The effects of the arbuscular mycorrhizal fungus Glomus deserticola ...

SYMBIOSIS (2009) 47, 133–140

©2009 Balaban, Philadelphia/Rehovot

ISSN 0334-5114

The effects of the arbuscular mycorrhizal fungus Glomus deserticola on growth of tomato plants grown in the presence of olive mill residues modified by treatment with saprophytic fungi E. Aranda1, I. Sampedro1, R. Díaz1, M. García-Sánchez1, C.A. Arriagada2, J.A. Ocampo1, and I. García-Romera1* 1

Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Prof. Albareda 1, Apdo. 419, 18008 Granada, Spain, Tel. +34-58-181600 (ext. 274), Fax. +34-58-129600, Email. [email protected]; 2 Universidad de la Frontera, Departamento de Ciencias Forestales, Av Fco Salazar 01145, Temuco, Chile (Received May 29, 2008; Accepted September 4, 2008) Abstract Olive oil extraction generates large amounts of olive mill residues (DOR) which may be used as fertilizer. The influence of arbuscular mycorrhizal (AM) on the phytotoxicity of dry olive residue (DOR) transformed with saprophytic fungi was studied. Aqueous extraction of DOR gave an (ADOR) fraction and an exhausted (SDOR) fraction, both of which had less phytotoxicity for tomato than the original DOR. The saprophytic fungi Trametes versicolor and Pycnoporus cinnabarinus further decreased the phytotoxicity of ADOR and SDOR on tomato. The decrease of phenols concentration and the differences in the level of laccase activity caused by these fungi suggest did not account fully for the reduced phytoxicity but the fact that the higher hydrolytic enzyme activity of P. cinnabarinus, paralleled the decrease of phytotoxicity, indicates that these enzymes seem to be involved. The AM fungus Glomus deserticola increased or exacerbated the beneficial effect of SDOR incubated with saprophytic fungi, in terms of dry weight of tomato plants. The percentage of root length colonized by G. deserticola strongly decreased in presence of DOR, but the level of mycorrhization was higher in presence of ADOR or SDOR. Our results suggest that the combination of aqueous extraction and incubation with saprophytic fungi will open the way for the use of olive oil extraction residues as organic amendment in agricultural soils. Keywords:

Arbuscular mycorrhizal, biofertilizer, dry olive mill residue, phytotoxicity, saprophytic fungi

1. Introduction Most agricultural soils in the Mediterranean region contain small amounts of organic matter which is one factor limiting plant growth and production (Brunetti et al., 2005). The quality and productivity of soils could be improved by the addition of organic matter but available material is scarce (Caravaca et al., 2003). The use of agrowastes might solve this problem. The extraction of olive oil generates large amounts of olive mill residues (DOR) which can be used as fertilizer, having a high content of organic matter, plant nutrients and the almost no heavy metals (Paredes et al., 1999). Addition of this waste to poor agricultural soils has been proposed (Abu-Zreig and Al-Widyan, 2002), but olive mill residues are phytotoxic. Uncontrolled use of *The author to whom correspondence should be sent.

these residues may cause serious environmental problems with unforeseen effects on the soil-plant system (Vassilev et al., 1997). The toxicity of olive mill residues toxicity is complex and related to phenols and cell wall fragments (oligosaccharides and glycoproteins) (Martin et al., 2002; Aranda et al., 2004; Isidori et al., 2005). Some ligninolytic and hydrolytic enzymes have been shown to be involved in the detoxification of olive mill dry residue (Aranda et al., 2004; 2006). Therefore, there is a need for guidelines in the management and use of these wastes. Biological, chemical, physical and physico-chemical treatments have been proposed; physical pre-treatments considerably decrease its phytotoxicity (Ginos et al., 2006), but to date none of these approaches offers an adequate solution to the toxicity problems (Cermola et al., 2004). Saprophytic fungi, by breaking down cellulosic materials to simple sugars, can provides energy sources for microorganisms including arbuscular mycorrhizal (AM)

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fungi (Radford et al., 1996). These fungi can mobilize nutrients, degrade phytotoxic substances and enable plants to optimise the use of available nutrients (Dix and Webster, 1995; Fracchia et al., 2000). It has been shown that phytotoxicity of DOR can be reduced by incubating it with saprophytic fungi for 20 weeks (Sampedro et al., 2004a). A shorter period of incubation can reduce the toxic effect of water-soluble substances in DOR on plants grown under hydroponic conditions (Aranda et al., 2006). Olive residues also change the activity and population of the rhizosphere microorganisms and this can influence phytotoxicity (Moreno et al., 1987; Pérez et al., 1992; Iyamuremye and Dick, 1996). There is evidence that AM fungi help their host plants to grow under polluted condition (Shetty et al., 1994) by increasing their supply of nutrients (mainly P) to plant (Querejeta et al., 1998), improving soil aggregation in eroded soils (Tisdall, 1991), and reducing hydric stress (Boyle and Hellenbrand, 1991). The ability to decrease phytotoxicity of toxic residues seems to depend on which AM endophytes are present (Volante et al., 2005). Some AM fungi appear to increase the action of toxic substances in olive residues (Martín et al., 2002). Synergistic effects between saprophytic fungi and AM on plant root colonization may affect the ability of AM fungi to increase host tolerance to heavy metals in soils (Fracchia et al., 2004; Martínez et al., 2004; Arriagada et al., 2004; 2005; Vogel-Mikus et al., 2005). The aim of this study was to determine the role of AM fungi in modifying the toxic effects of the water soluble and exhausted fractions from olive mill residues, transformed by saprophytic fungi, on the growth of tomato plants as assessed by their dry weight.

