Occurrence and localization of apocarotenoids in arbuscular mycorrhizal plant roots

The core structure of the yellow pigment from arbuscular mycorrhizal (AM) maize roots contains the apocarotenoids mycorradicin (an acyclic C14 polyene) and blumenol C cellobioside (a C13 cyclohexenone diglucoside). The pigment seems to be a mixture of different esterification products of these apocarotenoids. It is insoluble in water and accumulates as hydrophobic droplets in the vacuoles of root cortical cells. Screening 58 species from 36 different plant families, we detected mycorradicin in mycorrhizal roots of all Liliopsida analyzed and of a considerable number of Rosopsida, but also species were found in which mycorradicin was undetectable in mycorrhizal roots. Kinetic experiments and microscopic analyses indicate that accumulation of the yellow pigment is correlated with the concomitant degradation of arbuscules and the extensive plastid network covering these haustorium-like fungal structures. The role of the apocarotenoids in mycorrhizal roots is still unknown. The potential C40 carotenoid precursors, however, are more likely to be of functional importance in the development and functioning of arbuscules.     

The developmental ecology of mycorrhizal associations in mayapple, Podophyllum peltatum, Berberidaceae

Associations between plants and arbuscular mycorrhizal (AM) fungi are widespread and well-studied. Yet little is known about the pattern of association between clonal plants and AM fungi. Here we report on the pattern of mycorrhizal association within the rhizome systems of mayapple, Podophyllum peltatum. Mayapple is a long-lived understory clonal herb that is classified as obligately mycorrhizal. We found that while all mayapple rhizome systems maintained mycorrhizal associations, the percent colonization of roots by AM fungi differed among ramets of different age. The highest concentrations of AM fungi were in the roots of intermediate-aged ramets, while roots beneath the youngest ramet were not colonized. This pattern of ramet age or position-dependent colonization was observed in two separate studies; each conducted in a different year and at a different site. The pattern of AM fungal colonization of mayapple rhizome systems suggests that the mycorrhizal relationship is facultative at the ramet level. This conclusion is reinforced by our observation that augmentation of soil phosphate lowers root colonization by AM fungi. We also found that soil phosphate concentrations were depleted by ca. 1% under the same ramet positions where roots bore the highest AM fungal loads. Three non-exclusive hypotheses are proposed regarding the mechanisms that might cause this developmentally dependent pattern of mycorrhizal association.     

The co-occurrence of ectomycorrhizal,arbuscular mycorrhizal,and dark septate fungi in seedlings of four members of the Pinaceae

Although roots of species in the Pinaceae are usually colonized by ectomycorrhizal (EM) fungi, there are increasing reports of the presence of arbuscular mycorrhizal (AM) and dark septate endophytic (DSE) fungi in these species. The objective of this study was to determine the colonization patterns in seedlings of three Pinus (pine) species (Pinus banksiana, Pinus strobus, Pinus contorta) and Picea glauca x Picea engelmannii (hybrid spruce) grown in soil collected from a disturbed forest site. Seedlings of all three pine species and hybrid spruce became colonized by EM, AM, and DSE fungi. The dominant EM morphotype belonged to the E-strain category; limited colonization by a Tuber sp. was found on roots of Pinus strobus and an unknown morphotype (cf. Suillus–Rhizopogon group) with thick, cottony white mycelium was present on short roots of all species. The three fungal categories tended to occupy different niches in a single root system. No correlation was found between the percent root colonized by EM and percent colonization by either AM or DSE, although there was a positive correlation between percent root length colonized by AM and DSE. Hyphae and vesicles were the only AM intracellular structures found in roots of all species; arbuscules were not observed in any roots.     

Apocarotenoid biosynthesis in arbuscular mycorrhizal roots: contributions from methylerythritol phosphate pathway isogenes and tools for its manipulation

During colonization by arbuscular mycorrhizal (AM) fungi plant roots frequently accumulate two types of apocarotenoids (carotenoid cleavage products). Both compounds, C(14) mycorradicin and C(13) cyclohexenone derivatives, are predicted to originate from a common C(40) carotenoid precursor. Mycorradicin is the chromophore of the "yellow pigment" responsible for the long-known yellow discoloration of colonized roots. The biosynthesis of apocarotenoids has been investigated with a focus on the two first steps of the methylerythritol phosphate (MEP) pathway catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). In Medicago truncatula and other plants the DXS2 isogene appears to be specifically involved in the AM-mediated accumulation of apocarotenoids, whereas in the case of DXR a single gene contributes to both housekeeping and mycorrhizal (apo)carotenoid biosynthesis. Immunolocalization of DXR in mycorrhizal maize roots indicated an arbuscule-associated protein deposition, which occurs late in arbuscule development and accompanies arbuscule degeneration and breakdown. The DXS2 isogene is being developed as a tool to knock-down apocarotenoid biosynthesis in mycorrhizal roots by an RNAi strategy. Preliminary results from this approach provide starting points to suggest a new kind of function for apocarotenoids in mycorrhizal roots.     

