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SUMMARY Percent germination and length of hyphae of germinated Glomus mosseae spores, cultivated on water agar, decreased significantly in the presence of Aspergillus niger; this decrease was independent of any change in pH of the medium. Soluble and volatile compounds produced by A. niger significantly decreased percentage spore germination and the hyphal length of G. mosseae on water agar. The decrease caused by volatile compounds was significantly greater when A. niger was grown on malt extract agar. Shoot dry weights of maize and lettuce plants cultivated in soil in pots, and percentage arbuscular mycorrhizal (AM) root colonization of plants grown either in sand: vermiculite tubes inoculated with G. mosseae spores or in soil in pots with soil inoculum, were unaffected by A. niger when this saprobe was inoculated 2 wk after G. mosseae. Shoot dry weights and percentage AM colonization of plants decreased when the saprobic fungus was inoculated at the same time or 2 wk before G. mosseae. However, the metabolic activity resulting from AM colonization, measured as the percentage of mycelium showing succinate dehydrogenase activity, decreased in all treatments. The population of A. niger decreased when inoculated to the rhizosphere of plants at the same time as, or 2 wk after, G. mosseae, but not when it was inoculated 2 wk before G. mosseae. Our results show that G. mosseae decreases the saprobic fungal population through its effect on the plant, whereas A. niger, by the production of soluble or volatile substances, inhibits G. mosseae in its extramatrical stage.

The use of “alpeorujo” (dry olive residue) has been proposed as an organic amendment in order to enhance soil structure and to increase C storage in soils. The aim of this work is to study how aqueous alpeorujo (ADOR) extracts bioremediated with white-rot fungi and three representative phenolic acids present in this extract (protocatechuic, vanillic and caffeic acid) affect the growth of the arbuscular mychorrhizal fungus Rhizophagus custos in monoxenic culture. Our results show that ADOR decreased mycorrhization parameters; however, this negative effect ceased after ADOR bioremediation. Although protocatechuic and vanillic acids have drastic negative effects at high concentrations, these phenols enhance mycorrhization processes at low concentrations and caffeic acid negatively affects symbiosis at low concentrations. Finally, the capacity of root biomass to dissipate individual phenols was also estimated, in which mycorrhized roots improve phenol dissipation in the growth medium in the presence of different phenols. This study highlights the important role played by arbuscular mycorrhiza in protecting plants from phytotoxicity., This study was carried out thanks to financial support from the Ministry of Economy and Competitiveness (MINECO) within the framework of project AGL2012-32873. R. Reina gratefully acknowledges the support provided by the JAE-Pre Program which is co-financed by CSIC and the European Social Fund. E. Aranda thanks the Ministry of Economy and Competitiveness (MINECO) and FEDER funds for co-funding the Ramón y Cajal contract (RYC-2013-12481).

Phosphorus is one of the most important macronutrients required for plant growth. Plants have evolved many strategies for inorganic phosphorus (Pi) acquisition, including the symbiotic pathways, involving the formation of mycorrhiza. With regard to arbuscular mycorrhiza (AM), high Pi availability has long been known to negatively affect this association, although the underlying mechanism is unknown. In the present study, the interactive role played by ethylene and Pi in AM regulation was investigated in the tomato-Rhizophagus irregularis symbiosis. AM fungal colonization was analysed in epi, rin and NRO ethylene-responsive mutants under different Pi availability conditions, with a focus on the late stages of the interaction. Although Pi inhibited mycorrhizal parameters in the ethylene-insensitive rin mutant and wild-type cultivars, it did not alter the mycorrhization of the epi tomato mutant, which exhibits a constitutive ethylene-induced response. As with the colonization parameters, root ethylene content and the expression of AM-related and ethylene receptor 6 genes were inhibited by Pi in wild-type cultivars and rin mutants, but were unaffected or slightly activated in epi plants. The application of ethephon offsets the negative impact on the mycorrhizal development caused by the application of Pi. This compensation effect is dose dependent and was ineffective in the NRO mutant, which is more insensitive to the action of ethylene. Our results provide evidence that ethylene signalling negatively affects the suppressive effect of Pi on AM formation and suggests an overlap between this suppressive effect and the regulatory mechanism of Pi-starvation response pathway in plants mediated by ethylene., This study was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT) and the European Regional Development Fund (ERDF) through the Ministerio de Economía y Competitividad in Spain (AGL2011-25930; AGL2014-52298-P) as well as the Junta de Andalucía (Research Group BIO 260). We wish to thank the Tomato Genetics Resource Centre (TGRC) of the University of California for providing tomato seeds. We would also like to thank Michael O’Shea for proof-reading the document. R Torres de los Santos was supported by the Carolina foundation.

Arbuscular mycorrhizal fungi (AMF) establish an obligate mutualistic symbiosis with many plant species, increasing the uptake of phosphorous and other low-mobile nutrients by roots. In addition, AMF improve biotic and abiotic stress tolerance of host plants. Under these conditions, reactive oxygen species (ROS) increase occasional damage to proteins, lipids and DNA. Antioxidative enzymes in plants can play an important role in detoxifying ROS, thereby alleviating oxidative stress. In nursery practices, plants are subjected at least to two transplant conditions before being transplanted outside. It is important to achieve an optimal plant size to withstand environmental or other stresses when plants are transplanted into the field. The transplantation process can be considered a stress because plants have to adapt to new abiotic and biotic (rhizospheric) conditions. The aim of this study was to evaluate the effect of two Rhizophagus intraradices (N.C. Schenck & G.S. Sm.) C. Walker & A. Schüßler strains, GA5 and GC2, single and co-inoculated under two-transplant soil conditions, sterile and non-sterile, using cuttings of pomegranate (Punica granatum L.) as a model plant. These results showed that the GA5 single strain-inoculated plants improved growth and antioxidative enzyme responses to two transplant stress conditions. In conclusion, early mycorrhizal inoculation generates healthy plants that are more protected against environmental conditions, thereby improving plant transplant stress tolerance., The authors thank Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANCyPT) and Universidad de Buenos Aires (UBA) for financial support.

Background Solanum lycopersicum, an economically important crop grown worldwide, has been used as a model for the study of arbuscular mycorrhizal (AM) symbiosis in non-legume plants for several years and several cDNA array hybridization studies have revealed specific transcriptomic profiles of mycorrhizal tomato roots. However, a method to easily screen candidate genes which could play an important role during tomato mycorrhization is required. Results We have developed an optimized procedure for composite tomato plant obtaining achieved through Agrobacterium rhizogenes-mediated transformation. This protocol involves the unusual in vitro culture of composite plants between two filter papers placed on the culture media. In addition, we show that DsRed is an appropriate molecular marker for the precise selection of cotransformed tomato hairy roots. S. lycopersicum composite plant hairy roots appear to be colonized by the AM fungus Rhizophagus irregularis in a manner similar to that of normal roots, and a modified construct useful for localizing the expression of promoters putatively associated with mycorrhization was developed and tested. Conclusions In this study, we present an easy, fast and low-cost procedure to study AM symbiosis in tomato roots., This study was funded by grants from the Interministerial Commission of Science and Technology (CICYT) and the European Regional Development Fund (FEDER) through the Ministry of Economy, Industry and Competitiveness in Spain (AGL2014-52298-P). Tania Ho’s research fellowship was supported by the FPI-MINECO program. We are grateful to Dr. M Udvardi (Noble Research Institute) for generously providing the pUBIcGFP-DR vector. We also thank Dr. JA López-Ráez and Dr. A. Belver from the EEZ-CSIC for their help and critical comments; and M. Calvo-Polanco for encouraging us to publish this protocol. We also wish to thank Michael O’Shea for proof-reading the document.

