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We would like to thank farmers from Granada province for kindly permitting us to research their olive groves. We also wish to give special thanks to Mr. Antonio Leo´n, chief executive officer of Caja Rural in Granada, for encouraging this research and Toby Wakely for reviewing this manuscript in English., Data Availability Statement: All data are available from the GitHub repository (https://github.com/ jmrmcode/Verticillium-wilt-Dataset.git)., Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae, is one of the most harmful diseases in Mediterranean olive-growing areas. Although, the effects of both soil temperature and moisture on V. dahliae are well known, there is scant knowledge about what climatic drivers affect the occurrence of the pathogen on a large scale. Here, we investigate what climatic drivers determine V. dahliae occurrence in olive-growing areas in southern Spain. In order to bridge this gap in knowledge, a large-scale field survey was carried out to collect data on the occurrence of V. dahliae in 779 olive groves in Granada province. Forty models based on competing combinations of climatic variables were fitted and evaluated using information-theoretic methods. A model that included a multiplicative combination of seasonal and extreme climatic variables was found to be the most viable one. Isothermality and the seasonal distribution of precipitation were the most important variables influencing the occurrence of the pathogen. The isothermal effect was in turn modulated by the seasonality of rainfall, and this became less negative as seasonality increases. Thus, V. dahliae occurs more frequently in olive-growing areas where the day-night temperature oscillation is lower than the summer-winter one. We also found that irrigation reduced the influence of isothermality on occurrence. Our results demonstrate that long-term compound climatic factors rather than “primary” variables, such as annual trends, can better explain the spatial patterns of V. dahliae occurrence in Mediterranean, southern Spain. One important implication of our study is that appropriate irrigation management, when temperature oscillation approaches optimal conditions for V. dahliae to thrive, may reduce the appearance of symptoms in olive trees., Caja Rural Foundation of Granada

D14 and KAI2 receptors enable plants to distinguish between strigolactones (SLs) and karrikins (KARs), respectively, in order to trigger appropriate environmental and developmental responses. Both receptors are related to the regulation of arbuscular mycorrhiza (AM) formation and are members of the RsbQ-like family of α,β-hydrolases. DLK2 proteins, whose function remains unknown, constitute a third clade from the RsbQ-like protein family. We investigated whether the tomato SlDLK2 is a new regulatory component in the AM symbiosis. Genetic approaches were conducted to analyze SlDLK2 expression and to understand SlDLK2 function in AM symbiosis. We show that SlDLK2 expression in roots is AM-dependent and is associated with cells containing arbuscules. SlDLK2 ectopic expression arrests arbuscule branching and downregulates AM-responsive genes, even in the absence of symbiosis; while the opposite effect was observed upon SlDLK2 silencing. Moreover, SlDLK2 overexpression in Medicago truncatula roots showed the same altered phenotype observed in tomato roots. Interestingly, SlDLK2 interacts with DELLA, a protein that regulates arbuscule formation/degradation in AM roots. We propose that SlDLK2 is a new component of the complex plant-mediated mechanism regulating the life cycle of arbuscules in AM symbiosis., Tania Ho-Plágaro was supported by a research fellowship from the FPI-MINECO program. 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, industria y Competitividad in Spain (AGL2014-52298-P, AGL2017-83871-P), and Shanghai Center for Plant Stress Biology (Chinese Academy of Sciences) and the Chinese 1000 Talents program.

Histochemical staining and light microscopy-based techniques have been widely used to detect and quantify arbuscular mycorrhizal fungi (AMF) in roots. Here we describe a standardized method for staining of AMF in colonized roots, and we provide possible modifications to adjust the protocol according to particular requirements, such as the type of root material or the reduction of toxic products. In addition, we also summarize some of the most common ways to quantify arbuscular mycorrhizal colonization.