Transformed soybean (Glycine max) roots as a tool for the study of the arbuscular mycorrhizal symbiosis

Ri T-DNA transformed roots have been used effectively in studying the interaction between various plant hosts and arbuscular mycorrhizal (AM) fungi. We investigated the in vitro monoxenic symbiosis between the AM fungus Glomus intraradices and transformed soybean roots (TSRs). Comparisons were made between TSR system and plants of the same genotype. The extraradical fungal structures generated in vitro culture showed normal development. Straight runner hyphae branched into short simple branched absorbing structures and spores were initiated. AM symbiosis was confirmed by the presence of arbuscules and vesicles in cortical cells of the TSRs. The frequency of intraradical colonization in TSRs was higher than in plants grown in soil, whereas the intensity values of intraradical colonization in TSR cultures were similar to those in whole plants. These results show that TSR cultures were able to support the growth and characteristic development of G. intraradices.     

Mycorrhizal symbiosis stimulates endoreduplication in angiosperms

Symbiotic and parasitic relationships can alter the degree of endoreduplication in plant cells, and a limited number of studies have documented this occurrence in root cells colonized by arbuscular mycorrhizal (AM) fungi. However, this phenomenon has not been tested in a wide range of plant species, including species that are non-endopolyploid and those that do not associate with AM fungi. We grew 37 species belonging to 16 plant families, with a range of genome sizes and a range in the degree of endopolyploidy. The endoreduplication index (EI) was compared between plants that were inoculated with Glomus irregulare and plants that were not inoculated. Of the species colonized with AM fungi, 22 of the 25 species had a significant increase in endopolyploid root nuclei over non-mycorrhizal plants, including species that do not normally exhibit endopolyploidy. Changes in the EI were strongly correlated (R(2) = 0.619) with the proportion of root length colonized by arbuscules. No change was detected in the EI for the 12 non-mycorrhizal species. This work indicates that colonization by symbiotic fungi involves a mechanism to increase nuclear DNA content in roots across many angiosperm groups and is likely linked to increased metabolism and protein production.     

Polygalacturonase activity and location in arbuscular mycorrhizal roots of Allium porrum L.

Polygalacturonase activity and location were analysed in leek roots (Allium porrum L.) colonized by Glomus versiforme (Karst.) Berch, an arbuscular mycorrhizal (AM) fungus. Polygalacturonase activity in mycorrhizal roots did not differ quantitatively from that found in nonmycorrhizal roots on all of the four harvesting dates. Fractionation of mycorrhizal root extracts by ion-exchange chromatography showed that expression of polygalacturonase was specific to the mutualistic association. Immunofluorescence and immunogold experiments were carried out to locate the polygalacturonase in mycorrhizal roots using a polyclonal antibody raised against a Fusarium moniliforme endopolygalacturonase. Immunolabelling was observed all over the arbuscules (intracellular fungal structures) but particularly at the interface between the arbuscule and the plant membrane. Since pectins are located in this area, we suggest that polygalacturonase produced during the symbiosis could play a role in plant pectin degradation.     

Mycotrophy of Annona cherimola and the morphology of its mycorrhizae

The mycotrophic character of Annona cherimola (Magnoliales), a tropical/subtropical plantation crop of interest, is described for the first time. This crop seems to depend on mycorrhizae (arbuscular) for optimal growth, with Glomus deserticola being the most effective endophyte tested. Study of the morphology of the arbuscular mycorrhizae in Annona roots showed exclusively intracellular hyphal development, with cell-to-cell fungal passage and an abundance of arbuscules and coiled hyphae within cells. Intercellular distributive hyphae were not observed. The morphology and the pattern of spread of the mycorrhizal colonization were similar for the different endophytes involved and appeared to be dependent on the host root. Such features of mycorrhizal colonization are characteristic of host species lacking intercellular air channels and have been described for some species of ecological interest, but they are not commonly noted in the mycorrhizal literature, especially that dealing with crop species. Some ecophysiological consequences of this pattern of colonization are discussed.     

