Differences in the composition of arbuscular mycorrhizal fungal communities promoted by different propagule forms from a Mediterranean shrubland

As it is well known, arbuscular mycorrhizal (AM) colonization can be initiated from the following three types of fungal propagules: spores, extraradical mycelium (ERM), and mycorrhizal root fragments harboring intraradical fungal structures. It has been shown that biomass allocation of AM fungi (AMF) among these three propagule types varies between fungal taxa, as also differs the ability of the different AMF propagule fractions to initiate new colonizations. In this study, the composition of the AMF community in the roots of rosemary (Rosmarinus officinalis L., a characteristic Mediterranean shrub), inoculated with the three different propagule types, was analyzed. Accordingly, cuttings from this species were inoculated with either AMF spores, ERM, or colonized roots extracted from a natural soil. The AMF diversity within the rosemary roots was characterized using terminal restriction fragment length polymorphism (T-RFLP) of the small subunit (SSU) rDNA region. The AMF community established in the rosemary plants was significantly different according to the type of propagule used as inoculum. AMF taxa differed in their ability to initiate new colonizations from each propagule type. Results suggest different colonization strategies for the different AMF families involved, Glomeraceae and Claroideoglomeraceae colonizing mainly from colonized roots whereas Pacisporaceae and Diversisporaceae from spores and ERM. This supports that AMF taxa show contrasting life-history strategies in terms of their ability to initiate new colonizations from the different propagule types. Further research to fully understand the colonization and dispersal abilities of AMF is essential for their rational use in ecosystem restoration programs.     

Life-history strategies of arbuscular mycorrhizal fungi determine succession into roots of Rosmarinus officinalis L., a characteristic woody perennial plant species from Mediterranean ecosystems

Aim

Few studies have analyzed life-history strategies of arbuscular mycorrhizal fungi (AMF), in terms of the different propagule types they produce, and their ability to colonize new seedlings. The aim was to assess whether life-history strategies influence AMF successional dynamics and assemblages.

Methods

Rosemary (Rosmarinus officinalis L.) seedlings, grown in a mesocosm system, were colonized by either the AMF hyphae coming from a living rosemary plant, or from spores germinating in soil. The AMF community established in the plantlets was monitored every 3 months during 2 years, using terminal restriction fragment length polymorphism of genes coding for rDNA.

Results

The two different sources of AMF propagules resulted in a different initial community colonizing rosemary roots. AMF propagating from hyphae attached to living mycorrhizal-roots seemed to colonize faster and were season-dependent. AMF taxa originating from soil-borne propagules were most frequent over time and exhibit the dominant colonization strategy in this system. The evolution of the AMF community also revealed different strategies in succession.

Conclusions

AMF associated with rosemary evidenced contrasting life-history strategies in terms of source of inoculum for new colonization and hence survival. The observed successional dynamics of AMF have implications for understanding the ecological processes in Mediterranean environments and seasonality of colonization processes.     

Time-course study and partial characterization of a protein on hyphae of arbuscular mycorrhizal fungi during active colonization of roots

Material on the surface of hyphal walls of arbuscular mycorrhizal fungi (AMF) during active colonization of plant roots was detected by a monoclonal antibody. Pot-cultured isolates of Glomus, Acaulospora, Gigaspora, Scutellospora, and Entrophospora had immunofluorescent material (IM) on younger, thinner, intact hyphae, but IM was scant to absent on thicker, melanized or lysing hyphae. Colonization of corn (Zea mays L.), Sudangrass (Sorghum sudanense (Piper) Staph.) or red clover (Trifolium pratense L.) was examined during 5 months of plant growth by removing cores and performing an indirect immunoassay on roots with attached hyphae. Fresh spores of some Glomus spp. had IM on the outer layer of the spore wall. Abundant IM was seen on root hairs of plants colonized by some isolates, and some IM was detected on root surfaces of all plants examined even during early colonization. After cultures were dried, hyphae, roots and spores had little to no IM. Uninoculated control roots had very rare, small patches of IM. An immunoreactive protein was extracted from hyphae of Gigaspora and Glomus isolates by using 20mM citrate (pH 7.0) at 121°C for 90 min. Gel electrophoresis profiles indicated that all isolates tested had the same banding patterns. Lectin-binding of extracted protein is suggestive of a glycoprotein. The immunofluorescence assay can be used to examine root sections for active colonization by AMF, and the potential use of the protein to quantify AMF activity in soil is discussed.     

