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Aims: Many studies have reported beneficial effects of yeasts on the colonization and development of arbuscular mycorrhizae, thought a few studies have also shown neutral effects. All these studies have in common that the mechanism, by which yeasts and mycorrhizae interact, is little understood. Here, we explore how plant growth-promoting yeasts affect the colonization of tomato plants by beneficial mycorrhizal fungi. Methods: We tested the influence of the soil yeasts Candida saitoana, Tausonia pullulans, and Saccharomyces eubayanus on colonization of tomato roots by the mycorrhizal fungus Rhizophagus irregularis. We analyzed mycorrhizal parameters and the expression pattern of mycorrhiza-specific genes. In plants co-inoculated with S. eubayanus and R. irregularis, we measured the root accumulation pattern of jasmonic acid, oxo-phytodienoic acid, abscisic acid and salicylic acid, and the expression of genes related to plant hormone signaling and metabolism. Results: The three yeasts had distinct effects on mycorrhizal colonization: C. saitoana had no effect on mycorrhizal parameters, T. pullulans delayed mycorrhizal colonization at an early stage, and S. eubayanus slowed colonization down throughout the entire trial. In plants co-inoculated with S. eubayanus and R. irregularis, we observed a sustained increase in jasmonic acid and up-regulation of the JA biosynthesis related genes LOXD, OPR3, and AOS1. Conclusion: Co-inoculation with yeast affected mycorrhizal colonization and altered the expression pattern of mycorrhizal and plant defense-related genes. In particular, the yeast S. eubayanus modified plant defense hormones such as jasmonic acid, which is linked to mycorrhizal-induced resistance in tomato plants.

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.

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.

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.