Background Solanum lycopersicum, an economically important crop grown worldwide, has been used as a model for the study of arbuscular mycorrhizal (AM) symbiosis in non-legume plants for several years and several cDNA array hybridization studies have revealed specific transcriptomic profiles of mycorrhizal tomato roots. However, a method to easily screen candidate genes which could play an important role during tomato mycorrhization is required. Results We have developed an optimized procedure for composite tomato plant obtaining achieved through Agrobacterium rhizogenes-mediated transformation. This protocol involves the unusual in vitro culture of composite plants between two filter papers placed on the culture media. In addition, we show that DsRed is an appropriate molecular marker for the precise selection of cotransformed tomato hairy roots. S. lycopersicum composite plant hairy roots appear to be colonized by the AM fungus Rhizophagus irregularis in a manner similar to that of normal roots, and a modified construct useful for localizing the expression of promoters putatively associated with mycorrhization was developed and tested. Conclusions In this study, we present an easy, fast and low-cost procedure to study AM symbiosis in tomato roots.

The effect of the dual inoculation with arbuscular mycorrhizal (AM) and saprophytic fungi and a combination of wheat straw and sewage sludge residues were studied by determining their effect on dry weight of tomato and on chemical and biochemical properties of soil. Incubation of organic residue (sewage sludge combined with wheat straw) with saprophytic fungi and plant inoculation with mycorrhizal fungi was essential to study plant growth promotion. Soil application of organic residues increased the dry weight of tomato inoculated with Rhizophagus irregularis. The greatest shoot dry mass was obtained when the organic residues were incubated with Trichoderma harzianum and applied to AM plants. However, the greatest percentage of root length colonized with AM in the presence of the organic residues was obtained with inoculation with Coriolopsis rigida. The relative chlorophyll was greatest in mycorrhizal plants regardless of the presence of either saprophytic fungus. The presence of the saprophytic fungi increased soil pH as the incubation time increased. Soil nitrogen and phosphorus contents and acid phosphatase were stimulated by the addition of organic residues, and contents of N and P. Total N and P content in soil increased when the organic residue was incubated with saprobe fungi, but this effect decreased as the incubation period of the residue with saprobe fungi increased. The same trend was observed for soil β-glucosidase and fluorescein diacetate activities. The application of organic residues in the presence of AM and saprophytic fungi seems to be an interesting option as a biofertilizer to improve plant growth and biochemical parameters of soils.

The behaviour of tomato plants inoculated with arbuscular mycorrhizal (AM) fungi grown in the presence of aqueous extracts from dry olive residue (ADOR) was studied in order to understand how this symbiotic relationship helps plants to cope with oxidative stress caused by ADOR. The influence of AM symbiosis on plant growth and other physiological parameters was also studied. Tomato plants were inoculated with the AM fungus Funneliformis mosseae and were grown in the presence of ADOR bioremediated and non-bioremediated by Coriolopsis floccosa and Penicillium chrysogenum-10. The antioxidant response as well as parameters of oxidative damage were examined in roots and leaves. The data showed a significant increase in the biomass of AM plant growth in the presence of ADOR, regardless of whether it was bioremediated. The establishment and development of the symbiosis were negatively affected after plants were exposed to ADOR. No differences were observed in the relative water content (RWC) or PS II efficiency between non-AM and AM plants. The increase in the enzymatic activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6) and glutathione-S-transferase (GST; EC 2.5.1.18) were simultaneous to the reduction of MDA levels and H2O2 content in AM root growth in the presence of ADOR. Similar H2O2 levels were observed among non-AM and AM plants, although only AM plants showed reduced lipid peroxidation content, probably due to the involvement of antioxidant enzymes. The results highlight how the application of both bioremediated ADOR and AM fungi can alleviate the oxidative stress conditions, improving the growth and development of tomato plants.

The single application of either sewage sludge with high aluminium concentration, wheat straw or the mixture of both residues to soil did not increase the growth of Eucalyptus globulus Labill. However, inoculation with either the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis, the saprobe fungi Coriolopsis rigida (Berk. Et Mont.) Murrill and Phanerochaete chrysosporium Burds or the combination of each saprobe with the AM fungus increased both the P concentration and dry weight of E. globulus shoots. These effects were greater in the presence of wheat straw or sewage sludge, but were greatest in the presence of the mixed residue. Phanerochaete chrysosporium had the greatest effect on plant dry weight when co-inoculated with R. irregularis in the treatment with mixed residue. The co-inoculation of AM and saprobe fungi increased fluorescein diacetate and β-glucosidase activities in the bulk soil of E. globulus grown in the treatment with mixed residue. However, only the AM fungus increased dehydr...

The water regime affects a wide variety of physiological and biochemical processes in plants including an increased production of reactive oxygen species (ROS) capable of causing oxidative damage to proteins, DNA and lipids. Arbuscular mycorrhizal fungi (AMF) colonize a wide range of plant species though the ability of different AMF species to promote host growth or contribute to plant water deficit resistance varies. The first phase of olive tree cultivation takes place in a nursery where plants usually suffer stress by drying. Currently, olive production systems do not use of AMF to counteract this problem. To study the colonization strategies of two AMF strains and their efficiency with respect to growth and their effect on enzymatic activities, we inoculated them individually and co-inoculated then on olive plants under nursery growing conditions. The results showed the benefits generated by these fungi in terms of growth and survival rate. Co-inoculation, particularly, improved growth and reduced the damage due to water stress, partly as a result of the activation of the antioxidant defenses in the olive plant host. Fil: Bompadre, Maria Josefina. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Biodiversidad y Biologia Experimental. Laboratorio de Microbiologia del Suelo; Argentina Fil: Rios, Maria del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Colombo, Roxana. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Biodiversidad y Biologia Experimental. Laboratorio de Microbiologia del Suelo; Argentina Fil: Fernandez Bidondo, Laura. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Biodiversidad y Biologia Experimental. Laboratorio de Microbiologia del Suelo; Argentina Fil: Silvani, Vanesa Analia. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Biodiversidad y Biologia Experimental. Laboratorio de Microbiologia del Suelo; Argentina Fil: Pardo, Alejandro Guillermo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Laboratorio de Micologia Molecular; Argentina Fil: Ocampo, Juan Antonio. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidin; España Fil: Godeas, Alicia Margarita. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Biodiversidad y Biologia Experimental. Laboratorio de Microbiologia del Suelo; Argentina