2. Material and Methods Materials Dry olive residue (DOR) was collected from an “orujo” manufacturer (Sierra Sur S.A., Granada, Spain). The main characteristics of DOR determined by Sampedro et al. (2007) were as follows: total organic carbon (TCO), 58.5%; total nitrogen (TN), 1.87%; total phosphorus, 0.21%; lignin, 24.7%; cellulose, 18%; hemicellulose, 11.4%; total phenols, 2.65%; total lipids, 0.2%; ashes, 9.2%. The most abundant elements, in g kg–1 DOR were: K 30.5, Mg 3.8, Ca 13.6, Na 0.17, Fe 1.1, Cu 0.07, Zn 0.06, Mn 0.04. An aqueous extract from dry DOR was obtained by orbital-shaking this residue with distilled water in a proportion of 1:2 (w/v) for 8 h at room temperature. The suspension obtained was filtered through several layers of cheesecloth. The filtered (ADOR), and the solid residue obtained after the aqueous extraction (SDOR) was incubated or with saprophytic fungi. The toxicity of these

modified fractions was compared with control fractions that were not incubated with fungi. All fractions were tested to determine their phytotoxicity, phenol content and ligninolytic and hydrolytic activities. The two saprophytic fungi used were Trametes versicolor (A-1369) and Pycnoporus cinnabarinus (CECT 20448), obtained from the Culture Collection of Centro de Investigaciones Biológicas (CIB) of Madrid. Stock cultures were kept at 4ºC on 2% malt extract agar. The inoculum for ADOR was produced by growing the fungus under orbital shaking at 125 rpm and 28ºC on Czapek in the presence of 50% ADOR extracts for 15 days. The mycelium was collected by filtration, extensively washed with distilled water and homogenized. The fungi were grown in Erlenmeyer flask (250 ml) containing 70 ml of ADOR extract for 15 days at 28ºC with orbital shaking at 125 rpm. Each flask was inoculated with 0.45 g l–1 of each inoculum. The incubation process of SDOR was carried out in 500 ml Erlenmeyer flasks containing 125 g of steamsterilized SDOR, inoculated with four barley seeds previously colonized by the mycelium of each fungus for 14 days at 28ºC. Flasks were covered with sterile cotton plugs and incubated under stationary conditions at 28ºC for 20 weeks. Appropriate controls were prepared by incubating non-inoculated sterilized ADOR and SDOR under the same conditions used for fungal cultures. Phytotoxicity experiment The phytotoxicity experiments were carried out in pots with 300 g of soil that was steam-sterilized and mixed with sterilized quartz sand 1:1 by volume. The soil used was a grey loam obtained from the field of the Estación Experimental del Zaidín (Granada, Spain). The soil had a pH of 8.1 in a 1:1 soil:water ratio. The P, N, K, Fe, Mn, Cu and Zn contents were determined by the methods of Lachica et al. (1965). These were NaHCO3-extractable P 6.2 mg kg–1, N 2.5 mg kg–1, K 132 mg kg–1, Fe 9.6 g kg–1, Mn 110 mg g–1, Cu 5.8 mg g–1, Zn 5.7 mg kg–1. Glomus deserticola (Trappe, Bloss and Menge) from the Instituto de Investigaciones Agrobiológicas de Galicia (CSIC) was the AM fungus used in these experiments. The AM fungal inoculum was a root-and-soil inoculum consisting of rhizosphere soil containing spores and colonized root fragments of Sorghum vulgare. Eight grams of an inoculum that had previously achieved high levels of root colonization were added per pot. Plants grown in sterilized soil were given a filtrate of the soil (Whatman nº1 filter paper). The filtrate contained the common soil microorganisms, including bacteria and fungi but no propagules of AM fungi. Tomato (Lycopersicum esculentum L.) was used as the test plant. Seeds were sterilised, pregerminated and selected for uniformity prior to transplanting. Plants (one per pots) were grown in a greenhouse with supplementary light