Polyphosphate dynamics in mycorrhizal roots during colonization of an arbuscular mycorrhizal fungus

Inorganic polyphosphate (poly P) has been considered to be a translocatable form of phosphate (Pi) in arbuscular mycorrhizal fungi (AMF). Here we examined time-course changes in poly P content during the AMF colonization process. Onion (Allium cepa) plants were cultured with or without inoculation with Gigaspora margarita for 2-8 wk with periodic sampling. Poly P in the extracts, purified through gel filtration, was quantified by the reverse reaction of polyphosphate kinase. The length of poly P in mycorrhizal roots appeared to be shorter than in extraradical hyphae or in spores of the AMF, indicating that AMF depolymerize poly P before providing Pi to the host. The poly P content increased as colonization proceeded, and was highly correlated with the weight of the colonized roots. These results support the model that AMF supply Pi to the host through the poly P pool, and that the poly P content of a mycorrhizal root can be a good indicator of the Pi-supplying activity of AMF.     

Soil salinity delays germination and limits growth of hyphae from propagules of arbuscular mycorrhizal fungi

Colonisation of plant roots by some arbuscular mycorrhizal (AM) fungi is reduced in the presence of sodium chloride (NaCl), probably due to a direct effect of NaCl on the fungi. However, there appear to be differences between the fungi in their ability to colonise plants in the presence of NaCl. This experiment tested the hypothesis that propagules of different isolates and species of AM fungi from saline and nonsaline soils would differ in their ability to germinate and grow in the presence of NaCl in the soil solution. Spores or pieces of root colonised by a range of AM fungi were incubated between filters buried in soil to which NaCl had been added at concentrations of 0, 150 or 300 mM in the soil solution. At regular intervals, filters were removed from the soil and both the percentage of propagules which had germinated and the length of proliferating hyphae were determined. Germination of spores of AM fungi studied was delayed in the presence of NaCl, but the fungi differed in the extent to which germination was inhibited. Two isolates of Scutellospora calospora reached maximum germination in 300 mM NaCl, but neither of two isolates of Acaulospora laevis germinated in the presence of NaCl. Germination of spores of the other fungi, including some isolated from saline soil, fell between these extremes. For some fungi, the specific rate of hyphal extension was reduced by NaCl. For others, the specific rate of growth was similar in the presence of NaCl to that in the control treatment, but overall production of hyphae was reduced in the NaCl treatments because germination was reduced.     

An ecological view of the formation of VA mycorrhizas

In spite of the major advances in understanding the functioning of symbioses between plants and arbuscular mycorrhizal fungi, details of the ecology of mycorrhizal fungi are not well documented. The benefits of the association are related to the timing and extent of colonization of roots, and fungi differ in their contribution to plant growth and presumably to soil aggregation. Knowledge of the processes that lead to successful colonization of roots by beneficial fungi at appropriate times for the host plants will form the basis of guidelines for soil management to maximize the benefits from the symbiosis. Fungi differ in the manner and extent to which they colonize roots. They also differ in their capacity to form propagules. The importance of hyphae, spores and propagules within living or dead mycorrhizal roots also differs among species and for the same species in different habitats. The relationships between colonization of roots and propagule formation, and between propagule distribution and abundance and subsequent mycorrhiza formation, for different fungi in field environments, are not well understood. Methods for quantifying mycorrhizal fungi are not especially suitable for distinguishing among different fungi within roots. Consequenctly, the dynamics of colonization of roots by different fungi, within and between seasons, have been little studied. Research is required that focuses on the dynamics of fungi within roots as well as on changes in the abundance of propagules of different fungi within soil. Interactions between fungi during the colonization of roots, the colonization of soil by hyphae and sporulation are all poorly understood. Without knowledge of these processes, it will by difficult to predict the likely success of inoculation with introduced fungi. Such knowledge is also required for selecting soil management procedures to enhance growth and survival of key species within the population. The relative tolerance of various fungi to perturbations in their surroundings will provide a basis for identifying those fungi that are likely to persist in specific environments. The processes that influence mycorrhizal fungi in field soils can be identified in controlled studies. However, greater emphasis is required on studying these processes with mixed populations of fungi. The role played by diversity within populations of mycorrhizal fungi is virtually unexplored.     