Plant hormones have become appropriate candidates for driving functional plant mycorrhization programs, including the processes that regulate the formation of arbuscules in arbuscular mycorrhizal (AM) symbiosis. Here, we examine the role played by ABA/GA interactions regulating the formation of AM in tomato. We report differences in ABA and GA metabolism between control and mycorrhizal roots. Active synthesis and catabolism of ABA occur in AM roots. GAs level increases as a consequence of a symbiosis-induced mechanism that requires functional arbuscules which in turn is dependent on a functional ABA pathway. A negative interaction in their metabolism has been demonstrated. ABA attenuates GA-biosynthetic and increases GA-catabolic gene expression leading to a reduction in bioactive GAs. Vice versa, GA activated ABA catabolism mainly in mycorrhizal roots. The negative impact of GA3 on arbuscule abundance in wild-type plants is partially offset by treatment with ABA and the application of a GA biosynthesis inhibitor rescued the arbuscule abundance in the ABA-deficient sitiens mutant. These findings, coupled with the evidence that ABA application leads to reduce bioactive GA1, support the hypothesis that ABA could act modifying bioactive GA level to regulate AM. Taken together, our results suggest that these hormones perform essential functions and antagonize each other by oppositely regulating AM formation in tomato roots., This study was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT) and Fondos Europeos de Desarrollo Regional (FEDER) through the Ministerio de Economía y Competitividad in Spain (AGL2008-00742; AGL2011-25930) as well as the Junta de Andalucía (Research Group BIO 260). JM-R was supported by a research fellowship from the FPU-MICINN program. The work on hormone measurements was funded by a Grant Agency of the Czech Republic (grant no. 14-34792S) and by the Ministry of Education, Youth and Sports of the Czech Republic through the National Program for Sustainability I (Nr. LO1204).

Micromonospora is a Gram-positive bacterium that can be isolated from nitrogen-fixing root nodules from healthy leguminous plants and promotes plant growth. Selected strains of Micromonospora isolated in our laboratory from alfalfa root nodules showed in vitro antifungal activity against several pathogenic fungi. Moreover, root inoculation of tomato plants with these Micromonospora strains effectively reduced leaf infection by the fungal pathogen Botrytis cinerea, despite temporal and spatial separation between the inoculation of both microorganisms. Although Micromonospora improves plant growth and has antifungal activity against plant pathogens, the interaction between Micromonospora and AM fungi has not been studied before. Thus, the aim of the study was to work on the understanding of this interaction. The results obtained allow us to conclude that Micromonospora strains ALFpr18c and ALFb5 did not adversely affect the colonization and development of the AM fungi R. irregularis and F. mosseae on plant root. Micromonospora strain ALFpr18c increased the beneficial effect of the AM fungi on shoot dry weight of alfalfa and tomato when the AM fungi were established first on the roots. Therefore, it is possible to use Micromonospora ALFpr18c combined with AM fungi to improve plant growth. Micromonospora strains should be considered excellent plant probiotic bacteria as they combine plant growth-promoting activity with both direct antifungal activity against plant pathogens and the ability to prime plant immunity and did not adversely affect the colonization and development of the AM fungi.

Arbuscular mycorrhizal (AM) is a mutually beneficial interaction among higher plants and soil fungi of the phylum Glomeromycota. Numerous studies have pointed that jasmonic acid plays an important role in the development of the intraradical fungus. This compound belongs to a group of biologically active compounds known as oxylipins which are derived from the oxidative metabolism of polyunsaturated fatty acids. Studies of the regulatory role played by oxylipins in AM colonization have generally focused on jasmonates, while few studies exist on the 9-LOX pathway of oxylipins during AM formation. Here, the cDNA of Allene oxide synthase 3 (AOS3), a key enzyme in the 9-LOX pathway, was used in the RNA interference (RNAi) system to transform potato plants in order to suppress its expression. Results show increases in AOS3 gene expression and 9-LOX products in roots of wild type potato mycorrhizal plants. The suppression of AOS3 gene expression increases the percentage of root with mycorrhizal colonization at early stages of AM formation. AOS3 RNA interference lead to an induction of LOXA and 13-LOX genes, a reduction in AOS3 derived 9-LOX oxylipin compounds and an increase in jasmonic acid content, suggesting compensation between 9 and 13-LOX pathways. The results in a whole support the hypothesis of a regulatory role for the 9-LOX oxylipin pathway during mycorrhization., We wish to thank the Tomato Genetics Resource Centre (TGRC) of the University of California for providing tomato seeds. We would also like to thank Michael O’Shea for proof-reading the document. LC-MS/MS analyses were carried out by Dr. Lourdes Sánchez-Moreno at the Scientific Instrumentation Service of the Estación Experimental del Zaidín (CSIC), Granada, Spain. This study was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT) and Fondos Europeos de Desarrollo Regional (FEDER) through the Ministerio de Economía y Competitividad in Spain (AGL2011-25930; AGL2014-52298-P) as well as the Junta de Andalucía (Research Group BIO 260). R. J. León was supported by a research fellowship from the CSIC-JAE program.

Gaeumannomyces graminis var. tritici causes take-all disease, the most important root disease of cereal plants. Cereal plants are able to form a symbiotic association with soil-borne arbuscular mycorrhizal fungi which can provide bioprotection against soil-borne fungal pathogens. However, the bioprotective effect of arbuscular mycorrhizal fun gi against soil-borne fungal pathogens might vary. In the present study we tested the systemic bioprotective effect of the arbuscular mycorrhizal fungi Glomus mosseae, Glomus intraradices and Gigaspora rosea against the soilborne fungal pathogen Gaeumannomyces graminis var. tritici in a barley split-root system. Glomus intraradices, Glomus mosseae and Gigaspora rosea colonized the split-root system of barley plants at different levels; however, all arbuscular mycorrhizal fungi clearly reduced the level of root lesions due to the pathogen Gaeumannomyces graminis. Our data indicate that some arbuscular mycorrhizal fungi need high root colonization rates to protect plants against fungal pathogens, whereas others act already at low root colonization rates.

The saprophytic fungi Aspergillus niger, Coriolopsis rigida, Fusarium lateritium, F. oxysporum, Mucor racemosus, Paecilomyces farinosus, Penicillium chrysogenum, P. restrictum, Trametes versicolor, Trichoderma harzianum, T. pseudokoningii and T. viride were able to decrease the growth in vitro of Verticillium dahlie in the presence of aqueous extract of olive residue. The conidia number of V. dahliae decreased when grown on aqueous extract of olive residue, autoclaved or filtered through 0.45 micron filters after culture of the fungi. These results suggest not only the predominance of suppressive substances of a biological nature, but also the existence of non-biological inhibitory substances. The olive residue decreased the negative effect of V. dahliae on shoot and root dry weight of tomato (Solanum lycopersicum L.), by the antifungal compounds present in the olive residue and by the antifungal substances produced by the antagonistic saprophytic fungi grown in this residue.

The systemic effect of root colonization by the arbuscular mycorrhizal fungus (AMF) Glomus mosseae on the susceptibility of old and modern barley varieties to the soil-borne fungal pathogen Gaeumannomyces graminis var. tritici (Ggt) was studied in a split-root system. Plants were precolonized on one side of the split-root system with the AMF and thereafter the other side of the split-root system was inoculated with the pathogen. At the end of the experiment the level of bioprotection was estimated by quantifying lesioned roots and the determination of the root fresh weight. AM root colonization provided protection in some of the barley genotypes tested, but not in others. This protective effect seemed to vary in the oldest and the most modern barley variety tested.