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

1. Isolation and Culture of Arbuscular Mycorrhizal Fungi from Field Samples Dora Trejo-Aguilar and Jacob Banuelos 2. The Mycorrhizal Donor Plant (MDP) In Vitro Culture System for the Efficient Colonization of Whole Plants Ismahen Lalaymia and Stephane Declerck 3. A Whole-Plant Culture Method to Study Structural and Functional Traits of Extraradical Mycelium Cristiana Sbrana, Alessandra Pepe, Nuria Ferrol, and Manuela Giovannetti 4. Histochemical Staining and Quantification of Arbuscular Mycorrhizal Fungal Colonization Tania Ho-Plagaro, Maria Isabel Tamayo-Navarrete, and Jose Manuel Garcia-Garrido 5. Fluorescent Staining of Arbuscular Mycorrhizal Structures Using Wheat Germ Agglutinin (WGA) and Propidium Iodide Gennaro Carotenuto and Andrea Genre 6. Visualization of Arbuscular Mycorrhizal Fungal Extra-Radical Hyphae and Spores Vitality and Activity Pablo Cornejo and Humberto Aponte 7. Molecular Quantification of Arbuscular Mycorrhizal Fungal Root Colonization Martina Janouskova and Petra Caklova 8. Single-Spore-Extraction for Genetic Analyses of Arbuscular Mycorrhizal Fungi Dirk Redecker 9. High Throughput DNA Sequence-Based Analysis of AMF Communities Iris Marisa Maxaieie Victorino, Andrea Berruti, Alberto Orgiazzi, Samuele Voyron, Valeria Bianciotto, and Erica Lumini 10. Analysis of Arbuscular Mycorrhizal Fungal Communities by Terminal Restriction Fragment Length Polymorphism (TRFLP) Alvaro Lopez-Garcia 11. Bioinformatic Methods for the Analysis of High-Throughput RNA Sequencing in Arbuscular Mycorrhizal Fungi Francesco Venice, Alessandra Salvioli di Fossalunga, and Paola Bonfante 12. Arbuscular Mycorrhizal Fungal Gene Expression Analysis by Real-Time PCR Alessandro Silvestri, Jacob Perez-Tienda, and Juan Antonio Lopez-Raez 13. Laser Microdissection as a Useful Tool to Study Gene Expression in Plant and Fungal Partners in AM Symbiosis Raffaella Balestrini and Valentina Fiorilli 14. Detection of Arbuscular Mycorrhizal Fungal Gene Expression by In Situ Hybridization Bettina Hause and Natalia Requena 15. Functional Analyses of Arbuscular Mycorrhizal Fungal Genes in Yeast Elisabeth Tamayo, Tamara Gomez-Gallego, and Nuria Ferrol 16. Isotope Labelling to Study Phosphorus Uptake in the Arbuscular Mycorrhizal Symbiosis Carla Cruz-Paredes and Mayra E. Gavito 17. Recovery of Extra-Radical Fungal Peptides Amenable for Shotgun Protein Profiling in Arbuscular Mycorrhizae Ghislaine Recorbet, Pierre-Emmanuel Courty, and Daniel Wipf 18. Host-Induced Gene Silencing of Arbuscular Mycorrhizal Fungal Genes via Agrobacterium Rhizogenes-Mediated Root Transformation in Medicago truncatula Meike Hartmann, Stefanie Voss, and Natalia Requena 19. Application of Virus-Induced Gene Silencing to Arbuscular Mycorrhizal Fungi Tatsuhiro Ezawa, Hayato Maruyama, Yusuke Kikuchi, Kaede Yokoyama, and Chikara Masuta

Srivastava V., Mehrotra S., Mishra S. (eds) 2020 Hairy Root Cultures Based Applications. Rhizosphere Biology. pp. 237. ISBN: 978-981-15-4054-7, Arbuscular Mycorrhizal symbiosis is a mutualistic endosymbiosis widely distributed in the plant kingdom which has a significant impact on plant growth and health. Agrobacterium rhizogenes-mediated root transformation and composite plant generation have been described as a rapid method to assess gene functions in roots without the need for stable transformation plant production. We describe an optimized protocol for composite tomato plant obtaining achieved through A. rhizogenes-mediated transformation, and we also highlight key differences with other protocols that should be taken into account to adjust this method to the transformation of other plant species. This protocol has been adopted as a useful tool for localizing the promoter expression of genes putatively associated with mycorrhization or for functional analyses in mycorrhizal studies by reverse genetics., This work 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, industria y Competitividad in Spain (AGL2014-52298-P, AGL2017-83871-P).

