RocTest: A standardized method to assess the performance of root organ cultures in the propagation of arbuscular mycorrhizal fungi.

Over the past three decades, root organ cultures (ROCs) have been the gold standard method for studying arbuscular mycorrhizal fungi (AMF) under in vitro conditions, and ROCs derived from various plant species have been used as hosts for AM monoxenic cultures. While there is compelling evidence that host identity can significantly modify AMF fitness, there is currently no standardized methodology to assess the performance of ROCs in the propagation of their fungal symbionts. We describe RocTest, a robust methodological approach that models the propagation of AMF in symbiosis with ROCs. The development of extraradical fungal structures and the pattern of sporulation are modeled using cumulative link mixed models and linear mixed models. We demonstrate functionality of RocTest by evaluating the performance of three species of ROCs (Daucus carota, Medicago truncatula, Nicotiana benthamiana) in the propagation of three species of AMF (Rhizophagus clarus, Rhizophagus irregularis, Glomus sp.). RocTest produces a simple graphical output to assess the performance of ROCs and shows that fungal propagation depends on the three-way interaction between ROC, AMF, and time. RocTest makes it possible to identify the best combination of host/AMF for fungal development and spore production, making it an important asset for germplasm collections and AMF research.

Host identity influences nuclear dynamics in arbuscular mycorrhizal fungi.

The arbuscular mycorrhizal fungi (AMF) are involved in one of the most ecologically important symbioses on the planet, occurring within the roots of most land plants.1 Knowledge of even basic elements of AM fungal biology is still poor, with the discovery that AMF may in fact have a sexual life cycle being only very recently reported.2-5 AMF produce asexual spores that contain up to several thousand individual haploid nuclei6 of either largely uniform genotypes (AMF homokaryons) or nuclei originating from two parental genotypes2-5 (AMF dikaryons or heterokaryons). In contrast to the sexual dikaryons in the phyla Ascomycota and Basidiomycota,7,8 in which pairs of nuclei coexist in single hyphal compartments, AMF dikaryons carry several thousand nuclei in a coenocytic mycelium. Here, we set out to better understand the dynamics of this unique multinucleate condition by combining molecular analyses with advanced microscopy and modeling. Herein, we report that select AMF dikaryotic strains carry the distinct nucleotypes in equal proportions to one another, whereas others show an unequal distribution of parental nucleotypes. In both cases, the relative proportions within a given strain are inherently stable. Simulation models suggest that AMF dikaryons may be maintained through nuclear cooperation dynamics. Remarkably, we report that these nuclear ratios shift dramatically in response to plant host identity, revealing a previously unknown layer of genetic complexity and dynamism within the intimate interactions that occur between the partners of a prominent terrestrial symbiosis.

A Stimulatory Role for Cytokinin in the Arbuscular Mycorrhizal Symbiosis of Pea.

The arbuscular mycorrhizal (AM) symbiosis between terrestrial plants and AM fungi is regulated by plant hormones. For most of these, a role has been clearly assigned in this mutualistic interaction; however, there are still contradictory reports for cytokinin (CK). Here, pea plants, the wild type (WT) cv. Sparkle and its mutant E151 (Pssym15), were inoculated with the AM fungus Rhizophagus irregularis. E151 has previously been characterized as possessing high CK levels in non-mycorrhizal (myc-) roots and exhibiting high number of fungal structures in mycorrhizal (myc+) roots. Myc- and myc+ plants were treated 7, 9, and 11 days after inoculation (DAI) with synthetic compounds known to alter CK status. WT plants were treated with a synthetic CK [6-benzylaminopurine (BAP)] or the CK degradation inhibitor INCYDE, whereas E151 plants were treated with the CK receptor antagonist PI-55. At 13 DAI, plant CK content was analyzed by mass spectrometry. The effects of the synthetic compounds on AM colonization were assessed at 28 (WT) or 35 (E151) DAI via a modified magnified intersections method. The only noticeable difference seen between myc- and myc+ plants in terms of CK content was in the levels of nucleotides (NTs). Whereas WT plants responded to fungi by lowering their NT levels, E151 plants did not. Since NTs are thought to be converted into active CK forms, this result suggests that active CKs were synthesized more effectively in WT than in E151. In general, myc+ and myc- WT plants responded similarly to INCYDE by lowering significantly their NT levels and increasing slightly their active CK levels; these responses were less obvious in BAP-treated WT plants. In contrast, the response of E151 plants to PI-55 depended on the plant mycorrhizal status. Whereas treated myc- plants exhibited high NT and low active CK levels, treated myc+ plants displayed low levels of both NTs and active CKs. Moreover, treated WT plants were more colonized than treated E151 plants. We concluded that CKs have a stimulatory role in AM colonization because increased active CK levels were paralleled with increased AM colonization while decreased CK levels corresponded to reduced AM colonization.