Foliar mycoendophytome of an endemic plant of the Mediterranean biome (Myrtus communis) reveals the dominance of basidiomycete woody saprotrophs.

The true myrtle, Myrtus communis, is a small perennial evergreen tree that occurs in Europe, Africa, and Asia with a circum-Mediterranean geographic distribution. Unfortunately, the Mediterranean Forests, where M. communis occurs, are critically endangered and are currently restricted to small fragmented areas in protected conservation units. In the present work, we performed, for the first time, a metabarcoding study on the spatial variation of fungal community structure in the foliar endophytome of this endemic plant of the Mediterranean biome, using bipartite network analysis as a model. The local bipartite network of Myrtus communis individuals and their foliar endophytic fungi is very low connected, with low nestedness, and moderately high specialization and modularity. Similar network patterns were also retrieved in both culture-dependent and amplicon metagenomics of foliar endophytes in distinct arboreal hosts in varied biomes. Furthermore, the majority of putative fungal endophytes species were basidiomycete woody saprotrophs of the orders Polyporales, Agaricales, and Hymenochaetales. Altogether, these findings suggest a possible adaptation of these wood-decaying fungi to cope with moisture limitation and spatial scarcity of their primary substrate (dead wood), which are totally consistent with the predictions of the viaphytism hypothesis that wood-decomposing fungi inhabit the internal leaf tissue of forest trees in order to enhance dispersal to substrates on the forest floor, by using leaves as vectors and as refugia, during periods of environmental stress.

An improved method for Agrobacterium rhizogenes-mediated transformation of tomato suitable for the study of arbuscular mycorrhizal symbiosis.

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.

Gibberellin-Abscisic Acid Balances during Arbuscular Mycorrhiza Formation in Tomato.

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.

Plant 9-lox oxylipin metabolism in response to arbuscular mycorrhiza.

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.

A comparison of wild-type, old and modern tomato cultivars in the interaction with the arbuscular mycorrhizal fungus Glomus mosseae and the tomato pathogen Fusarium oxysporum f. sp. lycopersici.

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.

Altered pattern of arbuscular mycorrhizal formation in tomato ethylene mutants.

Although no specific role has been demonstrated for ethylene during Arbuscular Mycorrhizal (AM) symbiosis, recent results suggest its participation in the regulation of the AM. Analysis of arbuscular mycorrhizal colonization in the abscisic acid (ABA)-deficient tomato sitiens mutant has shown that ABA deficiency induced ethylene production. It has also been suggested that one of the mechanisms used by ABA to determine susceptibility to fungal infection is negative modulation of the ethylene pathway. In this study, we describe the pattern of mycorrhization in mutant plants with altered ethylene biosynthesis and/or perception pathways. Epinastic (epi) plants with increased ethylene response were unaffected in terms of mycorrhizal frequency, although this mutation had a considerable negative impact on the intensity of mycorrhizal root colonization. The negative impact of the mutation in epi plants on the intensity of mycorrhizal root colonization was associated with a transitory increase in the transcript level of the LeETR6 ethylene receptor gene. On the other hand, ripenenig-inhibitor (rin) tomato mutant plants were positively affected in relation to all the mycorrhizal colonization parameters measured, suggesting that, at least in tomato plants, the regulation of AM formation is mediated by the RIN pathway.

The effect of flavones and flavonols on colonization of tomato plants by arbuscular mycorrhizal fungi of the genera Gigaspora and Glomus.

No clear data are available on how flavonoids from different chemical groups affect root colonization by arbuscular mycorrhizal fungi (AMF) and whether flavonoids affecting the presymbiotic growth of AMF also affect root colonization by AMF. In the present work, we compared the effect of flavones (chrysin and luteolin) and flavonols (kaempferol, morin, isorhamnetin, and rutin) on root colonization (number of entry points and degree of root colonization) of tomato plants (Lycopersicum esculentum L.) with the effect of these flavonoids on the presymbiotic growth of these AMF, which has been reported in a recent study. With all tested AMF (Gigaspora rosea, Gigaspora margarita, Glomus mosseae, and Glomus intraradices) a correlation between the number of entry points and the percentage of root colonization was found. When the number of entry points was high, root colonization was also enhanced. Application of the flavones chrysin and luteolin and of the flavonol morin increased the number of entry points and the degree of colonization,whereas the flavonols kaempferol, isorhamnetin, and rutin showed no effect. These results show that in contrast to their effect on the presymbiotic growth of the AMF on the level of root colonization, the tested flavonoids do not exhibit a genus- and species-specificity. Moreover, comparison of our data with the data obtained by J.M. Scervino, M.A. Ponce, R. Erra-Bassells, H. Vierheilig, J.A. Ocampo, and A. Godeas. (2005a. J. Plant Interact. 15: 22-30) indicates that a positive effect on the hyphal growth of AMF does not necessarily result in an enhanced AM root colonization, further indicating that the mode of action of flavonoids at the level of root colonization is more complex.