Ferrol, N.; Lanfranco, L. (Eds.) 2020 Arbuscular Mycorrhizal Fungi. Methods and Protocols. Vol 2146. pp. 257. ISBN 978 1 0716-0602-5, Histochemical staining and light microscopy-based techniques have been widely used to detect and quantify arbuscular mycorrhizal fungi (AMF) in roots. Here we describe a standardized method for staining of AMF in colonized roots, and we provide possible modifications to adjust the protocol according to particular requirements, such as the type of root material or the reduction of toxic products. In addition, we also summarize some of the most common ways to quantify arbuscular mycorrhizal colonization., This work 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 (AGL2017-83871-P).

Ralstonia solanacearum is a devastating soil borne vascular pathogen that is able to infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably under-explored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease, but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed an easy method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 of tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relative short time and with small requirements of equipment and plant growth space.SUMMARYA versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

The formation and functioning of arbuscular mycorrhizal (AM) symbiosis are complex and tightly regulated processes. Transcriptional regulation mechanisms have been reported to mediate gene expression changes closely associated with arbuscule formation, where nutrients move between the plant and fungus. Numerous genes encoding transcription factors (TFs), with those belonging to the GRAS family being of particular importance, are induced upon mycorrhization. In this study, a screening for candidate transcription factor genes differentially regulated in AM tomato roots showed that more than 30% of known GRAS tomato genes are upregulated upon mycorrhization. Some AM-upregulated GRAS genes were identified as encoding for transcription factors which are putative orthologs of previously identified regulators of mycorrhization in other plant species. The symbiotic role played by other newly identified AM-upregulated GRAS genes remains unknown. Preliminary results on the involvement of tomato SlGRAS18, SlGRAS38, and SlGRAS43 from the SCL3, SCL32, and SCR clades, respectively, in mycorrhization are presented. All three showed high transcript levels in the late stages of mycorrhization, and the analysis of promoter activity demonstrated that SlGRAS18 and SlGRAS43 are significantly induced in cells containing arbuscules. When SlGRAS18 and SlGRAS38 genes were silenced using RNA interference in hairy root composite tomato plants, a delay in mycorrhizal infection was observed, while an increase in mycorrhizal colonization was observed in SlGRAS43 RNAi roots. The possible mode of action of these TFs during mycorrhization is discussed, with a particular emphasis on the potential involvement of the SHR/SCR/SCL3 module of GRAS TFs in the regulation of gibberellin signaling during mycorrhization, which is analogous to other plant developmental processes., 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 (AGL2014-52298-P, AGL2017-83871-P).