Evidence of differences between the communities of arbuscular mycorrhizal fungi colonizing galls and roots of Prunus persica infected by the root-knot nematode Meloidogyne incognita

Arbuscular mycorrhizal fungi (AMF) play important roles as plant protection agents, reducing or suppressing nematode colonization. However, it has never been investigated whether the galls produced in roots by nematode infection are colonized by AMF. This study tested whether galls produced by Meloidogyne incognita infection in Prunus persica roots are colonized by AMF. We also determined the changes in AMF composition and biodiversity mediated by infection with this root-knot nematode. DNA from galls and roots of plants infected by M. incognita and from roots of noninfected plants was extracted, amplified, cloned, and sequenced using AMF-specific primers. Phylogenetic analysis using the small-subunit (SSU) ribosomal DNA (rDNA) data set revealed 22 different AMF sequence types (17 Glomus sequence types, 3 Paraglomus sequence types, 1 Scutellospora sequence type, and 1 Acaulospora sequence type). The highest AMF diversity was found in uninfected roots, followed by infected roots and galls. This study indicates that the galls produced in P. persica roots due to infection with M. incognita were colonized extensively by a community of AMF, belonging to the families Paraglomeraceae and Glomeraceae, that was different from the community detected in roots. Although the function of the AMF in the galls is still unknown, we hypothesize that they act as protection agents against opportunistic pathogens.     

不同pH水平下两种菌根真菌对紫云英生长的影响及其相互作用

设计在不同pH水平（4.3、5.1、5.8、6.8）下两种VA菌根真菌Glomus mosseae和Gigaspora margarita对紫云英Astragalus sinicus进行单接种、混合接种及无接种对照的盆栽实验。对紫云英地上和地下部分生物量、根部侵染率、SDH和ALP酶活进行了检测。实验结果表明：紫云英的生长效应与VA菌根真菌的侵染率及两种酶活成明显相关性。土壤pH升高,单接种Glomus mosseae和混合接种的侵染率也随之升高,而单接种Gigaspora margarita的侵染率呈现     

Arbuscular mycorrhizal fungal propagules in a salt marsh

The tolerance of indigenous arbuscular mycorrhizal fungi (AMF) to stressful soil conditions and the relative contribution of spores of these fungi to plant colonization were examined in a Portuguese salt marsh. Glomus geosporum is dominant in this salt marsh. Using tetrazolium as a vital stain, a high proportion of field-collected spores were found to be metabolically active at all sampling dates. Spore germination tests showed that salt marsh spores were not affected by increasing levels of salinity, in contrast to two non-marsh spore isolates, and had a significantly higher ability to germinate under increased levels of salinity (20) than in the absence of or at low salinity (10). Germination of salt marsh spores was not affected by soil water levels above field capacity, in contrast to one of the two non-marsh spore isolates. For the evaluation of infectivity, a bioassay was established with undisturbed soil cores (containing all types of AM fungal propagules) and soil cores containing only spores as AM fungal propagules. Different types of propagules were able to initiate and to expand the root colonization of a native plant species, but spores were slower than mycelium and/or root fragments in colonizing host roots. The AM fungal adaptation shown by this study may explain the maintenance of AMF in salt marshes.     

The response of ectomycorrhizal fungal inoculum to long-term increases in nitrogen supply

The inoculum of ectomycorrhizal (EM) fungi was examined in a 16 y long nitrogen fertilization experiment maintained in a temperate oak savanna. To measure EM fungal inoculum, bur oak seedlings were grown in three types of bioassays: (i) intact soil cores that measure inoculum such as spores, mycelia and mycorrhizal roots; (ii) resistant propagule bioassays that measure inoculum types resistant to soil drying; and (iii) previously mycorrhizal root bioassays that measure the ability of EM fungi to colonize new roots from mycorrhizal roots. Colonization of bur oak seedlings was characterized by morphotyping and where necessary by restriction analysis and internal transcribed spacer (ITS) sequencing. Fourteen morphotypes were found in intact soil core bioassays with species of Cortinarius, Cenococcum and Russula abundant. Five morphotypes were found in resistant propagule bioassays with Cenococcum, a thelephoroid morphotype and a Wilcoxina-like ascomycete abundant and frequent. In intact soil core bioassays total percent root colonization and number of morphotypes were not affected by N supply in 2000 and 2001. However the composition of EM fungi colonizing oak seedling roots was different with increased N supply such that Russula spp. (primarily Russula aff. amoenolens) were most abundant at the highest level of N supply. Dominant Russula spp. did not colonize any roots in resistant propagule bioassays but did colonize oak seedling roots from previously mycorrhizal roots. Results suggest that in this savanna N supply can influence the kinds of inoculum propagules present and thereby might affect the dynamics of ectomycorrhizal communities by differentially influencing reproductive and colonization strategies.     