Solid-state cultures of Fusarium oxysporum transform aromatic components of olive-mill dry residue and reduce its phytotoxicity.

The present study mainly investigated the ability of solid-state cultures of the non-pathogenic Fusarium oxysporum strain BAFC 738 to transform aromatic components to reduce the phytotoxicity in olive-mill dry residue (DOR), the waste from the two-phase manufacturing process. Lignin, hemicellulose, fats and water-soluble extractives contents of DOR colonized by the fungus for 20 weeks were reduced by 16%, 25%, 71% and 13%, respectively, while the cellulose content increased by 25%. In addition, the ethyl acetate-extractable phenolic fraction of the waste was reduced by 65%. However, mass-balance ultra-filtration and size-exclusion chromatography experiments suggested that the apparent removal of that fraction, mainly including 2-(3,4-dihydroxyphenyl)ethyl alcohol and 2-(4-hydroxyphenyl)ethyl alcohol, was due to polymerization. Mn-peroxidase and Mn-independent peroxidase activities were found in F. oxysporum solid-state cultures, while laccase and aryl alcohol oxidase activities were not detected. Tests performed with seedlings of tomato (Lycopersicum esculentum L.), soybean (Glycine maximum Merr.), and alfalfa (Medicago sativa L.) grown on soils containing 6% (w/w) of bioconverted DOR (kg soil)(-1) showed that the waste's phytotoxicity was removed by 20 weeks-old fungal cultures. By contrast, the same material exhibited a high residual toxicity towards lettuce (Lactuca sativa L.).

Beneficial effect of saprobe and arbuscular mycorrhizal fungi on growth of Eucalyptus globulus co-cultured with Glycine max in soil contaminated with heavy metals.

The effects of saprobe and arbuscular mycorrhizal (AM) fungi on growth, chlorophyll and N, P and K content of Eucalyptus globulus Labill. growing in soil contaminated by heavy metals in the presence or absence of Glycine max were investigated. Glomus mosseae and Glomus deserticola increased dry weight, shoot length, total N, P and K concentration and the quantity of chlorophyll in E. globulus shoots. The protection of Eucalyptus by AM fungi against the action of the heavy metals was more evident when this plant grew as an intercrop with soybean than as a monoculture. The presence of the saprobe fungi Fusarium concolor and Trichoderma koningii further enhanced shoot dry weight, N, P and K content of AM Eucalyptus. The co-inoculation of Eucalyptus with Glomus deserticola and T. koningii was more effective for Cd uptake. In addition, Glomus deserticola enhanced the amount of Pb absorbed by Eucalyptus plants. We showed that it is important to select the most efficient AM and saprobe fungi to stimulate plant growth in heavy-metal-contaminated soil and that the combination of both plays an important role in metal tolerance of Eucalyptus plants.

Flavonoids exhibit fungal species and genus specific effects on the presymbiotic growth of Gigaspora and Glomus.

The effect of the flavonoids chrysin, isorhamnetin, kaempferol, luteolin, morin and rutin on pre-symbiotic growth, such as spore germination, hyphal length, hyphal branching and the formation of auxiliary cells and secondary spores, of the arbuscular mycorrhizal fungi Gigaspora rosea, G. margarita, Glomus mosseae and G. intraradices was studied. According to the effect on each fungal growth parameter, the tested compounds could be classified to be genus and/or species specific or specific, for a certain developmental stage of pre-symbiotic growth. A clear arbuscular mycorrhizal genus specific, and even species specific, effect of some flavonoids was observed. However, this specificity of a flavonoid could not be generalized but differs depending on the pre-symbiotic stage of the AM fungus. Moreover, our show that for a better understanding of the role of compounds in the AM symbiosis, studies should not be conducted only with one AM fungus looking at one fungal growth parameter such as spore germination or hyphal length, but should be wider, including several growth parameters and several AM fungi.

Arbuscular mycorrhizal colonization of tomato by Gigaspora and Glomus species in the presence of root flavonoids.