Arbuscular mycorrhizal (AM) formation enhances plant growth and fitness through improved uptake of water and mineral nutrients in exchange for carbon compounds to the AM fungus. The fungal structure for the reciprocal exchange of nutrients in the symbiosis is the arbuscule, and defence genes expressed in cells containing arbuscules could play a role in the control of hyphal spread and arbuscule formation in the root. We characterized and analyzed the Ptc52 gene from tomato (SlPtc52), a member of the gene family of non-heme oxygenases, whose function has been related to the lethal leaf spot 1 (Lls1) lesion mimic phenotype in plants which is sometimes associated with enhanced disease resistance. Sequence analysis of the SlPTC52 protein revealed conserved typical motifs from non-heme oxygenases, including a Rieske [2Fe-2S] motif, a mononuclear non-heme iron-binding motif and a C-terminal CxxC motif. The level of transcript accumulation was low in stem, flower and green fruits, and high in leaves. Although SlPtc52 expression was perceptible at low levels in roots, its expression increased concomitantly with AM fungus root colonization. Tomato non-mycorrhizal hairy roots expressing the GUS protein under the control of promoter SlPtc52 exhibited GUS activity in the endodermis, the apical meristem of the root tip and in the lateral root primordium. AM fungal colonization also resulted in intensive GUS activity that clearly corresponds to cortical cells containing arbuscules. SlPtc52 gene silencing led to a delay in root colonization and a decrease in arbuscular abundance, suggesting that SlPTC52 plays a regulatory role during AM symbiosis., We wish to thank the Tomato Genetics Resource Centre (TGRC) of the University of California for providing the tomato seeds. This study was supported by grants from the Interministerial Commission of Science and Technology (CICYT) and the European Regional Development Fund (FEDER) through the Spanish Ministry of the Economy and Competitiveness (AGL2014-52298-P; AGL2017-83871-P). J.A. Martín Rodríguez was supported by a research fellowship from the FPU-MICINN program. We also wish to thank Michael O’Shea for proofreading the manuscript.

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.

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.

SUMMARYArbuscular mycorrhizal (AM) fungi increase nitrogen (N) uptake by their host plants, but their role in plant N capture from soil organic material is still unclear. In particular, it is not clear if AM fungi compete with the host plant for the N coming from the decomposing organic matter (OM), especially when the AM extraradical mycelium (ERM) and plant roots share the same soil volume. The goal of the present research was to study the effects of AM fungi on wheat N capture after the addition of 15N-labelled OM to soil. Durum wheat (Triticum durum) was grown under controlled conditions in a sand:soil mix and the following treatments were applied: (1) AM inoculation with Glomus mosseae and uninoculated control; and (2) soil amended with 15N-enriched maize leaves and unamended soil. The addition of OM reduced plant growth and N uptake. The AM fungi increased both plant growth and N uptake compared with uninoculated control plants and the effect was enhanced when wheat was grown in soil amended with OM compared with the unamended control. Although AM fungi increased soil N mineralization rates and total plant N uptake, they strongly reduced wheat N recovery from OM, suggesting that AM fungi have marked effects on competition between plants and bacteria for the different N sources in soil.

The establishment of an Arbuscular Mycorrhizal symbiotic interaction (MA) is a successful strategy to substantially promote plant growth, development and fitness. Numerous studies have supported the hypothesis that plant hormones play an important role in the recognition and establishment of symbiosis. Particular attention has been devoted to jasmonic acid (JA) and its derivates, the jasmonates, which are believed to play a major role in AM symbiosis. Jasmonates belong to a diverse class of lipid metabolites known as oxylipins that include other biologically active molecules. Recent transcriptional analyses revealed upregulation of the oxylipin pathway during AM symbiosis in mycorrhizal tomato roots and point a key regulatory feature for oxylipins during AM symbiosis in tomato, particularly these derived from the action of 9-lipoxygenases (9-LOX). In this mini-review we highlight recent progress understanding the function of oxylipins in the establishment of the AM symbiosis and hypothesize that the activation of the 9-LOX pathway might be part of the activation of host defense responses which will then contribute to both, the control of AM fungal spread and the increased resistance to fungal pathogens in mycorrhizal plants.

