ABSTRACT
Lipid transfer from host plants to arbuscular mycorrhiza fungi was hypothesized for several years because sequenced arbuscular mycorrhiza fungal genomes lack genes encoding cytosolic fatty acid synthase ( Wewer et al., 2014 ; Rich et al., 2017 ). It was finally shown by two independent experimental approaches ( Jiang et al., 2017 ; Keymer et al., 2017 ; Luginbuehl et al., 2017 ). One approach used a technique called isotopolog profiling ( Keymer et al., 2017 ). Isotopologs are molecules, which differ only in their isotopic composition. For isotopolog profiling an organism is fed with a heavy isotope labelled precursor metabolite. Subsequently, the labelled isotopolog composition of metabolic products is analysed via mass spectrometry. The detected isotopolog pattern of the metabolite(s) of interest yields information about metabolic pathways and fluxes ( Ahmed et al., 2014 ). The following protocol describes an experimental setup, which enables separate isotopolog profiling of fatty acids in plant roots colonized by arbuscular mycorrhiza fungi and their associated fungal extraradical mycelium, to elucidate fluxes between both symbiotic organisms. We predict that this strategy can also be used to study metabolite fluxes between other organisms if the two interacting organisms can be physically separated.
ABSTRACT
ABSTRACT In vitro root cultivation techniques based on modified root systems are often used in studies on Arbuscular Mycorrhizal Fungi (AMF). It is a simplified but powerful tool to investigate AMF root colonization and development of the extraradical mycelium. The aim of this study was to establish and characterize the in vitro culture of a Cuban strain of Rhizophagus irregularis (INCAM 11) by using transformed chicory roots. For that, superficially disinfected propagules of R. irregularis were co-culture with the hairy transformed chicory roots on Modified Strullu and Romand (MSR) medium during five months. Spore germination was observed 3-5 days after surface disinfection. The first contact between AMF hyphae and roots occurred 1 - 3 days after germination and a significant production of extensive extraradical mycelium was observed. New spore formation started within 21 - 25 days. After 5 months, 2000 spores could be observed per plate which were able to germinate, colonize, establish and reproduce again spores when associated to young transformed roots of chicory. The most frequent associated microorganism to the in vitro culture of INCAM 11 was isolated and identified as Paenibacillus sp.
RESUMEN Las técnicas de cultivo de raíces in vitro basadas en sistemas de raíces modificadas se utilizan a menudo en los estudios sobre hongos micorrízicos arbusculares (HMA). Es una herramienta simplificada pero poderosa para investigar la colonización de las raíces de los HMA y el desarrollo del micelio extrarradical. El objetivo de este estudio fue establecer y caracterizar el cultivo in vitro de una cepa cubana de Rhizophagus irregularis (INCAM 11) utilizando raíces transformadas de achicoria. Para ello, propágulos de R. irregularis desinfectados superficialmente fueron co-cultivados con las raíces transformadas de achicoria en medio Strullu y Romand modificado (SRM) durante cinco meses. La germinación de las esporas se observó 3-5 días después de la desinfección superficial. El primer contacto entre las hifas y las raíces se produjo entre 1 y 3 días después de la germinación y se observó una producción significativa de micelio extrarradical. La formación de nuevas esporas comenzó entre 21 - 25 días. Después de 5 meses, se pudieron observar 2000 esporas por placa que fueron capaces de germinar, colonizar, establecer y reproducir nuevas esporas cuando se asociaron a raíces jóvenes transformadas de achicoria. El microorganismo asociado frecuentemente al cultivo in vitro de INCAM 11 fue aislado e identificado como Paenibacillus sp.