Characterization of Mycorrhizas Formed by Glomus sp. on Roots of Hypernodulating Mutants of Lotus japonicus

Lotus japonicus hypernodulating mutants, Ljsym78-1 and Ljsym78-2, by the arbuscular mycorrhizal fungus Glomus sp. was characterized. The mutants are defective in systemic autoregulation of nodulation and nitrate inhibition, and form an excess of nodules and lateral roots. The percent root length colonized by the arbuscular mycorrhizal fungi was significantly higher for the mutant than wild-type roots. Detailed assessment of the colonization indicated that the percentage of colonization by arbuscules was increased, but that by external hyphae, internal hyphae and vesicles was decreased, in the mutant roots compared with the wild-type. The succinate dehydrogenase activity of arbuscules, external hyphae and internal hyphae showed similar trends. In addition, the majority of individual arbuscules that formed on the mutant roots had a well-developed and seemingly tough morphology. The results suggest that mutation at the Ljsym78 locus positively stimulates the growth and activity of arbuscules, but leads to reduced growth and activity of hyphae. We report the first identification of Lotus japonicus mutants that show significantly increased arbuscule formation and termed these mutants Arb⁺⁺. Received 8 August 2000/ Accepted in revised form 19 October 2000     

Diversity and Spatial Structure of Belowground Plant–Fungal Symbiosis in a Mixed Subtropical Forest of Ectomycorrhizal and Arbuscular Mycorrhizal Plants

Plant–mycorrhizal fungal interactions are ubiquitous in forest ecosystems. While ectomycorrhizal plants and their fungi generally dominate temperate forests, arbuscular mycorrhizal symbiosis is common in the tropics. In subtropical regions, however, ectomycorrhizal and arbuscular mycorrhizal plants co-occur at comparable abundances in single forests, presumably generating complex community structures of root-associated fungi. To reveal root-associated fungal community structure in a mixed forest of ectomycorrhizal and arbuscular mycorrhizal plants, we conducted a massively-parallel pyrosequencing analysis, targeting fungi in the roots of 36 plant species that co-occur in a subtropical forest. In total, 580 fungal operational taxonomic units were detected, of which 132 and 58 were probably ectomycorrhizal and arbuscular mycorrhizal, respectively. As expected, the composition of fungal symbionts differed between fagaceous (ectomycorrhizal) and non-fagaceous (possibly arbuscular mycorrhizal) plants. However, non-fagaceous plants were associated with not only arbuscular mycorrhizal fungi but also several clades of ectomycorrhizal (e.g., Russula) and root-endophytic ascomycete fungi. Many of the ectomycorrhizal and root-endophytic fungi were detected from both fagaceous and non-fagaceous plants in the community. Interestingly, ectomycorrhizal and arbuscular mycorrhizal fungi were concurrently detected from tiny root fragments of non-fagaceous plants. The plant–fungal associations in the forest were spatially structured, and non-fagaceous plant roots hosted ectomycorrhizal fungi more often in the proximity of ectomycorrhizal plant roots. Overall, this study suggests that belowground plant–fungal symbiosis in subtropical forests is complex in that it includes “non-typical” plant–fungal combinations (e.g., ectomycorrhizal fungi on possibly arbuscular mycorrhizal plants) that do not fall within the conventional classification of mycorrhizal symbioses, and in that associations with multiple functional (or phylogenetic) groups of fungi are ubiquitous among plants. Moreover, ectomycorrhizal fungal symbionts of fagaceous plants may “invade” the roots of neighboring non-fagaceous plants, potentially influencing the interactions between non-fagaceous plants and their arbuscular-mycorrhizal fungal symbionts at a fine spatial scale.     

Early events in the life of apple roots: variation in root growth rate is linked to mycorrhizal and nonmycorrhizal fungal colonization

Early events of mycorrhizal and nonmycorrhizal fungal colonization in newly-emerging roots of mature apple (Malus domestica Borkh) trees were characterized to determine the relationship of these events to fine root growth rate and development. New roots were traced on root windows to measure growth and then collected and stained to quantify microscopically the presence of mycorrhizal and nonmycorrhizal fungal structures. Most new roots were colonized by either mycorrhizal or nonmycorrhizal fungi but none less 25 days old were ever internally colonized by both. Compared to nonmycorrhizal colonization, mycorrhizal colonization was associated with faster growing roots and roots that grew for a longer duration, leading to longer roots. While either type of fungi was observed in roots as soon as 3 days after root emergence, intraradical colonization by mycorrhizal fungi was generally faster (peaking at 7 to 15 days) than that by nonmycorrhizal fungi and often occurred more frequently in younger roots. Only 15 to 35% of the roots had no fungal colonization by 30 days after emergence. This study provides the first detailed examination of the early daily events of mycorrhizal and nonmycorrhizal fungal colonization in newly emerging roots under field conditions. We observed marked discrimination of roots between mycorrhizal and nonmycorrhizal fungi and provide evidence that mycorrhizal fungi may select for faster growing roots and possibly influence the duration of root growth by non-nutritional means.     