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 establishment of an arbuscular mycorrhizal (AM) symbiotic interaction is a successful strategy for the promotion of substantial plant growth, development, and fitness. Numerous studies have supported the hypothesis that plant hormones play an important role in the establishment of functional AM symbiosis. Particular attention has been devoted to jasmonic acid (JA) and its derivates, which are believed to play a major role in AM symbiosis. Jasmonates belong to a diverse class of lipid metabolites known as oxylipins that include other biologically active molecules. Recent transcriptional analyses revealed up-regulation of the oxylipin pathway during AM symbiosis in mycorrhizal tomato roots and indicate a key regulatory role for oxylipins during AM symbiosis in tomato, particularly those derived from the action of 9-lipoxygenases (9-LOXs). Continuing with the tomato as a model, the spatial and temporal expression pattern of genes involved in the 9-LOX pathway during the different stages of AM formation in tomato was analysed. The effects of JA signalling pathway changes on AM fungal colonization were assessed and correlated with the modifications in the transcriptional profiles of 9-LOX genes. The up-regulation of the 9-LOX oxylipin pathway in mycorrhizal wild-type roots seems to depend on a particular degree of AM fungal colonization and is restricted to the colonized part of the roots, suggesting that these genes could play a role in controlling fungal spread in roots. In addition, the results suggest that this strategy of the plant to control AM fungi development within the roots is at least partly dependent on JA pathway activation.

The effect of the arbuscular mycorrhizal symbiosis (AM) varies in plant cultivars. In the present study, we tested whether wild-type, old and modern tomato cultivars differ in the parameters of the AM interaction. Moreover, the bioprotective effect of AM against the soilborne tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol) was tested in the different cultivars. Ten tomato cultivars were inoculated with the arbuscular mycorrhizal fungus (AMF) Glomus mosseae alone or in combination with Fol. At the end of the experiment, AM root colonization, Fusarium infection, and the plant fresh weight was determined. The tomato cultivars differed in their susceptibility to AMF and Fol, but these differences were not cultivar age dependent. In all the cultivars affected by Fol, mycorrhization showed a bioprotective effect. Independent of the cultivar age, tomato cultivars differ in their susceptibility to AMF and Fol and the bioprotective effect of mycorrhization, indicating that the cultivar age does not affect the AM parameters tested in this study.

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.

It has been shown that salicylic acid (SA) acts as an endogenous signal molecule responsible for inducing abiotic stress tolerance in plants. The effect of SA and sodium chloride (NaCl) on growth, metabolite accumulation, oxidative stress and enzymatic and non-enzymatic antioxidant responses on common bean plants (Phaseolus vulgaris, cv. F-15) was studied. Results revealed that either SA or NaCl decrease, shoot, root and total plant dry weights. SA treatments decreased the contents of proline, and reduced forms of ascorbate and glutathione, however, the content of soluble sugars (TSS), thiobarbituric acid-reactive substances (TBARs) and oxidized ascorbate remained unaffected. On the other hand, salinity significantly reduced the levels of endogenous SA but increased the content of proline, soluble sugars, TBARs, ascorbate and glutathione, as well as all increasing the levels of antioxidant enzyme activities assayed, except CAT. The application of SA improved the response of common bean plants to salinity by increasing plant dry weight and decreasing the content of organic solutes (proline and TSS) and damage to the membrane (TBARs). Moreover, SA application under saline conditions decreased the levels of antioxidant enzyme activities POX, APX and MDHAR which could indicate successful acclimatization of these plants to saline conditions.