OLIVE RESIDUE BIOREMEDIATION BY AM AND SAPROPHYTIC FUNGI

provided by Sylvania incandescent and cold-white lamps, 400 nmol m–2 s–1 at 400–700 nm, with a 16/8 h day night cycle at 25/19ºC and 50% relative humidity. Plants were watered from below, and were fed with 10 ml nutrient solution per week (Hewitt, 1966). Two experiments were used: (1) Plants grown in sterilized soil and (2) plants grown in sterilized soil inoculated with 8 g of inoculum of G. deserticola. These experiments were carried out with four repetitions of each treatment. A dose of 4 kg m–2 (20 g kg–1) is recommended for farmyard manure applications (Ocampo et al., 1975). In our experiment DOR, ADOR and SDOR were applied to the 300 g mixture soil:sand mixture pots at concentrations of 0, 12.5 and 25 g kg–1. Plants were harvested after 4 weeks and their dry mass was determined. After the harvest fresh samples of roots were taken from the entire root system at random. The samples were cleared and stained using trypan blue in lactic acid (Phillips and Hayman, 1970), and the percentage of root length colonization was measured by the line-intersect method (Giovannetti and Mosse, 1980). Determination of phenolic content To extract phenols, 0.2 g of DOR or SDOR was incubated for 24 h in 10 ml distilled water/acetone mixture (1:1 v/v) under orbital shaking. The total phenolic content of extracts of DOR, ADOR and SDOR was estimated according to Riberau-Gayon (1968), using tannic acid as a standard and was expressed as g kg–1 of olive residue. Determination of extracellular enzyme production Extracellular enzymes were extracted from DOR and SDOR using a procedure described by Rejón-Palomares et al. (1996). ADOR and the extracts obtained from DOR and SDOR were assayed to determine endopolymethylgalacturonase (endo-PMG; EC 3.2.1.15), endoglucanase (endoGN; EC 3.2.1.4) and endoxyloglucanase (endo-XG) activities. All hydrolytic activities were assayed by the viscosity method (Rejón-Palomares et al., 1996) using carboxymethylcellulose (CMC), citrus pectin and xyloglucan from nasturtium seed [Tropaelum majus L.; extracted as described previously (McDougall and Fry, 1989)] as substrates. The reduction in viscosity was determined at 0–30 min intervals. The reaction mixture contained 1 ml of 0.5% substrate in 50 mM citrate-phosphate buffer (pH 5) with 0.2 ml enzyme. Viscosity reduction was determined at 37ºC. One unit of enzyme activity was expressed as U g of dry olive residue calculated as: U reciprocal of time in hours for 50% viscosity loss × 103 (Rejón-Palomares et al., 1996). Lacasse activity (EC 1.10.3.2) was assayed with 5 mM

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DMP (2,6-dimethoxyphenol) in 100 mM of acetate buffer pH 5.0 (ε469 = 27500 M–1 cm–1) (Saparrat et al., 2000). One unit of enzyme activity was expressed as the amount of enzyme releasing 1 µmol of oxidized product per minute. Statistical treatment of data Data obtained were subjected to ANOVA. The mean values of four replicate were compared using the Tukey test using a confidence level of 95%.

3. Results All the physical fractions of olive mill residues reduce the shoot dry weight related to that of plants grown in absence of DOR (Table 1). The phytotoxic effect of DOR was higher than that of ADOR or SDOR. The shoot dry weight was lower in presence of SDOR than in presence of ADOR. The phytotoxic effect of DOR on shoot dry weight of tomatoes inoculated with G. deserticola was greater than when the plants were grown in non AM inoculated soil. However, the shoot dry weight of plants grown in the presence of ADOR and SDOR was higher when plants were colonized with G. deserticola than when they were not AM colonized. The application of 12.5 g kg–1 of ADOR to soil inoculated with G. deserticola increased the shoot dry weight relative to that of plants grown in the absence of any residue (Table 1). The percentage of AM root length colonization of plants inoculated with G. deserticola decreased in presence of 25 g kg–1 of DOR, ADOR or SDOR (Table 1). However, the level of mycorrhization reached by tomato was higher in presence of 25 g kg–1 of ADOR or SDOR than in presence of 25 g kg–1 of DOR. The shoot dry weight of plants grown in presence of 12.5 g kg–1 of ADOR incubated with T. versicolor was higher than that of plants grown in absence of ADOR (Table 2). However, all doses of ADOR incubated with P. cinnabarinus increased the shoot dry weight relative to that of plants grown in absence of this residue. When ADOR incubated with T. versicolor was applied to plants inoculated with G. deserticola, the shoot dry weight was higher than that of non AM inoculated plants and similar to or higher than that of plants grown in absence of ADOR. However, the shoot dry weight of tomato inoculated with G. deserticola and grown in presence of ADOR incubated with P. cinnabarinus was lower than non AM inoculated plants and similar to plants grown in absence of ADOR (Table 3). A similar percentage of AM tomato root length was colonized by G. deserticola when plants were grown in presence of low doses of ADOR whether inoculated or not with the saprophytic fungi and when they were grown in absence of ADOR (Table 2). The incubation of ADOR with

