbuscular (Marschner, 1995) including that of metal mycorrhizal inoculum (GA) compared chelators that may reduce plant uptake of with a strain from collection of Glomus certain metals. The range of compounds claroideum (GC) on a metallophyte plant exuded is wide and could play a role in was evaluated. In this study, the negative plant metal tolerance (Hall, 2002). These effect of increasing Cu concentrations on compounds are greater in the rhizosphere mycorrhizal development was only of plants with a more developed root observed in GC-colonized plants but not system as occurred with A. niger-treated in those colonized by the autochthonous SB and AM inoculated plants (Rodríguez Cu adapted GA fungi. The main et al., 1999). Thus, the role of such characteristics of GA inoculum may be treatments alleviating HM toxicity in ascribed to the ability to maintain high plant possibly via exudates chelation may colonizing capacity under the highest Cu be important but it was not checked. An levels (500 ppm) assayed. Cu translocated indication related to this is the fact that from root to shoot under the highest Cu the metabolic (enzymatic) activity of level was highly reduced in GA colonized particular groups of rhizosphere plants (by 2.7 times) versus control. In microorganisms involved in nutrient addition, GA colonized plants cycling increased with the specific accumulated less Cu (80% in shoot and amendments applied to contaminated soil. 29% in roots) than non-AM plants.

362 Interactive effect of AM fungi organic amendments, Medina y Azcón

Concomitantly, as an index of Cu An autochthonous, Zn adapted, strain of tolerance, the antioxidant plant activity Glomus mosseae, was used and its was measured. The lowest values of interaction with amendments was glutathione reductase (GR), superoxide assessed on Trifolium repens growth, dismutase (SOD), ascorbate peroxidase nutrition and symbiotic (AM colonization (APX) and calatase (CAT) were found in and nodulation) values using a Zn GA colonized plants. These results (under contaminated soil (Medina et al., 2006). revision) suggest that to successfully The impact of such treatment on plant N, remediated Cu polluted sites it is essential P and Zn concentration was particularly to colonize metallophyte plants with evident when associated with G. mosseae. efficient and adapted AM fungi. The A. Total growth (four harvests) of AM plants niger-treated agro-waste application growing in SB + RP-treated soil was resulted to be a very important component about 28 times more than in non- for the less Cu tolerant non AM or GC mycorrhizal control plants. The effect of plant to survive under the highest Cu G. mosseae on shoot biomass ranged from concentration. 86% (without SB) to 1192% (with SB). There is a distinct efficiency of AM This growth improvement was the fungi for maintaining metal homeostasis consequence of increased N, P and K and buffering metal stress in plant itself nutrition and decreased Zn acquisition. through evolution of various Nevertheless, as consequence of an physiological and molecular mechanisms. enhanced plant biomass, Zn In fact, chelation of toxic metals with phytoextraction by these plants increased exudates, intracellular peptides by 1832% over untreated ones. (glutathiones, metallothioneins, HSPs) Nevertheless Whitfield et al.,( 2004a, b) and other non-proteinaceous compounds did not evidence that AM fungi reduced (polyphosphate granules), production of plant uptake of heavy metals, but antioxidant enzymes to reduced oxidative increased Zn uptake, while results from stress and transport proteins for metal Zhu et al. (2001), in agreement with those influx and efflux may be the mechanisms reported here, indicate that AM involved. They may be recommended as colonization exerted protective effect potential biotechnological tools for against plant Zn accumulation in a range remediation and reclamation of metal from 0 to 400 mg Zn kg-1. These authors, contaminated habitats. as it was observed in Medina et al. (2006) study, reported that mycorrhizal effect Influence of the application of organic cannot be explained simply by tissue amendments and AMF on Zn polluted dilution. In fact, Burleigh et al. (2002) soils recently reported that the expression of MtZIP2 gene (a plant Zn transporter) was The detrimental effect of Zn excess in the down-regulated in the roots of ecosystem is due to its long persistence. mycorrhizal plants and associated with a Soil application of amendments has reduced Zn level within the host plant resulted in a practical method because of tissues. its advantages in increasing soil fertility in González-Guerrero et al. (2005) heavy metal-degraded soil. Metabolizable observed increased transcript levels of a C compounds from this SB amendment putative Zn transporter gene (GintZnT1) must be applied to the microbes to ensure of the CDF family in the mycelium of their growth and activity. Glomus intraradices under Zn exposure,

