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

Abstract

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.

FIG. 1.

Percentages of successful anastomoses with respect to total hyphal contacts in the different Glomus mosseae isolates. Bars represent 95% confidence limits.

FIG. 2.

Percentage of hyphal contacts leading to hyphal intersection with no cellular response (dark gray bars), type A interaction between hyphae (open bars), and type B interaction between hyphae (light gray bars). See text for description of interactions of types A and B.

FIG. 3.

Light micrographs showing incompatible interactions between hyphae belonging to geographically different isolates of the AM fungus G. mosseae, after SDH and trypan blue staining. (a) Development of multiple hyphal branches (IN101C) growing towards branch initiation sites (arrows) on the recipient hypha (AZ225C). Note hyphal swellings and consecutive retraction septa (arrowheads) produced prior to any physical contact between the hyphae. Scale bar = 35 μm. (b) Multiple hyphal branches (AZ225C) showing precontact protoplasm retraction and localized thickening of hyphal tips (arrow). Scale bar = 9 μm. (c) Change of growth direction in an approaching hypha (BEG25) accompanied by protoplasm withdrawal and septum formation. Scale bar = 17 μm. (d) Retraction septa developed by an approaching hypha (IN101C) after incompatible interaction (type A). Scale bar = 9 μm.

FIG. 4.

Type B incompatible interactions between geographically different isolates of the AM fungus G. mosseae. Scale bar = 9 μm. (a) Epifluorescence image, after Calcofluor and DAPI staining, showing wall thickening of a hyphal swelling developed by the approaching hypha (AZ225C) on a lateral branch initial of the contacted one (IN101C). (b) Light micrograph, after SDH and trypan blue staining, showing protoplasm withdrawal and septum formation in an approaching hypha (IN101C) after incompatible contact with a branch initial (SY710).

FIG. 5.

MDH profiles of different G. mosseae isolates and of G. caledonium (IMA2), included for comparison. Lane 1, BEG25; lane 2, IN101C; lane 3, AZ225C; lane 4, SY710; lanes 5 and 6, IMA1; lane 7, IMA2.

FIG. 6.

UPGMA cluster analysis of soluble protein profiles of four geographically different isolates of G. mosseae and of G. coronatum (IMA3), included for comparison.

FIG. 7.

RFLP patterns produced by restriction digestions of the ITS region of the rDNA of four geographically different isolates of G. mosseae. Isolates were digested with DpnII (top) and with TaqI (bottom). Lane 1, AZ225C; lane 2, BEG25; lane 3, IMA1; lane 4, IN101C; lanes M, size markers.

FIG. 8.

Dendrogram produced from cluster analysis of RFLP-ITS profiles, pooling data from all restriction enzymes, of four geographically different isolates of G. mosseae and of G. coronatum (IMA3), included for comparison.