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Table 1. Shoot dry weight and percentage of AM colonized root length in tomato (Lycopersicum esculentum L.) inoculated or not with G. deserticola, in presence of dry olive mill residue (DOR), aqueous extract of dry olive mill residue (ADOR) and solid residue obtained after the aqueous extraction (SDOR). Treatments

Amount of olive residue (g kg–1)

Control DOR

0 12.5 25 12.5 25 12.5 25

ADOR SDOR

Shoot dry weight (mg) Without AM G. deserticola 567.3 207.4 53.8 445.8 333.2 305.7 92.3

f,1 c,2 b,2 e,1 d,1 d,1 a,1

843.4 120.2 16.2 960.8 648.5 672.3 189.7

e,2 b,1 a,1 f,2 d,2 d,2 c,2

% Root length colonization 45.2 42.7 27.7 48.6 36.4 54.6 38.7

c c a c b c b

Column values followed by the same letter or row values followed by the same number are not significantly different as assessed by the Tukey test (P≤0.05). Data are de mean of four replicate samples. Table 2. Shoot dry weight and percentage of AM colonized root length of tomato (Lycopersicum esculentum L.) inoculated or not with G. deserticola, in presence of aqueous extract of dry olive mill residue (ADOR) incubated with Trametes versicolor and Pycnoporus cinnabarinus. Treatments


Control ADOR ADOR + T. versicolor ADOR + P. cinnabarinus

0 12.5 25 12.5 25 12.5 25


b,1 b,1 a,1 c,1 a,1 c,2 c,2

281.4 320.2 190.2 464.8 264.5 273.3 266.7

b,2 c,2 a,2 d,2 b,2 b,1 b,1


b b a a,b a a,b a

Column values followed by the same letter or row values followed by the same number are not significantly different as assessed by the Tukey test (P≤0.05). Data are the means of four replicate samples. Table 3. Shoot dry weight and percentage of AM colonized root length in tomato (Lycopersicum esculentum L.) inoculated or not with G. deserticola, in presence of solid residue obtained after the aqueous extraction of dry olive mill residue (SDOR) incubated with Trametes versicolor and Pycnoporus cinnabarinus. Treatments


Control SDOR SDOR + T. versicolor SDOR + P. cinnabarinus

0 12.5 25 12.5 25 12.5 25


d,1 b,1 a,1 c,1 b,1 d,2 c,2

590.2 196.5 76.6 280.1 146.6 293.3 183.3

d,2 b,2 a,2 c,2 b,2 c,1 b,1


c c b c c a a

Column values followed by the same letter or row values followed by the same number are not significantly different as assessed by the Tukey test (P≤0.05). Data are the means of four replicate samples.

the saprophytic fungi did not change the percentage of tomato root colonized by G. deserticola. All saprophytic fungi tested decreased the toxicity caused by the application of SDOR on tomatoes (Table 3). The application of 12.5 g kg–1 of SDOR incubated with P. cinnabarinus increased the shoot dry weight of plants

grown, the weight being similar to that of plants grown in absence of SDOR. The shoot dry weight of tomato inoculated with G. deserticola and grown in presence of SDOR incubated with T. versicolor was similar to that of non AM inoculated plants. However, the shoot dry weight of tomato inoculated with G. deserticola and grown in

OLIVE RESIDUE BIOREMEDIATION BY AM AND SAPROPHYTIC FUNGI

presence of SDOR incubated with P. cinnabarinus was lower than that of non AM inoculated plants. G. deserticola increased the dry weight of tomatoes grown in presence of SDOR incubated with the saprophytic fungi compared with that of plants grown in presence of SDOR not incubated with saprophytic fungi (Table 3). The application of SDOR inoculated with T. versicolor to soil increased the percentage of root length colonization by G. deserticola (Table 3). The application of SDOR inoculated with P. cinnabarinus decreased the percentage of AM root length colonization of tomato to a lower level than when this residue was not incubated with the saprophytic fungus (Table 3). Of the 28–39 g kg–1 phenol content of DOR, the physical fractions ADOR and SDOR only contain 18.4 and 14.1 g kg–1 of phenols, respectively. The phenol content of ADOR was reduced by T. versicolor and P. cinnabarinus to 7.2 and 9.4 g kg–1, respectively (Table 4). Both saprophytic fungi show laccase activity when they were incubated in ADOR and T. versicolor had higher laccase activity than P. cinnabarinus (Table 5). Very low endo-PMG activity was observed in ADOR incubated with T. versicolor and P. cinnabarinus. Higher endo-GN and endo-XG activities was observed in ADOR incubated with P. cinnabarinus than with T. versicolor (Table 5).