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indicating a possible role of this gene compartmentalized growth system, product in Zn homeostasis and protection consisting of a root compartment (RC) against Zn stress. and two hyphal compartments (HCs). The The amendments and AM influence of Aspergillus niger-treated colonization had the expected effect, DOC on intraradical and extraradical AM reducing metal concentration in shoot fungi development was investigated in biomass that allowed an enhancement of this experimental system. In addition, the plant growth and, consequently, a higher viability and infectivity of the detached phytoextraction of this metal from extraradical mycelium in plants, contaminated soil. Thus, these treatments designated as receptor plants, grown in can be considered as successful the HC after removal of the RC was biotechnological tools for the recovery of studied. Results showed that AM Zn polluted soils. intraradical and extraradical development in the RC was not eliminated by Cd Influence of organic amendment contamination, which suggests a certain application and AMF on Cd polluted level of Cd tolerance as reported in a soils previous work (Vivas et al., 2006a). Furthermore, the arbuscular richness (a, Cadmium (Cd) is a heavy metal (HM) A) was increased in plants grown in A. dispersed in natural and agricultural niger-treated DOC-amended soils. This environments mainly through human parameter shows the functioning of the activities such as mining, refining, AM symbiosis, as it is considered to be municipal waste incinerators and fossil the interactive structural link between the fuel combustion sources. In comparison plant and the fungus. Plants growing in A. with other HMs, Cd solubility in soils niger-treated DOC-amended soils seem to and its toxicity to plants and animals are be more Cd-tolerant than in the absence high (Medina et al., 2005; Seregin and of A. niger-treated DOC. Probably, the A. Ivanov, 2001). niger-treated DOC effect with respect to Medina et al. (2005) reported a increasing the arbuscule content was an positive interaction between the important part of the tolerance amendment A. niger-treated SB and G. mechanism. mosseae for improving Trifolium repens Both the amendment and the AM growth and nutrition in a Cd contaminated fungus increased shoot and root biomass soil. and nodulation in both the non- Studies done by our research group contaminated and Cd contaminated have shown the positive effect of the soils. application of A. niger- treated DOC to a The applied treatments also improved desertified Mediterranean soil, in terms of nodule formation by an inoculated improving both soil characteristics and Rhizobium, that was highly depressed in plant growth (Medina et al., 2004a, b). this Cd-contaminated soil. Medina et al. (2010b) also tested the The absence of nodules in non- effectiveness of this amendment in mycorrhizal and non-amended plants interaction with AM fungi for reclamation grown in Cd-contaminated soil indicates of Cd-contaminated soils since in non- that the inoculated Rhizobium was highly contaminated soils, the tested effects can sensitive to the high concentration of be explained by an increase in nutrient available Cd in the soil (Biró et al., 1995) availability. For this study in Cd- and that it was not able to colonise roots contaminated soil was used a in non-amended soil. However, the