Verticillium wilt (VW) is caused by the soilborne pathogen, Verticillium dahliae. The fact that available control measures are not completely effective, together with an increasing spread of the highly virulent defoliating pathotype (D) in the last two decades, makes VW one of the most threatening olive tree disease. For better management, epidemiology and spatial patterns of VW were investigated in an important olive-growing area in southern Spain. A sampling survey was conducted from 2002 to 2005 in 873 olive orchards, which were assessed for the presence of V. dahliae . PCR assays were used to identify the highly virulent defoliating (D) and nondefoliating (ND) pathotypes of V. dahliae . Ordinary kriging, a geostatistical tool that uses regression techniques for interpolation of spatial data, was applied to obtain predictive V. dahliae incidence maps and to evaluate the correlation between distance from the river and VW. Prevalence in olive orchards was 14.1%, with a disease incidence of 1.9% and mean incidence per olive orchard of 9.5%. Prevalence of the ND pathotype was higher (10.7%) than for the D pathotype (3.4%). VW was three times more prevalent in irrigated than in non-irrigated orchards (relative risk = 3.01) and there was a close association between the ND pathotype and irrigated orchards (relative risk = 2.03). Spatial pattern analysis showed several factors involved in the spread of the pathogen. VW was more prevalent in the north-west, suggesting the spreading of the disease from other bordering areas known to be contaminated, in southwest Spain. The D pathotype had a very restricted and aggregated distribution, suggesting the introduction of this pathotype in new olive orchards areas. The incidence map generated by kriging displayed an increase in north, northeast and south areas with numerous subareas with probabilities equalling or exceeding 53%, showing the effect of intensive agriculture (high plant density and irrigation) on incidence of the disease. Assessment of rivers-pathogen relationship revealed an association of VW with the distance to the nearest river, indicating that soils through the rivers' axis and previously cropped with horticultural crops susceptible to V. dahliae , have implications for the establishment of new olive orchards and the occurrence of the disease.

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.

In recent years, the spread of Verticillium wilt in olive orchards, caused by the soil-borne pathogen Verticillium dahliae, is often related to intensive modern farming of highly productive cultivars, planted at high densities, usually irrigated, and under a mechanised system. The effects of agricultural factors associated with olive orchards were investigated in an important olive-growing area in southern Spain, as tools in predicting outbreaks of the disease. A stratified double-sampling technique was designed to determine the number of olive orchards needed to survey. A sampling survey was conducted from 2002 to 2005 in 873 olive orchards randomly selected, the owners were interviewed for details of agronomic factors, and orchards were inspected for the presence or absence of the disease. Polymerase chain reaction assays were carried out for identifying V. dahliae pathotypes. Pathogen prevalence showed a significant linear correlation with the mean plant density (r 2 = 0.93), associated predominantly with a less virulent non-defoliating pathotype (r 2 = 0.96). Overall, irrigation × high density caused disease incidence to peak in super-high-density olive-tree-planting systems. Olive orchards that had V. dahliae, however, did not differ in pathogen prevalence regardless of the olive cultivars. Young olive orchards were significantly more affected by V. dahliae than were old ones, particularly orchards with trees 8 to 12 years old. Irrigation increased pathogen prevalence and disease incidence in very young orchards (

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.

The systemic effect of root colonization by the arbuscular mycorrhizal fungus Glomus mosseae on infection of barley by Gaeumannomyces graminis var. tritici (Ggt) was studied. In split-root systems of barley one side was inoculated with G. mosseae and the other side was inoculated with Ggt. Root infection by Ggt was systemically reduced when barley plants showed high degrees of mycorrhizal root colonization, whereas a low mycorrhizal root colonization exhibited no effect on Ggt infection. Our results show a clear systemic bioprotectional effect depending on the degree of root colonization by the mycorrhizal fungus. At a higher mycorrhizal colonization rate the concentration of salicylic acid (SA) was increased in roots colonized by the mycorrhizal fungus but no systemic increase of SA could be measured in non-mycorrhizal roots of mycorrhizal plants, indicating that the systemic bioprotectional effect against Ggt is not mediated by salicylic acid.

© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust. For survival, plants have to efficiently adjust their phenotype to environmental challenges, finely coordinating their responses to balance growth and defence. Such phenotypic plasticity can be modulated by their associated microbiota. The widespread mycorrhizal symbioses modify plant responses to external stimuli, generally improving the resilience of the symbiotic system to environmental stresses. Phytohormones, central regulators of plant development and immunity, are instrumental in orchestrating plant responses to the fluctuating environment, but also in the regulation of mycorrhizal symbioses. Exciting advances in the molecular regulation of phytohormone signalling are providing mechanistic insights into how plants coordinate their responses to environmental cues and mycorrhizal functioning. Here, we summarize how these mechanisms permit the fine-tuning of the symbiosis according to the ever-changing environment., We apologize to all those colleagues whose work has not been cited because of space limitations, but have greatly contributed to our understanding of phytohor mone functions in mycorrhizas. This work was supported by grants AGL2012-39923 and AGL2011-25930 from the Spanish Ministry of Science an d Innovation (MINCIN). We thank Rocio Tor res for pictures of mycorrhizas and Drs J. M. Barea and A. Biere for their critical reading of the manuscript.