Table 4. Phenol content (g kg–1) of aqueous extract (ADOR) and solid residue obtained after the aqueous extraction of the dry olive mill residue (SDOR) incubated with Trametes versicolor and Pycnoporus cinnabarinus. Treatment Without fungi T. versicolor P. cinnabarinus

Olive mill residue ADOR SDOR 18.4 b 7.2 a 9.4 a

14.1 b 4.2 a 4.4 a

Column values followed by the same letter for each residue are not significantly different as assessed by the Tukey test (P≤0.05). Data are the means of five replicate samples.

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Phenol content of SDOR was reduced by T. versicolor and P. cinnabarinus by about 69% (Table 4). Higher laccase activity was detected in SDOR incubated with P. cinnabarinus than with T. versicolor. No endo-PMG activity was observed in SDOR incubated with the saprophytic fungi. P. cinnabarinus had higher endo-GN and endo-XG activities than T. versicolor when they were incubated in SDOR (Table 5).

4. Discussion Olive mill residue have phytotoxic effects that are particularly evident during the germination and seedling development of higher plants (Casa et al., 2003; Sampedro et al., 2004b). It is generally accepted that olive residue phytotoxicity is largely due to the presence of water soluble substances, although other DOR constituents such as volatile fatty acids, aldehydes and alcohols also have negative effect on plant growth (Komilis et al., 2005). In the present study both tested saprophytic fungi eliminated similar quantity of phenols from both ADOR and SDOR. However, they differed in their ability to decrease the toxicity of both residues on tomato grown in soil. These results indicated that the decrease of total phenol concentration achieved by saprophytic fungi was not closely related to the decrease in ADOR and SDOR toxicity. The differences in the level of the phenol degrading enzymes laccase between T. versicolor and P. cinnabarinus incubated either in ADOR or in SDOR suggest that saprophytic fungi degrade different phenol compounds in the residues, which may result in different effects on plant growth. However, a close correlation between the level of these enzymes produced by each saprophytic fungus and the decrease in the phytotoxicity of the physical fractions of DOR was not found. This indicates that the phenolic compounds, degraded by these enzymes, are not the only factors implicated in the phytotoxicity of ADOR and SDOR. Substances other than phenols such as cell wall fragments (oligosaccharides) and glycoproteins have toxic effects on plant health (Bucheli et al., 1990).

Table 5. Laccase, endopolymethylgalacturonsase (endo-PMG), endoglucanase (endo-GN) and endoxiloglucanase (endo-XG) activities of aqueous extract (ADOR) and solid residue (SDOR) obtained after the aqueous extraction of the dry olive mill residue incubated with Trametes versicolor and Pycnoporus cinnabarinus. Residue

Saprophytic fungi

Ligninolytic activities (mUI/g of DOR) Laccase

ADOR

T. versicolor P. cinnabarinus T. versicolor P. cinnabarinus

1600 600 5 400

SDOR

b a a b

Hydrolytic activities (U/g of DOR) Endo-PMG Endo-GN Endo-XG 0 5 0 0

50 310 2000 2500

a b a b

70 100 2700 8000

a b a b

Column values followed by the same letter for each residue and group of enzymatic activity are not significantly different as assessed by the Tukey test (P≤0.05). Data are the means of five replicate samples.

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Hydrolytic enzymes have been found to be implicated in the reduction of the toxicity of ADOR (Aranda et al., 2004). The higher endo-GN and endo-XG activities produced by P. cinnabarinus paralleled with the decrease of phytotoxicity caused by this fungus in ADOR and SDOR reinforce this idea. The results obtained in the present study, confirm our previous observations that AM fungi may facilitate the action or transfer of toxic substances from DOR to plants (Martín et al., 2002). However, G. deserticola decreased the sensitivity of plant to the phytotoxicity of the physical fractions (ADOR and SDOR) of this residue and this AM fungus also considerably improved the growth of tomato in presence of low doses of ADOR. The effect of AM fungi on plant sensitivity to the toxins in the different olive mill residue fractions is not known. However, the level of phytotoxic substances in soil, such as heavy metals and pesticides (Ocampo, 1993; Khan et al., 2000), may be implicated in the increase of sensitivity and/or resistance of plants to olive residues conferred by the AM fungi. In our experiments we observed that the beneficial effect of ADOR and SDOR incubated with P. cinnabarinus on the shoot dry weight of tomato was eliminated by G. deserticola in presence of ADOR, whereas it was increased or not affected by the AM fungus when ADOR or SDOR were incubated with P. cinnabarinus and T. versicolor. Therefore these effects cannot be solely attributed to the level of phenols. The establishment of AM symbiosis seems to be greatly affected by the concentration of toxic compounds in the soil (Leadir et al., 1997; Medina et al., 2005). As was previously observed, the percentage of root length colonized by G. deserticola strongly decreased in presence of DOR (Martín et al., 2002). It has been suggested that DOR does not affect AM fungi directly and its negative effect are mediated by the plant (Martín et al., 2002). The effect of the physical fractions of DOR on AM colonization also seems to be mediated through the plant. In fact the decrease of AM root length colonization of tomato caused by ADOR and SDOR fractions paralleled the decrease of the plant dry weight. However, the physical fraction of DOR incubated with the saprophytic fungi T. versicolor and P. cinnabarinus had different effects on the percentage of AM colonization of tomato depending more on the physical fraction and on the saprophytic fungi type than on effects through the plant. We conclude this because no relationship was observed between effects on AM root colonization and plant dry weight. The mechanisms implicated in the harmful or beneficial effect of saprophytic fungi exudates on AM fungal colonization of roots is very complex (Fracchia et al., 2004). The laccase activity of P. cinnabarinus produced in SDOR could participate in the removal of some of the monomeric phenols such as lutein and rutin from this residue as has been observed in DOR (Sampedro et al., 2004a). Beneficial or negative effect of