Trypan blue as a fluorochrome for confocal laser scanning microscopy of arbuscular mycorrhizae in three mangroves

Roots of three mangroves, Acanthus ilicifolius, Ceriops tagal and Excoecaria agallocha, collected from forests of the Sundarbans of India were stained with trypan blue to observe arbuscular mycorrhizal colonization. Spores of arbuscular mycorrhizal fungi isolated from rhizospheric soil, collected together with the root samples, also were stained for testing the suitability of the dye as a fluorochrome. Confocal laser scanning microscopy images were constructed. A. ilicifolius and E. agallocha exhibited “Arum” type colonization with highly branched arbuscules, whereas C. tagal showed “Paris” type association with clumped and collapsed arbuscules. We demonstrated that trypan blue is a suitable fluorochrome for staining arbuscular mycorrhizal fungal spores, fungal hyphae, arbuscules and vesicles, which presumably have a considerable amount of surface chitin. It appears that as the integration of chitin into the fungal cell wall changes, its accessibility to trypan blue dye also changes.     

The differential behavior of arbuscular mycorrhizal fungi in interaction with Astragalus sinicus L. under salt stress

Three arbuscular mycorrhizal (AM) fungi (Glomus mosseae, Glomus claroideum, and Glomus intraradices) were compared for their root colonizing ability and activity in the root of Astragalus sinicus L. under salt-stressed soil conditions. Mycorrhizal formation, activity of fungal succinate dehydrogenase, and alkaline phosphatase, as well as plant biomass, were evaluated after 7 weeks of plant growth. Increasing the concentration of NaCl in soil generally decreased the dry weight of shoots and roots. Inoculation with AM fungi significantly alleviated inhibitory effect of salt stress. G. intraradices was the most efficient AM fungus compared with the other two fungi in terms of root colonization and enzyme activity. Nested PCR revealed that in root system of plants inoculated with a mix of the three AM fungi and grown under salt stress, the majority of mycorrhizal root fragments were colonized by one or two AM fungi, and some roots were colonized by all the three. Compared to inoculation alone, the frequency of G. mosseae in roots increased in the presence of the other two fungal species and highest level of NaCl, suggesting a synergistic interaction between these fungi under salt stress.     

Mycorrhization alleviates benzo[a]pyrene-induced oxidative stress in an in vitro chicory root model

Among chemicals that are widely spread both in terrestrial and aquatic ecosystems, benzo[a]pyrene is a major source of concern. However, little is known about its adverse effects on plants, as well as about the role of mycorrhization in protection of plant grown in benzo[a]pyrene-polluted conditions. Hence, to contribute to a better understanding of the adverse effects of polycyclic aromatic hydrocarbons on the partners of mycorrhizal symbiotic association, benzo[a]pyrene-induced oxidative stress was studied in transformed Cichorium intybus roots grown in vitro and colonized or not by Glomus intraradices. The arbuscular mycorrhizal fungus development (colonization, extraradical hyphae length, and spore formation) was significantly reduced in response to increasing concentrations of benzo[a]pyrene (35–280 μM). The higher length of arbuscular mycorrhizal roots, compared to non-arbuscular mycorrhizal roots following benzo[a]pyrene exposure, pointed out a lower toxicity of benzo[a]pyrene in arbuscular mycorrhizal roots, thereby suggesting protection of the roots by mycorrhization. Accordingly, in benzo[a]pyrene-exposed arbuscular mycorrhizal roots, statistically significant decreases were observed in malondialdehyde concentration and 8-hydroxy-2′-desoxyguanosine formation. The higher superoxide dismutase activity detected in mycorrhizal chicory roots could explain the benzo[a]pyrene tolerance of the colonized roots. Taken together, these results support an essential role of mycorrhizal fungi in protecting plants submitted to polycyclic aromatic hydrocarbon, notably by reducing polycyclic aromatic hydrocarbon-induced oxidative stress damage.     