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.

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.

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.

Using a genomic clone encoding a rice lipid transfer protein, LTP1, we analysed the activity of the 5′ region of the Ltp1 gene in transgenic rice (Oryza sativaL.) during plant development and under pathogen attack. The −1176/+13, −556/+13 and −284/+13 regions of the promoter were fused upstream from the uidA reporter gene and nos 3′ polyadenylation signal, resulting in the pΔ1176Gus, pΔ556Gus and pΔ284Gus constructs which were transferred to rice by microprojectile bombardment. Histochemical and fluorometric GUS assays and in situ detection of uidA transcripts in transgenic homozygous lines harbouring the pΔ1176Gus construct demonstrated that the Ltp1 promoter is preferentially active in aerial vegetative and reproductive organs and that both specificity and level of expression are regulated during organ development. In leaf sheath, GUS activity which is initially strictly localized in the epidermis of growing tissue, becomes restricted to the vascular system in mature tissues. In expanded leaf blade, expression of the uidA gene was restricted to the cutting level suggesting inducibility by wounding. Strong activity was detected in lemma and palea, sterile glumes, and immature anther walls and microspores but not in female reproductive organs. No GUS activity was detected during seed embryo maturation whereas the uidA gene was strongly expressed at early stages of somatic embryogenesis in scutellum tissue. The Ltp1 transcripts were found to strongly accumulate in response to inoculation with the fungal agent of the blast disease, Magnaporthe grisea,in two rice cultivars exhibiting compatible or incompatible host-pathogen interactions. Analysis of pΔ1176Gus leaf samples inoculated with the blast fungus demonstrated that the Ltp1promoter is induced in all cell types of tissues surrounding the lesion and notably in stomata guard cells. In plants harbouring the Ltp1 promoter deletion construct pΔ556Gus, activity was solely detected in the vascular system of mature leaves whereas no uidA gene expression was observed in pΔ284Gus plants. These observations are consistent with the proposed role of LTP1 in strenghtening of structural barriers and organ protection against mechanical disruption and pathogen attack.

Summary The plant signal compounds jasmonic acid or salicylic acid were applied as abiotic elicitors to leaves of glucosinolate-containing members of the Tropaeolaceae (Tropaeolum majus) and Caricaceae (Carica papaya) and to leaves of the glucosinolate-free plant cucumber. Both compounds are known to induce the accumulation of glucosinolates in Brassica plants. In roots of glucosinolate-containing plants the two compounds enhanced glucosinolate levels or new glucosinolates were accumulated. In all plants treated with jasmonic acid a reduction of root colonization by the arbuscular mycorrhizal fungus Glomus mosseae was observed. No such effect occurred after the salicylic acid treatment. In addition, members of the glucosinolate-containing Tropaeolaceae family were inoculated with the arbuscular mycorrhizal fungus (AMF), and later on the glucosinolate content was determined in roots of mycorrhizal and in roots of non-mycorrhizal plants. Root colonization by the AMF resulted in a large increase of the glucosinolate content, however, the glucosinolate levels in mycorrhizal and in non-mycorrhizal plants showed no effect on root colonization by the AMF. From our results we concluded that the glucosinolate levels can not generally be linked to the nonhost status of glucosinolate-containing plants, however, a role of specific glucosinolates in the expression of the non-host status of glucosinolate-containing plants can not be excluded. Moreover, we found that the application of jasmonic acid highly suppresses mycorrhization and this suppression is not a glucosinolate-dependent mechanism.