these flavonoids on AM colonization of root depending on flavonoids concentration and on AM fungal specie have been observed (Vierheilig et al., 1998; Scervino et al., 2007). Close relationships between the percentage of AM colonization and its effect on plant growth are not commonly found (García-Garrido et al., 2000; AdrianoAnaya et al., 2006). Indeed in our experiments no relationship was observed between the percentage of AM colonization and plant dry weight of tomato affected by the physical fractions of DOR incubated with the saprophytic fungi. In general the application of ADOR and SDOR incubated with the saprophytic fungi for 2 and 8 weeks respectively increased the shoot dry weight of tomato, which indicates that a combination of physical and biological treatments may open the way for management and use of these wastes as organic amendments for agricultural soils. On a large scale, water extraction followed by composting using natural saprophytic fungi may yield a commercial compost of agricultural value. However, as the AM fungi may either increase or eliminate the beneficial effects of supplementing soils with olive mill residues that have been incubated with saprophytic fungi, the role of microorganisms must be taken into account in agrowaste applications. Acknowledgements The authors wish to thank Dr. María Jesus Martinez, CIB, CSIC, for providing the saprophytic fungus. Professor D.H.S. Richardson is thanked for help with English and grammar in a draft version of this paper. Financial support for this study was provided by the Comision Interministerial de Ciencia y Tecnología (CICYT) through project AGL 2004-00036. This work was partially supported by the Fondecyt 1060390 grant. REFERENCES Abu-Zreig, M. and Al-Widyan, M. 2002. Influence of olive mills solid waste on soil hydraulic properties. Communications in Soil Science and Plant Analysis 33: 505–517. Adriano-Anaya, M.L., Salvador-Figueroa, M., Ocampo, J.A., and García-Romera, I. 2006. Hydrolytic enzymes production in maize (Zea mays) and sorghum (Sorghum bicolor) roots inoculated with Gluconacetobacter diazotrophicus and Glomus intraradices. Soil Biology and Biochemistry 38: 879–886. Aranda, E., Sampedro, I., Ocampo, J.A., and García-Romera, I. 2004a. Contribution of hydrolytic enzymes produced by saprobe fungi on the decrease of plant toxicity caused by water-soluble substances of olive mill dry residue. Applied Microbiology and Biotechnology 64: 132–135. Aranda, E., Sampedro, I., Ocampo, J.A., and García-Romera, I. 2006. Phenolic removal of olive-mill dry residues by laccase activity of white-rot fungi and its impact on tomato plant growth. International Biodeterioration and Biodegradation 58: 176–179.