364 Interactive effect of AM fungi organic amendments, Medina y Azcón

detrimental Cd effect on nodule formation fact, in polluted zones decrease the typical was compensated by microbially-treated biodiversity of such area resulting in the DOC or AM colonisation. degradation of the natural ecosystem. The positive interaction between the Such unfavourable conditions for plant microbiologically treated DOC and the growth require applying methods for AM fungus resulted in the highest plant improving nutrient balance, microbial yield, which can be explained by activity and soil quality (García et al., enhanced nutrient acquisition and 1999). In polluted multicontaminated arbuscular richness as well as by Cd areas, plants are more dependent on immobilisation in amended soils. microbial activity (Moreno-Ortego et al., However, A. niger-treated DOC had 1999) since plant establishment and no effect on the extraradical mycorrhizal further development is seriously limited mycelium development. Although Cd in there. (Baker et al., 1995; Puppi et al., decreased, AM hyphal length density, 1994) symbiotic infectivity was similar in However, no clear information is receptor plants grown in non- available about the effect on soil contaminated and contaminated soil, thus biological characteristics of the confirming the AM fungal inoculum application of treated agrowastes potential. which include alternative Joner and Leyval (1997) demonstrated carbohydrate sources affecting the that hyphae are less sensitive than roots environmental structure and function of with respect to heavy metal toxicity. microbial community compared to 3- Similarly, Janouskova and Vosatka conventional PO 4 fertilization used in (2005) observed that carrot roots grown in parallel. monoxenic cultures were more sensitive The microbial inoculants and to Cd than extraradical Glomus amendment application favoured plant intraradices and Gigaspora margarita growth and the phytoextraction process in mycelium. the multicontaminated soil. The Environmental conditions in Cd inoculation with AM fungi in A. niger contaminated soils (having low levels of treated SB amended soil increased plant 3- -1 nutrients and organic matter and high growth similarly to PO4 (100 μg kg ) toxic contaminant levels) are addition, and both treatments matched in unfavourable for the growth and activity P acquisition. However, bacterial of the indigenous microorganisms. A. biodiversity (estimated by denaturing niger-treated DOC contains high levels of gradient gel electrophoresis of amplified available carbon and nutrients and the 16S rDNA sequences) was more microbial fermentation by A. niger stimulated by the presence of the AM 3- decreases the phenolic compound fungus than by PO 4 fertilization. toxicity, as Vassilev et al. (2006) The treated SB amendment plus AM reported. inoculation increased the microbial diversity by 233% and also changed Influence of organic amendment (by 215%) the structure of the application and AMF on multi- bacterial community (Azcón et al., contaminated soil: plant and biological 2009a, b). soil properties In general, the highest enzymatic activities were related to the stimulating The exploitation of natural resources effect of root exudates. Nevertheless, the causes important ecological problems. In quality of such exudates seems more

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important than their quantity, according to Following a similar trend, dehydrogenase some results. and β-glucosidase activities reached the Soil quality is related to the highest values in A. niger- treated SB + rhizosphere microbial groups. but few AM treated soils. 3- studies have considered the dual effect of An interesting result is that PO 4 the mycorrhizal fungi colonization and fertilization and single AM inoculation treated SB amendment on the structure of (used in parallel) similarly promoted plant the bacterial communities and diversity biomass, but only AM inoculation (Maliszewska-Kordybach and Smreczak, increased microbial diversity in the 2003; Zhang et al., 2006). Enzymatic presence of SB amendment (Azcón et al., activities are considered as major factors 2009b). contributing to overall soil microbial A. niger-treated SB amendment may activity and soil quality (Nannipieri, be a suitable tool for increasing and 1994). The increase of enzymatic changing the bacterial community in activities in soils is involved in an rhizosphere of the multicontaminated soil. increase in the availability of nutrients to Results show that the application of the plants, which, in turn, have a positive treated SB agrowaste not only favored influence on soil fertility (García et al., plant development, but also the microbial 1997). properties of this multicontaminated soil, Organic matter from the such as biodiversity and increased mineralization of A. niger-treated SB can dominance index. be used as C and energy sources for activities of soil microorganisms (Bowen and Rovira, 1999). This beneficial effect CONCLUSION was confirmed by the enhancement of the dehydrogenase, β-glucosidase and phosphatase values which are indicative Results reported here are indicative of the of greater microbial activities (Alef et al., positive effect of the amendments and 1995; Ceccanti and García, 1994; García beneficial microorganisms, such as AM et al., 1997). fungi, on the microbial soil status and the Values of β-glucosidase activity that relevance of such biotechnological indicate carbohydrates transformation, management on bioremediation in single showed that SBW + AM fungus increased or multi-contaminated soils as well as on this hydrolytic activity which is important revegetation of semi-arid sites. as energy source for rhizospheric Mechanisms that govern the interaction in microorganisms. the microbial processes in these semi-arid In these polluted zones hydrolase and/or contaminated soils may be activities were increased by the considered in future proposals for application of organic products. Soil reclamation strategies of degraded soils. It enzymatic activities, which are indicative is interesting to understand the main of biological performance in the microbial activities and mechanisms rhizosphere, were also improved by involved in these positive effects. applied treatments. We tested that According to these results, phosphatase activity in the rhizosphere management practices involving organic was 1257% higher by the application of amendments and microbial (AM) A. niger-treated SB plus AM fungus than inoculation seem a promising option for in control non-treated rhizosphere soil. re-afforestation of threatened sites, since