We investigated the relationship between ABA and ethylene regulating the formation of the arbuscular mycorrhiza (AM) symbiosis in tomato (Solanum lycopersicum) plants and tried to define the specific roles played by each of these phytohormones in the mycorrhization process. We analysed the impact of ABA biosynthesis inhibition on mycorrhization by Glomus intraradices in transgenic tomato plants with an altered ethylene pathway. We also studied the effects on mycorrhization in sitiens plants treated with the aminoethoxyvinyl glycine hydrochloride (AVG) ethylene biosynthesis inhibitor and supplemented with ABA. In addition, the expression of plant and fungal genes involved in the mycorrhization process was studied. ABA biosynthesis inhibition qualitatively altered the parameters of mycorrhization in accordance with the plant's ethylene perception and ethylene biosynthesis abilities. Inhibition of ABA biosynthesis in wild-type plants negatively affected all the mycorrhization parameters studied, while tomato mutants impaired in ethylene synthesis only showed a reduced arbuscular abundance in mycorrhizal roots. Inhibition of ethylene synthesis in ABA-deficient sitiens plants increased the intensity of mycorrhiza development, while ABA application rescued arbuscule abundance in the root's mycorrhizal zones. The results of our study show an antagonistic interaction between ABA and ethylene, and different roles of each of the two hormones during AM formation. This suggests that a dual ethylene-dependent/ethylene-independent mechanism is involved in ABA regulation of AM formation.

We examined whether the reduced mycorrhization of abscisic acid (ABA)-deficient tomato mutants correlates with their incapacity in ABA biosynthesis and whether this effect is dependent on ethylene production. The mycorrhization of notabilis and sitiens mutants, which have different ABA deficiencies and an excess of ethylene production, was analyzed. Comparative analysis of the ABA-deficient tomato mutants showed both quantitative and qualitative differences in the pattern of arbuscular mycorrhiza (AM) colonization between the two tomato mutant phenotypes. The sitiens mutant showed a great limitation in fungal colonization (mycorrhizal intensity and arbuscule formation) well correlated with their incapacity in ABA biosynthesis. The notabilis plants, which maintained normal ABA levels in roots under our experimental conditions, appeared to be less affected in their capacity for AM formation, and only a decrease in mycorrhizal intensity was noted at the end of the mycorrhization process. Blockage of ABA formation after tungstate application resulted in a reduction in mycorrhization of wild-type tomato plants. The transcript accumulation of the mycorrhiza-responsive LePT4 gene (tomato phosphate transporter) was clearly associated with the ABA content and mycorrhiza development in roots, as the tungstate treatment in wild-type plants and the inherent ABA deficiency in sitiens mutants led to a complete abolishment of their expression. Our results suggest that the decrease in arbuscular abundance in mycorrhizal sitiens roots is directly associated with their ABA biosynthesis deficiency, and the accumulation of ethylene, as a consequence of ABA deficiency in the mutants, primarily affects mycorrhizal intensity.

Summary Pea (Pisum sativum) is an important grain legume crop whose cultivation in the Mediterranean basin and West Asia is severely constrained by infection by the weedy root parasite Orobanche crenata (crenate broomrape). Pea is a false host of other species such as O. minor, O. foetida and Phelipanche aegyptiaca. In the present experiments, we demonstrate that seed germination of the Orobanche and Phelipanche species examined is reduced in the presence of root exudates from pea plants colonised by arbuscular mycorrhizal (AM) fungi, Glomus mosseae and G. intraradices. Reduced germination was due to the effect of the AM fungi and not to the microbial populations potentially present in the soil inoculum. This protective effect of mycorrhizae was noticeable after 35 days of AM colonisation for O. crenata, O. foetida and P. aegyptiaca, but at least 45 days of colonisation were needed to get some reduction of O. minor germination.

The presence of high concentrations of arsenic (As) decreased the shoot and root dry weight, chlorophyll and P and Mg content of Eucalyptus globulus colonized with the arbuscular mycorrhizal (AM) fungi Glomus deserticola or G. claroideum, but these parameters were higher than in non-AM plants. As increased the percentage of AM length colonization and succinate dehydrogenase (SDH) activity in the root of E. globulus. Trichoderma harzianum, but not Trametes versicolor, increased the shoot and root dry weight, chlorophyll content, the percentage of AM root length colonization and SDH activity of E. globulus in presence of all As concentrations applied to soil when was inoculated together with G. claroideum. AM fungi increased shoot As and P concentration of E. globulus to higher level than the non-AM inoculated controls. The contribution of the AM and saprobe fungi to the translocation of As from root to shoot of E. globulus is discussed.

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 fungiTrametes versicolor andPycnoporus 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 ofP. cinnabarinus, paralleled the decrease of phytotoxicity, indicates that these enzymes seem to be involved. The AM fungusGlomus 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 byG. 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.

The role of the jasmonate signalling pathway in modulating the establishment of the arbuscular mycorrhiza (AM) symbiosis between tomato plants and Glomus intraradices fungus was studied. The consequences of AM formation due to the blockage of the jasmonate signalling pathway were studied in experiments with plant mutants impaired in JA perception. The tomato jai-1 mutant (jas- monic acid insensitive 1) failed to regulate colonization and was more susceptible to fungal infection, showing acceler- ated colonization. The frequency and the intensity of fungal colonization were greatly increased in the jai-1 insensitive mutant plants. In parallel, the systemic effects on mycorrh- ization due to the activation of the jasmonate signalling pathway by foliar application of MeJA were evaluated and histochemical and molecular parameters of mycorrhizal intensity and efficiency were measured. Histochemical deter- mination of fungal infectivity and fungal alkaline phosphatase activity reveal that the systemic application of MeJA was effective in reducing mycorrhization and mainly affected fungal phosphate metabolism and arbuscule formation, ana- lyzed by the expression of GiALP and the AM-specific gene LePT4, respectively. The results of the present study clearly show that JA participates in the susceptibility of tomato to infection by arbuscular mycorrhizal fungi, and it seems that arbuscular colonization in tomato is tightly controlled by the jasmonate signalling pathway.

We studied the influence of olive mill dry residue (DOR) treated with saprobe fungi on growth of tomato and alfalfa colonized by Glomus deserticola . The application of 25 g kg −1 of dry DOR to soil decreased the shoot and root dry weight of tomato and alfalfa. Plants were more sensitive to the toxicity of DOR when colonized with the arbuscular mycorrhizal (AM) fungi. The sensitivity of both plants to the toxicity of DOR differed according to whether they were colonized by G. deserticola or by indigenous AM fungi. The phytotoxicity of DOR towards tomato and alfalfa was decreased by incubation the residue before planting with saprobe fungi for 20 wk. The beneficial effects of AM fungi on plant growth added with DOR incubated with saprobe fungi depend of the type of the plant and AM fungi. The contribution of AM fungi to the beneficial effect of DOR incubated with saprobe fungi varied according to the type of the plant and AM fungi. G. deserticola increased the shoot and root dry weight of plants when they were grown in the presence of DOR incubated with saprobe fungi for 20 wk. The beneficial effect of saprobe fungi on the dry weight and the level of AM mycorrhization of plants seem to be related to the decrease caused by these fungi in the phenol concentration in DOR. However, the toxicity of DOR due to substances other than phenols can not be ignored. The use of certain saprobe and AM fungi allows the possibility of using DOR as an organic fertilizer.