OLIVE RESIDUE BIOREMEDIATION BY AM AND SAPROPHYTIC FUNGI Arriagada, C., Herrera, M.A., García-Romera, I., and Ocampo, J.A. 2004. Tolerance to Cd of soybean (Glycine max) and eucalyptus (Eucalyptus globulus) inoculated with arbuscular mycorrhizal and saprope fungi. Symbiosis 36: 285–301. Boyle, C.D. and Hellenbrand, K.E. 1991. Assessment of the effect of mycorrhizal fungi on drought tolerance of conifer seedlings. Canadian Journal of Botany 69: 1764–1771. Brunetti, G., Plaza, C., and Senesi, N. 2005. Olive pomace amendment in Mediterranean conditions: effect on soil and humic acid properties and wheat (Triticum turgidum L.) yield. Journal of Agricultural and Food Chemistry 53: 6730–6737. Bucheli, P., Doares, S.H., Albersheim, P., and Darvill, A. 1990. Host-pathogen interactions. XXXVI. Partial purification and characterization of heat-labile molecules secreted by the rice blast pathogen that solubilize plant cell wall fragments that kill plant cells. Physiology and Molecular Plant Pathology 36: 159– 173. Caravaca, F., Figueroa, D., Alguacil, M.M., and Roldán, A. 2003. Application of composted urban residue enhanced the performance of afforested shrub species in a degraded semiarid land. Bioresource Technology 90: 65–70. Casa, R., D’Annibale, A., Pieruccetti, F., Stazi, S.R., Sermanni, G.G., and Lo Cascio, B. 2003. Reduction of the phenolic components in olive-mill wastewater by an enzymatic treatment and its impact on durum wheat (Triticum durum Desf.) germinability. Chemosphere 50: 959–966. Cermola, F., DellaGreca, M., Iesce, M.R., Montella, S., Pollio, A., and Temussi, F. 2004. A mild photochemical approach to the degradation of phenols from olive oil mill wastewater. Chemosphere 55: 1035–1041. Dix, N.J. and Webster, J. 1995. Fungal Ecology. Chapman and Hall, England. Fracchia, S., García-Romera, I., Godeas, A., and Ocampo, J.A. 2000. Effect of the saprophytic fungus Fusarium oxysporum on arbuscular mycorrhizal colonization and growth of plants in greenhouse and field trials. Plant and Soil 223: 175–184. Fracchia, S., Sampedro, I., Scervino, J.M., García-Romera, I., Ocampo, J.A., and Godeas, A. 2004. Influence of saprobe fungi and their exudates on arbuscular mycorrhizal symbioses. Symbiosis 36: 169–182. García-Garrido, J.M., Tribak, M., Rejón-Palomares, A., Ocampo, J.A., and García-Romera, I. 2000. Hydrolytic enzymes and ability of arbuscular mycorrhizal fungi to colonize roots. Journal of Experimental Botany 51: 1443–1448. Ginos, A., Manios, T., and Mantzavinos, D. 2006. Treatment of olive mill effluent by coagulation-flocculation-hydrogen peroxide oxidation and effect on phytotoxicity. Journal of Hazard Material 133: 135–142. Giovannetti, M. and Mosse, B. 1980. An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. New Phytologist 84: 489–500. Hewitt, E.J. 1966. Sand water culture methods used in the study of plant nutrition. Commonwealth Agriculture Bureau, Technical communication, Farmham Royal, Bucks, UK, No. 22. Isidori, M., Lavorgna, M., Nardelli, A., and Parrella, A. 2005. Model study of the effect of 15 phenolic olive mill wastewater constituents on seed germination and Vibrio fischeri metabolism. Journal of Agricultural and Food Chemistry 53: 8414–8417. Iyamuremye, F. and Dick, R.P. 1996. Organic amendments and phosphorus sorption by soils. Advances in Agronomy 56: 139– 185. Khan, G., Kuek, C., Chaudhry, T.M., Khoo, C.S., and Hayes, W.J. 2000. Role of plants, mycorrhizae and phytochelators in heavy

139

metal contaminated land remediation. Chemosphere 41: 197– 207. Komilis, D.P., Karatzas, E., and Halvadakis, C.P. 2005. The effect of olive mill wastewater on seed germination after various pretreatment techniques. Journal of Environmental Management 74: 339–348. Lachica, M., Recalde, L., and Esteban, E. 1965. Métodos analíticos utilizados en la Estación Experimental del Zaidín. Anales de Edafología y Agrobiología 24: 589–610. Leadir, L., Fries, M., Pacovsky, R.S., Safir, G.R., and Siquiera, J.O. 1997. Plant growth and arbuscular mycorrhizal fungal colonization affected by exogenously applied phenolic compounds. Journal of Chemistry and Ecology 23: 1755–1767. Martín, J., Sampedro, I., García-Romera, I., García-Garrido, J.M., and Ocampo, J.A. 2002. Arbuscular mycorrhizal colonization and growth of soybean (Glycine max) and lettuce (Lactuca sativa) and phytotoxic effects of olive mill residues. Soil Biology and Biochemistry 34: 1769–1775. Martínez, A., Obertello, M., Pardo, A., Ocampo, J.A., and Godeas, A. 2004. Interaction between Trichoderma pseudokoningii strains and the arbuscular mycorrhizal fungi Glomus mosseae and Gigaspora rosea. Mycorrhiza 14: 79–84. Medina, A., Vassilev, N., Barea, J.M., and Azcón, R. 2005. Application of Aspergillus niger-treated agrowaste residue and Glomus mosseae for improving growth and nutrition of Trifolium repens in a Cd-contamined soil. Journal of Biotechnology 116: 369–378. McDougall, G.J. and Fry, S.C. 1989. Structure-activity relationships for oligosaccharides with anti-auxin activity. Plant Physiologist 89: 883–887. Moreno, E., Perez, J., Ramos-Cormenzana, A., and Martinez, J. 1987. Antimicrobial effect of wastewaters from olive oil extraction plants selecting soil bacteria after incubation with diluted waste. Microbios 51: 169–174. Ocampo, J.A. 1993. Influence of Pesticides on VA mycorrhiza. In: Pesticide-Plant Pathogen Interactions in Crop Production: Beneficial and Deleterious Effects. Altman, J., ed. CRC Press, Boca-Raton, Florida, pp. 214–216. Ocampo, J.A., Barea, J.M., and Montoya, E. 1975. Interaction between Azotobacter and phosphobacteria and their establishment in the rhizosphere as affected by soil fertility. Canadian Journal of Microbiology 21: 1160–1165. Paredes, C., Cegarra, J., Roig, A., Sánchez-Monedero, M.A., and Bernal, M.P. 1999. Characterization of olive-mill wastewater (alpechín) and its sludge for agricultural purposes. Bioresource Technology 67: 111–115. Perez, J., De la Rubia, T., Moreno, J., and Martinez, J. 1992. Phenolic content and antibacterial activity of olive oil waste waters. Environmental Toxicology and Chemistry 11: 489–495. Phillips, J.M. and Hayman, D.S. 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society 55: 158–161. Querejeta, J.I., Roldan, A., Albaladejo, J., and Castillo, V. 1998. The role of mycorrhizae, site preparation, and organic amendment in the afforestation of a semi-arid Mediterranean site with Pinus halepensis. Forest Science 44: 203–211. Radford, A., Stone, P.J., and Taleb, F. 1996. Cellulase and amylase complexes. In: Biosynthesis and Biodegradation of Cellulose. Haigler, C.H. and Weimer, P.J., eds. Marcel Dekker, New York, pp. 535–597. Ribereau-Gayon, P. 1968. Les Composes Phenoliques des Vegetaux. Dumond, Paris.

