Divergence of Funneliformis mosseae populations over 20 years of laboratory cultivation, as revealed by vegetative incompatibility and molecular analysis.

Arbuscular mycorrhizal fungi (AMF) are widespread, important plant symbionts. They absorb and translocate mineral nutrients from the soil to host plants through an extensive extraradical mycelium, consisting of indefinitely large networks of nonseptate, multinucleated hyphae which may be interconnected by hyphal fusions (anastomoses). This work investigated whether different lineages of the same isolate may lose the ability to establish successful anastomoses, becoming vegetatively incompatible, when grown separately. The occurrence of hyphal incompatibility among five lineages of Funneliformis mosseae, originated from the same ancestor isolate and grown in vivo for more than 20 years in different European locations, was assessed by systematic detection of anastomosis frequency and cytological studies. Anastomosis frequencies ranged from 60 to 80% within the same lineage and from 17 to 44% among different lineages. The consistent detection of protoplasm continuity and nuclei in perfect fusions showed active protoplasm flow both within and between lineages. In pairings between different lineages, post-fusion incompatible reactions occurred in 6-48% of hyphal contacts and pre-fusion incompatibility in 2-17%. Molecular fingerprinting profiles showed genetic divergence among lineages, with overall Jaccard similarity indices ranging from 0.85 to 0.95. Here, phenotypic divergence among the five F. mosseae lineages was demonstrated by the reduction of their ability to form anastomosis and the detection of high levels of vegetative incompatibility. Our data suggest that potential genetic divergence may occur in AMF over only 20 years and represent the basis for detailed studies on the relationship between genes regulating anastomosis formation and hyphal compatibility in AMF.

Genetic and phenotypic diversity of geographically different isolates of Glomus mosseae.

In this work, we combined morphological taxonomy and molecular methods to investigate the intraspecific diversity of Glomus mosseae, whose global distribution has been reviewed by a survey of scientific literature and Web-available records from international germplasm collections (International Culture Collection of Vesicular Arbuscular Mycorrhizal Fungi and International Bank of Glomeromycota). We surveyed 186 publications reporting the occurrence of G. mosseae from at least 474 different sites from 55 countries throughout all continents, producing a geographical map of their distribution. The relationships among G. mosseae isolates originating from Europe (United Kingdom), the United States (Arizona, Florida, and Indiana), Africa (Namibia), and West Asia (Syria) were analyzed. The level of resolution of internal transcribed spacer (ITS) sequences strongly supports the morphological species definition of G. mosseae. An ITS - restriction fragment length polymorphism assay with the enzyme HinfI yielded a unique profile for all G. mosseae isolates, allowing a straightforward identification of this morphospecies. Genetic variability among G. mosseae isolates was revealed by the inter-simple-sequence repeat (ISSR) - polymerase chain reaction: the magnitude of genetic divergence shown by the investigated geographical isolates was higher than 50%, consistent with previous data on vegetative compatibility and functional diversity. The variability of ISSR patterns suggests that intraspecific diversity is much higher than that foreseen by morphology and rDNA regions, and should be further investigated by using other genes, such as those related to functional diversity.

At the root of the wood wide web: self recognition and non-self incompatibility in mycorrhizal networks.

Arbuscular mycorrhizal (AM) fungi are mutualistic symbionts living in the roots of 80% of land plant species, and developing extensive, below-ground extraradical hyphae fundamental for the uptake of soil nutrients and their transfer to host plants. Since AM fungi have a wide host range, they are able to colonize and interconnect contiguous plants by means of hyphae extending from one root system to another. Such hyphae may fuse due to the widespread occurrence of anastomoses, whose formation depends on a highly regulated mechanism of self recognition. Here, we examine evidences of self recognition and non-self incompatibility in hyphal networks formed by AM fungi and discuss recent results showing that the root systems of plants belonging to different species, genera and families may be connected by means of anastomosis formation between extraradical mycorrhizal networks, which can create indefinitely large numbers of belowground fungal linkages within plant communities.

Patterns of below-ground plant interconnections established by means of arbuscular mycorrhizal networks.

• The ability of arbuscular mycorrhizal (AM) networks originating from plants of different species, genera and families to become interconnected by means of hyphal anastomoses was assessed. • An in vivo two-dimensional experimental model system was used to reveal the occurrence of linkages between contiguous mycorrhizal networks spreading from Allium porrum root systems and those originating from Daucus carota, Gossypium hirsutum, Lactuca sativa, Solanum melongena, colonized by Glomus mosseae. • Percentages of hyphal contacts leading to anastomosis between extraradical networks originating from different plant species ranged from 44% in the pairing A. porrum-S. melongena to 49% in A. porrum-G. hirsutum. DAPI and Sytox stainings detected nuclei in the middle of fusion bridges connecting different mycorrhizal networks. • Present data suggest that, by means of anastomoses, AM fungal mycelium would potentially create an indefinitely large network interconnecting different plants in a community, and that, in the absence of sexual recombination, the intermingling of nuclei in extraradical mycelium may provide endless opportunities for the exchange of genetic material.

Genetic diversity of isolates of Glomus mosseae from different geographic areas detected by vegetative compatibility testing and biochemical and molecular analysis.

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.