The role of abscisic acid (ABA) during the establishment of the arbuscular mycorrhiza (AM) was studied using ABA sitiens tomato (Lycopersicon esculentum) mutants with reduced ABA concentrations. Sitiens plants and wild-type (WT) plants were colonized by Glomus intraradices. Trypan blue and alkaline phosphatase histochemical staining procedures were used to determine both root colonization and fungal efficiency. Exogenous ABA and silver thiosulfate (STS) were applied to establish the role of ABA and putative antagonistic cross-talk between ABA and ethylene during AM formation, respectively. Sitiens plants were less susceptible to the AM fungus than WT plants. Microscopic observations and arbuscule quantification showed differences in arbuscule morphology between WT and sitiens plants. Both ABA and STS increased susceptibility to the AM fungus in WT and sitiens plants. Fungal alkaline phosphate activity in sitiens mutants was completely restored by ABA application. * The results demonstrate that ABA contributes to the susceptibility of tomato to infection by AM fungi, and that it seems to play an important role in the development of the complete arbuscule and its functionality. Ethylene perception is crucial to AM regulation, and the impairment of mycorrhiza development in ABA-deficient plants is at least partly attributable to ethylene.

Dry olive mill residue (DOR) is a solid waste arising from the olive oil two-phase extraction system. The objective of the present study was to investigate the impact of an aerobic treatment of DOR with selected lignin-degrading fungi on both organic matter evolution and residual phytotoxicity of the waste. To this aim, several white-rot fungi, including Phlebia radiata, Coriolopsis rigida, Phanerochaete chrysosporium, Pycnoporus cinnabarinus, Poria subvermispora and Pleurotus pulmonarius were inoculated under axenic conditions for 2 and 20 weeks. The chemical composition and phytotoxicity of DOR were scarcely affected by fungi after 2 weeks incubation. By contrast, both a significant depletion of phenolic compounds and a partial removal of phytoxicity towards Lycopersicum esculentum plants were generally obtained after 20 weeks. The most effective fungus in degrading lignin, total phenols and in removing phytotoxicity was C. rigida. A high correlation was observed between phytotoxicity and phenols added to soil with the waste.

We studied the influence of the laccase activity of two white-rot fungi on the toxic effect of water-soluble substances from dry residues of olives (ADOR) on tomato plants. Pycnoporus cinnabarinus and Coriolopsis rigida decreased the phenol content of ADOR to 73% after 15 days. P. cinnabarinus and C. rigida produced laccase activity after 5 and 15 days, respectively, and the highest activity in both fungi was detected at 20 days. The treatment of ADOR with these white-rot fungi decreased the phytotoxicity of this residue on tomato plants. A close relationship was found between the amount of laccase produced, the decrease in phenol content of ADOR by the saprobic fungi, decrease of phytotoxicity of ADOR, and the increase in dry weight of tomato plants. These results show that phenol removal by the laccase activity of white-rot fungi can be important in the elimination of phytotoxic substances present in olive-mill dry residues.

Two strains of Gluconacetobacter diazotrophicus (Pal 5, UAP5541) and the arbuscular mycorrhizal fungus Glomus intraradices increased both the shoot and root dry weight of sorghum 45 days after inoculation, whereas they had no effect on the shoot and root dry weight of maize. Co-inoculation (Gluconacetobacter diazotrophicus plus Glomus mosseae) did not increase the shoot and root dry weight of either plant. There was a synergistic effect of Gluconacetobacter diazotrophicus on root colonization of maize by Glomus intraradices, whereas an antagonistic interaction was observed in the sorghum root where the number of Gluconacetobacter diazotrophicus and the colonization by Glomus intraradices were reduced. Plant roots inoculated with Gluconacetobacter diazotrophicus and Glomus intraradices, either separately or together, significantly increased root endoglucanase, endopolymethylgalacturonase and endoxyloglucanase activities. The increase varied according to the plant. For example, in comparison with non-inoculated plants, there were higher endoglucanase (+328%), endopolymethylgalacturonase (+180%) and endoxyloglucanase (+125%) activities in 45-day old co-inoculated maize, but not in 45-day old sorghum. The possibility is discussed that hydrolytic enzyme activities were increased as a result of inoculation with Gluconacetobacter diazotrophicus, considering this to be one of the mechanisms by which these bacteria may increase root colonization by AM fungi.

The application of Pb inhibited the development of mycelia of the saprobe fungi Fusarium concolor and Trichoderma koningii and the hyphal length of the arbuscular mycorrhizal fungi (AM) Glomus mosseae and G. deserticola in vitro. The application to soil of 1500 mg kg−1 of Pb decreases the dry weight, total N, P, Mg and Fe concentration and chlorophyll content of the shoot of E. globulus no inoculated with AM fungi. However, G. deserticola increased the dry weight, total nutrient concentration and chlorophyll content of the shoot, and the percentage of AM root length colonization and the succinate dehydrogenase activity of AM mycelia of E. globulus in presence of 1500 mg kg−1 of Pb, and these increases were higher when G. deserticola was inoculated together with T. koningii. The application to soil of 3000 mg kg−1 of Pb decreased the shoot dry weight and AM colonization of E. globulus in all treatments tested. Pb was accumulated in the stem more than in the leaves of E. globulus. In presence of 1500 mg kg−1 of Pb the highest accumulation of this metal in the stem took place when E. globulus was colonized with G. deserticola. In conclusion, the possibility to increase Lead accumulation in stem is very attractive for phytoextraction function, the saprobe fungi, AM and their interaction may have a potential role in elevating phytoextraction efficiency and stimulate plant growth under adverse conditions such as lead contaminated soil.

The flavonoids 5,6,7,8,9-hydroxy chalcone, 3,7-hydroxy-4′methoxy flavone, 5,6,7,8-hydroxy-4′-methoxy flavone and 3,5,6,7,4′-hydroxy flavone can be detected only in non-mycorrhizal roots of white clover, but not in mycorrhizal roots, whereas the flavonoids acacetin, quercetin and rhamnetin are only present in mycorrhizal roots. We tested the effect of several concentrations of these compounds on spore germination, hyphal growth, hyphal branching, formation of clusters of auxiliary cells and of secondary spores of the arbuscular mycorrhizal fungi Gigaspora rosea, Gigaspora margarita, Glomus mosseae and Glomus intraradices. Our results indicate that depending on the flavonoid, the tested compounds are involved at different stages in the regulation of mycorrhization. This hypothesis is strengthened by their differing effect on several AM fungal growth parameters. Furthermore, our study provides more data on the AM fungus genus/species specificity of flavonoids.

White clover (Trifolium repens) plants were grown in the presence or absence of the arbuscular mycorrhizal fungus Glomus intraradices. Flavones, 4',5,6,7,8-pentahydroxy-3-methoxyflavone and 5,6,7,8-tetrahydroxy-3-methoxyflavone, as well as two flavones 3,7-dihydroxy-4'-methoxyflavone and 5,6,7,8-tetrahydroxy-4'-methoxyflavone never previously reported in plants, were isolated. The known 3,5,6,7,8-pentahydroxy-4'-methoxyflavone, 2',3',4',5',6'-pentahydroxy-chalcone, 6-hydroxykaempferol, 4',5,6,7,8-pentahydroxyflavone and 3,4'-dimethoxykaempferol were also obtained. Analysis of extracts obtained from roots and shoots revealed that the compositions of the flavonoid mixtures varied with growing conditions. Quercetin, acacetin and rhamnetin accumulated in roots of inoculated plants, whereas they were not detected in non-inoculated plants.