140

E. ARANDA ET AL.

Rejón-Palomares, A., García-Garrido, J.M., Ocampo, J.A., and García-Romera, I. 1996. Presence of xyloglucan-hydrolyzing glucanases (xyloglucanases) in arbuscular mycorrhizal symbiosis. Symbiosis 21: 249–261. Sampedro, I., Aranda, E., Martín, J., García-Garrido, J.M., GarcíaRomera, I., and Ocampo, J.A. 2004a. Saprobic fungi decrease plant toxicity caused by olive mill residues. Applied Soil Ecology 26: 149–156. Sampedro, I., Romero, C., Ocampo, J.A., Brenes, M., and GarcíaRomera, I. 2004b. Removal of monomeric phenols in dry mill olive residue by saprobic fungi. Journal of Agricultural and Food Chemistry 52: 4487–4492. Sampedro, I., Marinari, S., D’Annibale, A., Grego, S., Ocampo, J.A., and García-Romera, I. 2007. Organic matter evolution and partial detoxification in two-phase olive mill waste colonized by white rot fungi. International Biodeterioration and Biodegradation 60: 116–125. Saparrat, M.C.N., Martínez, M.J., Tournier, H.A., Cabello, M.N., and Arambarri, A.M. 2000. Production of ligninolytic enzymes by Fusarium solani strains isolated from different substrate. World Journal of Microbiology and Biotechnology 16: 199–803. Scervino, J.M., Ponce, M.A., Erra-Bassells, R., Vierheilig, H., Ocampo, J.A., and Godeas, A. 2005. Flavonoids exhibit fungal species and genus specific effects on the presymbiotic growth of Gigaspora and Glomus. Mycological Research 109: 789–794.

Shetty, K.G., Hetrick, B.A.D., Figge, D.A.H., and Schwab, A.P. 1994. Effects of mycorrhizae and other soil microbes on revegetation of heavy metal contaminated mine spoil. Environmental Pollution 86: 181–188. Tisdall, J.M. 1991. Fungal hyphae and structural stability of soil. Austrian Journal of Soil Research 29: 729–743. Vassilev, N., Fenice, M., Federici, F., and Azcón, R. 1997. Olive mill waste water treatment by immobilized cells of Aspergillus niger and its enrichment with soluble phosphate. Process Biochemistry 32: 617–620. Vierheilig, H., Bago, B., Albrecht, C., Poulin, M.J., and Piche, Y. 1998. Flavonoids and arbuscular mycorrhizal fungi. In: Flavonoids in the Living System, Manthey, J. and Buslig, B., eds., Plenum Press, New York, pp. 9–33. Vogel-Mikus, K., Drobne, D., and Regvar, M. 2005. Zn, Cd and Pb accumulation and arbuscular mycorrhizal colonisation of pennycress Thlaspi praecox Wulf. (Brassicaceae) from the vicinity of a lead mine and smelter in Slovenia. Environmental Pollution 133: 233–242. Volante, A., Lingua, G., Cesaro, P., Cresta, A., Puppo, M., Ariati, L., and Berta, G. 2005. Influence of three species of arbuscular mycorrhizal fungi on the persistence of aromatic hydrocarbons in contaminated substrates. Mycorrhiza 16: 43–50.