The effect of flavonoids isolated from arbuscular mycorrhizal (AM) colonized and noncolonized clover roots on the number of entry points and percentage of root colonization of tomato (Lycopersicum esculentum L.) by Gigaspora rosea, Gi margarita, Glomus mosseae and G. intrarradices symbionts was determined. With fungi of both genera, a correlation between the number of entry points and the percentage of root colonization was found in the presence of some of the tested flavonoids. The flavonoids acacetin and rhamnetin, present in AM clover roots, inhibited the formation of AM penetration structures and the AM colonization of tomato roots, whereas the flavonoid 5,6,7,8,9-hydroxy chalcone, which could not be detected in AM clover root, inhibited both parameters. The flavonoid quercetin, which was present in AM clover roots, stimulated the penetration and root colonization of tomato by Gigaspora. However, the flavonoids 5,6,7,8-hydroxy-4'-methoxy flavone and 3,5,6,7,4'-hydroxy flavone, which was not found in AM clover root, increased the number of entry points and the AM colonization of tomato roots by Gigaspora. These results indicated that flavonoids could be imnplicated in the process of regulation of AM colonization in plant root, but its role is highly complex and depend not only on flavonoids, but also on AM fungal genus or even species.

Flavonoids from shoots, roots and roots exudates of Brassica alba.

Analysis of extracts obtained from shoots, roots and exudates of Brassica alba revealed the presence of 3,5,6,7,8-pentahydroxy-4'-methoxy flavone in shoots, as well as 2',3',4',5',6'-pentahydroxy chalcone and 3,5,6,7,8-pentahydroxy flavone in roots and exudates. Apigenin was also found in the shoots and roots, but not in the root exudates.

Flavonoids from shoots and roots of Trifolium repens (white clover) grown in presence or absence of the arbuscular mycorrhizal fungus Glomus intraradices.

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.

Removal of monomeric phenols in dry mill olive residue by saprobic fungi.

The dry olive residue (DOR) obtained from the olive oil extraction process has toxic components against plants and microorganism growth, particularly monomeric phenols. In this investigation nine saprobic fungi were found to be capable of completely removing these phenols from the solid after 20 weeks of growth, although the rate depended on the type of fungi and phenol. Results showed that most of the fungi tested first eliminated o-diphenols and then non-o-diphenols. However, some fungi did not follow this trend. Phanerochaete chrysosporium first removed hydroxytyrosol and tyrosol and later their glucosides and, in contrast, Paecylomyces farinosus hydrolyzed hydroxytyrosol and tyrosol glucosides at the first stage, 2 weeks of growth, and then eliminated all monomeric phenols. The behavior of this fungus seems of great interest for recovering phenolic antioxidants from the DOR. Similarly, differences in DOR decolorization capacity among the fungi tested were also observed. Coriolopsis rigida showed the highest capacity, followed by Phebia radiata, Pycnoporus cinnabarinus, and Pha. chrysosporium. Therefore, both decolorization and monomeric phenol elimination pointed out that saprobic fungi could be used to detoxify the DOR obtained from the two-phase system of the olive oil extraction process.

Interactions between Trichoderma pseudokoningii strains and the arbuscular mycorrhizal fungi Glomus mosseae and Gigaspora rosea.

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 CFUs 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.

Regulation of the plant defence response in arbuscular mycorrhizal symbiosis.

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.

Production of xyloglucanolytic enzymes by Trichoderma viride, Paecilomyces farinosus, Wardomyces inflatus, and Pleurotus ostreatus.

Different conditions of culture medium, incubation time, concentration and surfactant were tested to determine xyloglucanase activity. Trichoderma viride, Paecilomyces farinosus, Wardomyces inflatus and Pleurotus ostreatus showed increased xyloglucanase activities when the fungi grown on microcrystalline cellulose as the sole carbon source. Endoxyloglucanase activity increased with the growth of the fungi and reached a peak on day 14 of incubation, practically 95% of the activity was associated with the extracellular fraction. Precipitation with ammonium sulfate was the best concentration method for detection of endoxyloglucanase activity of the fungi. Endoxyloglucanase activity of the fungi was increased by 4 fold with the use of the non-ionic surfactant Tween 20. Six and three bands of xyloglucanase activities were observed in T. viride and P. ostreatus, respectively, whereas both P. farinosus and W. inflatus presented only one xyloglucanase activity band. These results indicate the presence of several xyloglucanases in the saprophytic fungi examined.