We studied the influence of dry olive mill residue (DOR), as affected by saprobic fungi on the growth of soybean ( Glycine max ) and tomato ( Lycopersicum esculentum ). The application of 60 g kg −1 of DOR decreased the dry weight of tomato and soybean plants mainly because of its phenolic content. The phytotoxicity of DOR decreased when it was incubated in the presence of almost all of the saprobic fungi tested. All saprobic fungi tested in our experiments were able to decrease the phenol content of DOR, and a correlation between the decrease in the phenol content of DOR by saprobic fungi and the increase in the dry weight of plants was found. However, the toxicity of these residues to either tomato or soybean plants did not decrease in the presence of all saprobic fungi in spite of a decrease in phenolic compounds. Some of the saprobic fungi tested eliminate completely the phytotoxicity of DOR. F. oxysporum 738 and F. lateritum were capable of transforming the DOR, after incubation for 20 weeks, so that the tomato plants to which this transformed DOR was applied increased significantly in shoot dry weight compared with the tomato plants which did not receive DOR. These results open the possibility of the use of DOR as organic fertilizer.

The interaction between Trichoderma pseudokoningii (Rifai) 511, 2212, 741A, 741B and 453 and the arbuscular mycorrhizal fungi Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe BEG12 and Gigaspora rosea Nicolson & Schenck BEG9 were studied in vitro and in greenhouse experiments. All T. pseudokoningii strains inhibited the germination of G. mosseae and Gi. rosea except the strain 453, which did not affect the germination of Gi. rosea. Soluble exudates and volatile substances produced by all T. pseudokoningii strains inhibited the spore germination of G. mosseae. The germination of Gi. rosea spores was inhibited by the soluble exudates produced by T. pseudokoningii 2212 and 511, whereas T. pseudokoningii 714A and 714B inhibited the germination of Gi. rosea spores by the production of volatile substances. The strains of T. pseudokoningii did not affect dry matter and percentage of root length colonization of soybean inoculated with G. mosseae, except T. pseudokoningii 2212, which inhibited both parameters. However, all T. pseudokoningii strains decreased the shoot dry matter and the percentage of AM root length colonization of soybean inoculated with Gi. rosea. The saprotrophic fungi tested seem to affect AM colonization of root by effects on the presymbiotic phase of the AM fungi. No influence of AM fungi on the number of CPUs of T. pseudokoningii was found. The effect of saprotrophic fungi on AM fungal development and function varied with the strain of the saprotrophic species tested. Fil: Martinez, Alicia Elba. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; Argentina Fil: Obertello, Mariana. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pardo, Alejandro Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Ocampo, Juan A.. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; España Fil: Godeas, Alicia Margarita. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Wild type, transgenic NahG tobacco plants with reduced levels of salicylic acid (SA) and transgenic CSA (constitutive SA biosynthesis) tobacco plants with enhanced SA levels were inoculated with the arbuscular mycorrhizal fungi (AMF) Glomus mosseae or Glomus intraradices. In a time course study the effect of SA content on root colonization by the fungal symbiont was determined. Throughout the experiment in NahG plants an enhanced root colonization level could be detected, whereas in CSA plants mycorrhization was reduced. At the end of the experiment with Glomus mosseae, root colonization was similar in wild type and in transgenic plants (NahG and CSA plants), indicating that enhanced SA levels in plants can have an effect on delay AMF root colonization, but do not affect the symbiotic potential of plants in terms of changes in maximal threshold of root colonization. Compared to non-mycorrhizal plants, in mycorrhizal wild type and NahG the SA concentration was reduced. The role of SA in the regulation of mycorrhization is discussed.

The effect of the soil yeast, Rhodotorula mucilaginosa LBA, on Glomus mosseae (BEG n°12) and Gigaspora rosea (BEG n°9) was studied in vitro and in greenhouse trials. Hyphal length of G. mosseae and G. rosea spores increased significantly in the presence of R. mucilaginosa. Exudates from R. mucilaginosa stimulated hyphal growth of G. mosseae and G. rosea spores. Increase in hyphal length of G. mosseae coincided with an increase in R. mucilaginosa exudates. No stimulation of G. rosea hyphal growth was detected when 0.3 and 0.5 ml per petri dish of yeast exudates was applied. Percentage root length colonization by G. mosseae in soybean (Glycine max L. Merill) and by G. rosea in red clover (Trifolium pratense L. cv. Huia) was increased only when the soil yeast was inoculated before G. mosseae or G. rosea was introduced. Beneficial effects of R. mucilaginosa on arbuscular mycorrhizal (AM) colonization were found when the soil yeast was inoculated either as a thin agar slice or as a volume of 5 and 10 ml of an aqueous solution. R. mucilaginosa exudates (20 ml per pots) applied to soil increased significantly the percentage of AM colonization of soybean and red clover.

We studied the influence of olive mill dry residue (DOR) on growth of soybean (Glycine max ) and lettuce (Lactuca sativa ) colonized with the arbuscular fungi Glomus mosseae or G. deserticola. The DOR has 6 mg g 21 of soluble phenolic compounds and the application of 2.5 g kg 21 of DOR (15 mg kg 21 of phenolic content) to soil decreased the growth of plants colonized with the AM fungi. The application to the soil of 10 g kg 21 of DOR (60 mg kg 21 of phenolic content) decreased the dry weight of nonarbuscular mycorrhizal (AM) plants. The application of DOR decreased the percentage of AM colonization of plants except those were inoculated with G. mosseae or G. deserticola 4 weeks before the application of DOR. No effect of the residue on the colonization of these previously AM inoculated plants was observed. DOR had no effect neither on the fungal succinate dehydrogenase activity nor on the most probable number of AM propagules in all treated soils. The obtained results showed than AM fungi increased the phytotoxicity of DOR in lettuce and soybean. q 2002 Elsevier Science Ltd. All rights reserved.

The response of plants to arbuscular mycorrhizal fungi involves a temporal and spatial activation of different defence mechanisms. The activation and regulation of these defences have been proposed to play a role in the maintenance of the mutualistic status of the association, however, how these defences affect the functioning and development of arbuscular mycorrhiza remains unclear. A number of regulatory mechanisms of plant defence response have been described during the establishment of the arbuscular mycorrhizal symbiosis, including elicitor degradation, modulation of second messenger concentration, nutritional and hormonal plant defence regulation, and activation of regulatory symbiotic gene expression. The functional characterization of these regulatory mechanisms on arbuscular mycorrhiza, including cross-talk between them, will be the aim and objective of future work on this topic.

We studied the possibility that the antagonistic saprobe fungi Coriolopsis rigida, Trametes versicolor, Fusarium lateritium, Penicillium chrysogenum and Verticillium dahliae-2379 may control V. dahliae disease through the inhibition of the activity of hydrolytic enzymes produced by the pathogen. These saprobe fungi were able to decrease the growth in vitro of V. dahliae. The exudates produced by these fungi seemed to be the main factor responsible for their antagonistic effect. The exudates of all the fungi tested had endopolymethylgalacturonase, endoglucanase and endoxyloglucanase activities. Exudates of P. chrysogenum and V. dahliae-2379 had higher hydrolytic activities than those of V. dahliae. The saprobe V. dahliae-2379 did not decrease the growth of the pathogen V. dahliae but decreased its harmful effect on the growth of tomato. However, no inhibition of the activities of the hydrolytic enzyme of the pathogenic V. dahliae by the no pathogenic V. dahliae-2379 or by the other saprobe fungi was observed. Nevertheless, the protection of tomato by the five antagonistic saprobe fungi against V. dahliae disease could be carried out by the induction of plant defense by the hydrolytic enzymes produced by these fungi.