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they positively affect soil quality and Alguacil, M. M., Hernández, J.A., Caravaca, fertility, the formation of symbiotic F., Portillo, B., Roldán, A. 2003b. Antioxidant structures (nodulation and AM enzyme activities in shoots from three mycorrhizal shrub species afforested in a colonization) which improve plant degraded semi-arid soil. Physiol. Plant. 118, 562- nutrition and growth as well as plant 570. tolerance to stress conditions caused by Alguacil, M. M., Lumini, E., Roldán, A., HM and/or drought. Salinas-García, J. R., Bonfante, P., Bianciotto, Application of genetic engineering V. 2008b. The impact of tillage practices on could prove an asset for efficiency arbuscular mycorrhizal fungal diversity in increment and better adaptability of subtropical crops. Ecol. Appl. 18, 527-536. fungal symbionts through alterations in Augé, R. M. 2004. Arbuscular mycorrhizae and molecular pathways leading to high metal soil/plant water relations. Can. J. Soil Sci. 84, tolerance and/or detoxification, besides 373-381. incorporating the promising genes Azcón, R., Medina, A., Roldán, A., Biró, B., responsible for overproduction of metal Vivas, A. 2009a. Significance of treated chelating agents for enhanced metal agrowaste residue and autochthonous inoculates binding at target sites. (Arbuscular mycorrhizal fungi and Bacillus cereus) on bacterial community structure and Therefore, it is of great importance to phytoextraction to remediate heavy metals inoculate metal hyper-accumulator plants contaminated soils. Chemosphere 75, 327-334. with efficient and effective mycorrhizal Azcón, R., Perálvarez, M. C., Biró, B., Roldán, fungal strains better adapted to a A., Ruíz-Lozano, J.M. 2009b. Antioxidant particular set of conditions and/or host activities and metal acquisition in mycorrhizal plant to expedite the process of metal plants growing in a heavy-metal remediation and successful restoration of multicontaminated soil amended with treated degraded ecosystems. lignocellulosic agrowaste. Appl. Soil Ecol. 41, 168-177. Azcón, R., Ruíz-Lozano, J. M., Rodríguez, R. 2001. Differential contribution of arbuscular REFERENCES mycorrhizal fungi to plant nitrate uptake (N15) under increasing N supply to the soil. Can. J. Bot. 79, 1175-1180. Alef, K., Nannipieri, P., Trasar-Cepeda, C. Bååth, E. 1989. Effects of heavy metals in soil 1995. Phosphatase activity. In: K. Alef, P. on microbial proceses and populations. Water Air Nannipieri (eds). Methods in Applied Soil Soil Pollut. 47, 335-379. Microbiology and Biochemistry. Academic Press, London, pp: 335-336. Baker, A., Sprent, J. I., Wilson, J. 1995. Effects of sodium chloride and mycorrhizal infection on Alguacil, M. M., Caravaca, F., Azcón, R., the growth and nitrogen fixation of Prosopis Pera, J., Díaz, G., Roldán, A. 2003a. juliflora Symbiosis 19, 39-51. Improvements in soil quality and performance of mycorrhizal Cistus albidus L. seedlings resulting Barea, J. M., Azcón, R., Azcón-Aguilar, C. from addition of microbially treated sugar beet 2002a. Mycorrhizosphere interactions to improve residue to a degraded semiarid Mediterranean plant fitness and soil quality. Anton. Leeuw. Int. soil. Soil Use Manage. 19, 277-283. J. G. 81, 343-351. Alguacil, M. M., Caravaca, F., Azcón, R., Barea, J. M., Gryndler, M., Lemanceau, P., Roldán, A. 2008a. Changes in biological activity Schüepp, H., Azcón, R. 2002b. The rhizosphere of a degraded Mediterranean soil after using of mycorrhizal plants. In: S. Gianinazzi, H. microbially-treated dry olive cake as a biosolid Schüepp, J. M. Barea, K. Haselwandter (eds). amendment and arbuscular mycorrhizal fungi. Mycorrhiza Technology in Agriculture: from Eur. J. Soil Biol. 44, 347-354. Genes to Bioproducts. Birkhäuser Verlag, Basel, Switzerland, pp: 1-18.

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