© 2014 Scandinavian Plant Physiology Society. Gibberellins (GAs) are key regulators of plant growth and development and recent studies suggest also a role during arbuscular mycorrhizal (AM) formation. Here, complementary approaches have been used to obtain a clearer picture that correlates AM fungal development inside roots with GA metabolism. An extensive analysis of genes associated with GA metabolism as well as a quantification of GA content in roots was made. Application of GA3 and its biosynthesis inhibitor prohexadione calcium (PrCa) combined with a GA-constitutive response mutant (procera) were used to determine whether fungal colonization is altered by the level of these hormones or by changes in the GA-signaling pathway. The increased levels of specific GAs from the 13-hydroxylation pathway in mycorrhizal roots correlate closely with the increased expression of genes coding enzymes from the GA biosynthetic trail. The imbalance of GAs in tomato roots caused by exogenous applications of GA3 or PrCa affects arbuscules in both negative and positive ways, respectively. In addition, procera plants were adversely affected by the mycorrhization process. Our findings demonstrate that an imbalance in favor of an increased amount of GAs negatively affects the frequency of mycorrhization and particularly the arbuscular abundance in tomato mycorrhizal roots and the results point out that AM formation is associated with a change in the 13-hydroxylation pathway of GAs., We wish to thank the Tomato Genet- ics Resource Centre (TGRC) of the University of California for providing tomato seeds. We would also like to thank Isabel Tamayo for her help with the plant experiments and Michael O’Shea for proof-reading the document. Financial support for this study was provided by grants from the Comisión Interministerial de Ciencia y Teconlogía (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). JA Martín Rodríguez was supported by fellowships under the FPU-MICINN program. The authors also acknowledge financial support of Ministry of Education, Youth and Sports of the Czech Republic (LK21306), EU funding from the Operational Program Research and Development for Innovations (ED0007/01/01) and Internal Grant Agency of Palack y University (PrF_2013_023).

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.

In a split root system with barley plants, one half of the divided root system had been colonized by the arbuscular mycorrhizal fungi Glomus mosseae, G. intraradices or Gigaspora rosea. After extensive root colonization by the fungi, the other half of the divided root system was inoculated with G. mosseae. Prior colonization of one half of the split root system with any of the three fungi resulted in a clear suppression of colonization by G. mosseae in the other half of the root system. As carbohydrates are one of the principal elements of plant growth, AM colonization, which is supposed to present a carbon sink, on one half of the split root system, could affect root growth and thus root fresh weight, on the non-mycorrhizal half of the root system. Besides, the improved P-status of mycorrhizal plants has been suggested to affect further AM root colonization. Our experiment showed that neither the root fresh weight, nor the P-concentration differed significantly in split root systems of non-mycorrhizal control plants and in split root systems of half mycorrhizal plants. Thus the suppressional effect in prior AM colonized plants on subsequent AM root colonization seemed not to be linked to a competition of the AM fungi for carbohydrates or an altered P-status. A systemic suppression of further mycorrhization in mycorrhizal roots, which is not AM fungal species or genera specific is discussed.

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.

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 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.

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.

A protocol is described for simultaneous histochemical detection of LacZ and Gus activity in plant tissues after microprojectile bombardment. The suitability of two different Gus substrates (Salmon-glcA, Magenta-β-d-glcA) is compared. This detection system is used to assay the number of cells expressing either or both reporter gene. This technique is used as a qualitative assay to demonstrate the tissue specificity of a Hrgp promoter in maize embryos, and to measure ABA responsiveness of a Lea promoter in Arabidopsis. The promoter to be studied is linked to the gus reporter gene and the lacZ reporter gene linked to the CaMV 35S promoter is used as a constitutive internal control. The use of an internal control drastically reduces the data variation inherent to microprojectile bombardment.

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.