The capacity of roots to sense soil physicochemical parameters plays an essential role in maintaining plant nutritional and developmental functions under abiotic stress. These conditions generate reactive oxygen species (ROS) in plant tissues causing oxidation of proteins and lipids among others. Some plants have developed adaptive mechanisms to counteract such adverse conditions such as symbiotic association with arbuscular mycorrhizal fungi (AMF). AMF enhance plant growth and improve transplant survival by protecting host plants against environmental stresses. The aim of this study was to evaluate the alleviation of transplanting stress by two strains of Rhizophagus irregularis (GC2 and GA5) in olive. Our results show that olive plants have an additional energetic expense in growth due to an adaptative response to the growing stage and to the mycorrhizal colonization at the first transplant. However, at the second transplant the coinoculation improves olive plant growth and protects against oxidative stress followed by the GA5-inoculation. In conclusion, a combination of two AMF strains at the beginning of olive propagation produces vigorous plants successfully protected in field cultivation even with an additional cost at the beginning of growth. Fil: Bompadre, Maria Josefina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pergola, Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fernandez Bidondo, Laura. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Colombo, Roxana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Silvani, Vanesa Analia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pardo, Alejandro Guillermo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Micología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ocampo, Juan Antonio. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; España Fil: Godeas, Alicia Margarita. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Strigolactones have recently been suggested to be phytohormones that are present in all plants. Strigolactones are released by roots into the rhizosphere, stimulating the seed germination of parasitic plants such as Striga spp. and Orobanche spp. and play a crucial role in the interaction between plants and symbiotic arbuscular mycorrhizal fungi. By applying different concentrations of the synthetic strigolactone analogue GR24 to alfalfa (Medicago sativa) inoculated with Sinorhizobium meliloti we could show that in alfalfa nodulation is positively affected by the presence of the strigolactone analogue GR24. Moreover, we could show that this increased nodulation cannot be linked with a stimulatory effect of GR24 on the growth or the expression of nod genes of S. meliloti. Putative mechanisms operating in the plant in response to the addition of GR24 and leading to increased nodule formation by rhizobia are discussed.

The expression of a lipid transfer protein (LTP) gene is regulated in Oryza sativa roots in response to colonization by the mycorrhizal fungus Glomus mosseae. Transcript levels increased when the fungus forms appressoria and penetrates the root epidermis and decreased at the onset of the intercellular colonization of the root cortex. The analysis of histochemical GUS staining in transgenic rice plants carrying the Ltp/Gus construct confirm the induction of LTP: gene associated with fungal appressoria formation and penetration area. The induction of Ltp gene expression coincided in time with a transient increase in the expression of a phenylalanine ammonia-lyase (PAL:) gene and a transient accumulation of salicylic acid (SA) in the mycorrhizal roots. The expression of LTP: and PAL: was induced in rice roots after treatment with SA and Pseudomonas syringae indicating that both genes could be implicated in the plant defence response. The exogenous application of SA to rice interacting with the mycorrhizal fungus did not affect appressoria formation but, instead, resulted in a transient delay of root mycorrhization. Nevertheless, although LTP: maintained a prolonged SA-induced expression level, mycorrhizal formation could still proceed.

biology, ecological specificity and symbiotic activity (Giovannetti and Gianinazzi-Pearson, 1994). There are The production of hydrolytic enzymes from external many reports of inter- and intraspecific differences in the mycelia associated with roots and colonized soybean efficiency of AM fungi in terms of plant growth and roots (Glycine max L.) inoculated with different arbus- protection (Harley and Smith, 1983; Sieverding, 1991; cular-mycorrhizal (AM) fungi of the genus Glomus, and Ruiz-Lozano and Azcon, 1995; Ruiz-Lozano et al., 1995). the possible relationship between these activities and The physiological basis for these variations is poorly the capacity of the AM fungi to colonize plant roots known. The ability of mycorrhizas to increase plant was studied. There were differences in root coloniza- growth can in most cases be explained by an increased tion and plant growth between the Glomus strains, phosphorus uptake. The hyphal spread of AM fungi is and also between two isolates of G. mosseae. an important factor influencing the phosphorus supply to Hydrolytic activities in the root and external mycelia the host plant (Jakobsen et al., 1992). Mycorrhizal fungi associated with roots differed in the AM fungi tested. may differ in their capacity to develop an external hyphal Correlations were only found between the endoxylog- system regardless of their capacity to colonize the root lucanase activity of the external mycelia associated cortex (Graham et al., 1982). The morphological proper- with roots of the AM fungi tested and the percentage ties and spatial distribution of the external hyphae in soil root colonization or plant growth. However, hydrolytic and differences in hyphal uptake, translocation capacities activities of roots colonized by the different endo- and metabolic activity seem to play an important role in phytes correlated with those of external mycelia. The the efficiency of AM fungi ( Kothari et al., 1991; Jakobsen hydrolytic activities were not qualitatively different et al., 1992). because the endoxyloglucanase from AM colonized Differences in the ability of mycorrhizal fungi to roots and the external mycelia did not show a high enhance phosphorus uptake and growth of the host plant, degree of polymorphism in the different species of even between species for which the extent of root coloniza- fungus tested. The possible role of the hydrolytic activ- tion is similar, may be due to functional differences in ity of external hyphae of AM fungi was discussed as a the host-fungal interface. Different band patterns of factor affecting fungal ability to colonize the root and enzymatic activities in AM fungi belonging to the same influence plant growth.

Catalase and ascorbate peroxidase enzymatic activities were examined during the interaction between Nicotiana tabacum and the arbuscular mycorrhizal Glomus mosseae. Transient enhancements of both enzymatic activities were detected in the inoculated plant roots coinciding in time with the stage of appressoria formation in the root surface. The analysis of free salicylic acid content in roots revealed that the increases in enzymatic activities were coincident in time with the accumulation of SA in inoculated roots. These data indicate that the first reaction of the root cells to the invasion of arbuscular mycorrhizal fungi is a defence response.

The analysis of SA accumulation in roots of plant symbiotic mutants revealed two independent phenomena associated with the inability of either the plant or the microsymbiont to form a compatible symbiosis. SA accumulation in roots of the wild type and symbiosis-resistant P2 (Nod - , Myc - ) Pisum sativum genotypes was induced upon interaction with Glomus mosseae. The amplitude of this accumulation was higher in P2 plants and increased with time, an effect that was not observed in roots of the wild-type P. sativum genotype. Likewise, Rhizobium leguminosarum wild type or a mutant blocked in Nod factor biosynthesis induced SA accumulation in P2, whereas SA accumulation in roots of the wild-type plant was dependent on the inability of the bacterium to produce Nod factors. These results suggest that the sym30 gene, which is mutated in P2 plants, could be implicated in a common pathway that leads to the suppression of an SA-dependent defence mechanism in legume plants against Rhizobium and endomycorrhizal fungi, thus allowing establishment of symbiosis.