Establishment of mycorrhizal symbiosis in<Emphasis Type="Italic">Gentiana verna</Emphasis>

The last lowland locality ofGentiana verna in the Czech Republic is a calcareous grassland near Rovná at Strakonice in South Bohemia. This locality was the subject of a recovery programme that included support of the remaining population by micropropagation. The plants were inoculated with arbuscular mycorrhizal fungi (AMF) after their transfer toex vitro and the effect of AMF on their establishment and survival was studied. Although the conventional method of inoculation ofG. verna using spores or colonized root segments as an inoculum source resulted in no or negligible root colonization, the transplantation of gentians to the locality Rovná was successful and the plants became colonized with AMF very rapidly in the field. Successful mycorrhization of gentians under experimental conditions occurred only via the extraradical mycelial network established by neighbouring mycorrhizal plant species (nurse plant effect). Different nurse plant species formed different morphological types of mycorrhiza when inoculated with the same fungal isolate. Gentians always had theParis type of root colonization with intracellular hyphal loops and swellings. Intercellular hyphae, arbuscules and vesicles were not observed. No evidence for a positive growth response was found inG. verna.     

Positive association between mycorrhiza and foliar endophytes in Poa bonariensis, a native grass

The interaction between mycorrhiza and leaf endophytes (Neotyphodium sp.) was studied in three Poa bonariensis populations, a native grass, differing significantly in endophyte infection. The association between endophytes and mycorrhizal fungi colonisation was assessed by analysing plant roots collected from the field. We found that roots from endophyte-infected populations showed a significantly higher frequency of colonisation by mycorrhizal fungi and that soil parameters were not related to endophyte infection or mycorrhiza colonization. In addition, we did not observe significant differences in the number of AM propagules in soils of the three populations sites. We also report the simultaneous development of Paris-type and Arum-type mycorrhiza morphology within the same root systems of P. bonariensis. The co-occurrence of both colonisation types in one and the same root system found in the three populations, which differed in Neotyphodium infection, suggests that foliar endophytes do not determine AM morphology. The percentage of root length colonised by different types of fungal structures (coils, arbuscules, longitudinal hyphae and vesicles) showed significant and positive differences in arbuscular frequency associated with endophyte infection, whereas the much smaller amounts of vesicles and hyphal coils did not differ significantly.     

Influence of temperature on colonization of spring barleys by vesicular arbuscular mycorrhizal fungi

The effects of three soil temperatures on growth of spring barleys (Hordeum vulgare L.) and on their root colonization by vesicular arbuscular mycorrhizal (VAM) fungi from agricultural soils in Montana (USA) or Syria at different inoculum concentrations were tested in soil incubators in the greenhouse. The number of mycorrhizal plants as well as the proportion and intensity of roots colonized increased with higher soil temperatures. VAM fungi from Montana, primarily Glomus macrocarpum, were cold tolerant at 11°C while those from Syria, primarily G. hoi, were heat tolerant at 26°C. Inoculum potential of Montana VAM fungi was higher than Syrian VAM fungi in cool soils. Harmal, selected from Syrian barley land races, had the highest colonization by mycorrhizal fungi of the cultivars tested.Journal Series Paper: J-2532 Montana Agricultural Experiment Station.     

The mycorrhizal status and colonization of 26 tree species growing in urban and rural environments

Urban environments are highly disturbed and fragmented ecosystems that commonly have lower mycorrhizal fungal species richness and diversity compared to rural or natural ecosystems. In this study, we assessed whether the mycorrhizal status and colonization of trees are influenced by the overall environment (rural vs. urban) they are growing in. Soil cores were collected from the rhizosphere of trees growing in urban and rural environments around southern Ontario. Roots were extracted from the soil cores to determine whether the trees were colonized by arbuscular mycorrhizal fungi, ectomycorrhizal fungi, or both, and to quantify the percent colonization of each type of mycorrhizal fungi. All 26 tree species were colonized by arbuscular mycorrhizal fungi, and seven tree species were dually colonized by arbuscular mycorrhizal and ectomycorrhizal fungi. Overall, arbuscular mycorrhizal and ectomycorrhizal fungal colonization was significantly (p < 0.001) lower in trees growing in urban compared to rural environments. It is not clear what ‘urban’ factors are responsible for the reduction in mycorrhizal fungal colonization; more research is needed to determine whether inoculating urban trees with mycorrhizal fungi would increase colonization levels and growth of the trees.