We studied the influence of the arbuscular mycorrhizal (AM) non-host plants Stellaria media (Caryophyllaceae), Chenopodium album and Spinaceae oleracea (Chenopodiaceae), Brassica campestris , B. nigra , Capsella bursa-pastoris and Sisymbrium altissimum (Brassicaceae), Juncus balticus (Juncaceae), Urtica dioica (Urticaceae) and of the AM host plant Taraxacum officinale (Asteraceae) on the colonization of Pisum sativum by the AM fungus Glomus mosseae . None of the non-host plants tested were colonized by Glomus mosseae . Older non-host plants competed with P. sativum . No inhibition of AM colonization was observed in host plants that were grown in the same pot and at the same time as non-host plants. However, when non-host plants were grown for 30 d before P. sativum , they inhibited mycorrhizal colonization of the latter. In a split pot system the presence of U. dioica on the left-side decreased AM colonization of P. sativum roots on the left-side, but not on the right-side. Non-host plants decreased the inoculum potential of G. mosseae and in some cases the percentage AM colonization of host plants cultivated after non-host plants. These results indicate that roots of non-host species have factors that seem to affect the AM fungus before it establishes in the root of host plants.

The effect of xyloglucan on spore germination, hyphal length and mycorrhizal colonization of alfalfa plants was studied. The presence of high concentrations of xyloglucan in the rooting medium inhibited mycorrhizal colonization in plants inoculated with Glomus mosseae. Intermediate xyloglucan concentrations had no effect on arbuscular mycorrhizal (AM) colonization, but a low concentration increased mycorrhization of host plants. The effects of these doses on spore germination and hyphal length of G. mosseae were similar to those observed for mycorrhizal colonization. Production of xyloglucanase was assayed during colonization by the AM fungus G. mosseae in lettuce and onion. Endoxyloglucanase activity peaked 15 d after inoculation, whereas exoxyloglucanase activity peaked at 30 and 50 d. Extracts from external mycelia of G. mosseae showed endo- and exoxyloglucanase activities. Some of the endoxyloglucanase activities detected in AM colonized plant roots may be derived from the AM fungus, as endoxyloglucanase proteins found in the external mycelia of G. mosseae and in mycorrhizal root extracts showed similar electrophoretic mobility. These results suggest that xyloglucanase is involved in the process of colonization of plants by G. mosseae.

The aqueous biphasic system (ABS) method using PEG and ammonium sulphate improved the recovery of spores from tropical soils as compared with the sucrose gradient method. The ABS was not affected by low temperatures. The optimal recovery of spores was achieved when 100% PEG and 50% ammonium sulphate concentration and PEG8000 instead of PEG4000 were used. Under these conditions an optimal interface formation which facilitates the localization and recovery of spores was obtained. Both phases of the ABS are aqueous and did not affect either the viability of spores or the level of root colonization reached by these spores. Soil composition did not affect the number of spores obtained by the ABS method either.

The saprophytic fungi Wardomyces inflatus (Marchal) Hennebert, Paecilomyces farinosus (Holm G G. roseum, T. pseudokoningiiand T. harzianum had no effect on germination; and P. farinosus and SDM-54 increased the percentage of spore germination of G. mosseae after 4 d. Wardomyces inflatus significantly decreased hyphal length of spores which germinated, but no other saprophytic fungi affected hyphal growth. Trichoderma pseudokoningii, T. harzianum , P. farinosus and SDM-54 increased the number of auxiliary cells formed by G. mosseae. The effect of saprophytic fungi on arbuscular mycorrhizal colonization of soybean was studied in a greenhouse trial. The percentage of soybean root length colonized was decreased by W. inflatus, unaffected by SDM-54 and T. harzianum, and increased by P. farinosus. Gliocladium roseumdecreased root length colonized when plants were 12 wk old, and T. pseudokoningii increased colonization of roots when plants were 4 wk old. Antagonistic, synergistic and neutral actions of G. mosseae upon the saprophytic fungi were observed. The population of T. harzianum decreased and the populations of T. pseudokoningii and SDM54 increased in the presence of G. mosseae. Our results indicate a complex interaction between G. mosseae and associated saprophytic fungi.

Vesicular-arbuscular mycorrhizal (VAM) fungi interconnect the root systems of adjacent plants, and mediate the transfer of nutrients between them. The objective of this study was to determine if the VAM fungus Glomus deserticola Trappe, Bloss and Menge enhanced such a transfer in a crop-weed association when one associated plant was selectively weakened by an herbicide. Wheat (Triticum aestivum L.) or perennial ryegrass (Lolium perenne L.) plants were grown with or without VAM fungi either in monocultures, or together as an intercrop for 7 weeks, when the herbicide diclofop was applied as a soil drench at dose rates of 0%, 10%, 50%, 100% and 1000% of the field recommendation (0.9 kg ha−1). Seed yield at harvest (13 weeks) was significantly greater for the + VAM than for the − VAM intercropped wheat plants at all dose rates. When grown in monoculture, wheat yield was greater in the − VAM plants. Shoot growth of intercropped + VAM wheat was enhanced, while that of ryegrass was inhibited. In monoculture, the plant dry masses of both − VAM wheat and ryegrass were greater than those of the + VAM plants, while in intercrop the + VAM wheat plants fared better. The data showed that (1) the herbicide inhibited root colonization by G. deserticola in wheat but not in ryegrass, and (2) when grown together, wheat growth and yield were enhanced and ryegrass growth was inhibited in + VAM herbicide-treated associations. We interpret the findings as a change in interplant source-sink relations upon treatment with diclofop: ryegrass roots became sources of nutrients to the tolerant wheat roots (stronger sinks), and the transfer process was enhanced by the VAM mycelium common to both.

The effect of inoculation with the saprophytic fungi Alternaria alternata or Fusarium equiseti on maize (Zea mays) and lettuce (Lactuca sativa) with or without arbuscular mycorrhizal (AM) colonization by Glomus mosseae was studied in a greenhouse trial. Plant dry weights of non-AM-inoculated maize and lettuce were unaffected by the presence of A. alternata and F. equiseti. In contrast, A. alternata and F. equiseti decreased plant dry weights and mycorrhization when inoculated to the rhizosphere before G. mosseae. The saprophytic fungi inoculated 2 weeks after G. mosseae did not affect the percentage of root length colonized by the AM endophyte, but did affect its metabolic activity assessed as succinate dehydrogenase activity. Although F. equiseti inoculated at the same time as G. mosseae did not affect mycorrhization of maize roots, its effect on AM colonization of lettuce roots was similar to that with A. alternata. In the rhizosphere of both plants, the population of saprophytic fungi decreased significantly, but was not affected by the presence of G. mosseae. Our results suggest that there may have been a direct effect of the saprophytic fungi on the mycorrhizal fungi in the extramatrical phase of the latter, and when the AM fungus was established in the root the AM fungus was less affected by the saprophytic fungi.

An arbuscular endoglucanase (EC 3.2.1.4) was purified to homogeneity from roots of onion ( Allium cepa cv. Babosa) colonized by the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe. The stepwise purification procedure consisted of Filtron concentration (10 kDa), anion-exchange chromatography, anion-exchange fast protein liquid chromatography, and electroelution from polyacrylamide gels. Pure endoglucanase had a specific activity of 2500 units mg −1 protein, and was purified 198-fold with a yield of 0.6 μg enzyme g −1 root. The endoglucanase has a relative molecular weight of about 27 kDa, and behaves as a monomer in its native form.