不同pH水平下两种菌根真菌对紫云英生长的影响及其相互作用

设计在不同pH水平(4.3、5.1、5.8、6.8)下两种VA菌根真菌Glomus mosseae和Gigaspora margarita对紫云英Astragalus sinicus进行单接种、混合接种及无接种对照的盆栽实验.对紫云英地上和地下部分生物量、根部侵染率、SDH和ALP酶活进行了检测.实验结果表明:紫云英的生长效应与VA菌根真菌的侵染率及两种酶活成明显相关性.土壤pH升高,单接种Glomus mosseae和混合接种的侵染率也随之升高,而单接种Gigaspora margarita的侵染率呈现出先上升后下降的趋势.本实验设计了特异性扩增Glomus mosseae和Gigaspora margarita的引物gml和gigl,在混合接种实验中,nested PCR扩增结果显示:在低pH水平下(4.3-5.1)大多数根段为Gigaspora margarita所侵染,在高pH水平下(5.8-6.8)Glomusmosseae表现出较强的竞争力,但并没有检测到两种VA真菌存在于同一条侵染根段;对比单接种实验,在低pH水平下,Glomus mosseae显著抑制了Gigaspora margarita的侵染,而在高pH水平下Gigasporamargarita明显促进Glomus mosseae的侵染.     

Mycorrhizae and succession in plantings of beachgrass in sand dunes

A survey of arbuscular mycorrhizal fungi (AMF), arbuscular mycorrhizae (AM), and hyphal networks of AMF was carried out in sand dune sites of different successional stages in the Province Lands Area of Cape Cod National Seashore, Massachusetts. The study focused on large-scale plantings (each of 12–20 ha) of American beachgrass (Ammophila breviligulata) aged 0–7 yr and five adjacent natural dune areas. Sample sites ranged in vegetative cover from barren to forested. Spores of 17 species of AMF were recovered from the dunes. Over the successional sequence, there were increases in the richness and spore populations of the AMF community, the extent of colonization of A. breviligulata roots, and the mycorrhizal inoculum potential of the soil. Unvegetated sites lacked propagules of AMF, but roots of planted culms of A. breviligulata (which carried propagules of AMF) became mycorrhizal in <1 yr after planting. Spores were recovered from previously AMF-free sites that had been planted with beachgrass for 47 wk, and five species of AMF sporulated in sites <6 yr old. Significant hyphal networks were not present in any of the planted areas (<6 yr old at the time of sampling), but did occur in natural areas. The rate of invasion of areas planted to A. breviligulata by later successional plant species may in part depend upon the establishment of a vigorous network of hyphae of AMF in a site.     

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.     

The composition of arbuscular mycorrhizal fungal communities differs among the roots,spores and extraradical mycelia associated with five Mediterranean plant species

Arbuscular mycorrhizal fungi (AMF) are essential constituents of most terrestrial ecosystems. AMF species differ in terms of propagation strategies and the major propagules they form. This study compared the AMF community composition of different propagule fractions – colonized roots, spores and extraradical mycelium (ERM) – associated with five Mediterranean plant species in Sierra de Baza Natural Park (Granada, Spain). AMF were identified using 454 pyrosequencing of the SSU rRNA gene. A total of 96 AMF phylogroups [virtual taxa (VT)] were detected in the study site, including 31 novel VT. After per‐sample sequencing depth standardization, 71 VT were recorded from plant roots, and 47 from each of the spore and ERM fractions. AMF communities differed significantly among the propagule fractions, and the root‐colonizing fraction differed among host plant species. Indicator VT were detected for the root (13 Glomus VT), spore (Paraglomus VT281, VT336, Pacispora VT284) and ERM (Diversispora VT62) fractions. This study provides detailed evidence from a natural system that AMF taxa are differentially allocated among soil mycelium, soil spores and colonized root propagules. This has important implications for interpreting AMF diversity surveys and designing applications of AMF in vegetation restoration.     

Colonization potential of in vitro-produced arbuscular mycorrhizal fungus spores compared with a root-segment inoculum from open pot culture

 A reliable inoculum, free from other microorganisms, to produce arbuscular mycorhizal (AM) plants is of the greatest importance when studying the interaction between AM plants and soil microorganisms. We investigated the colonization of leeks from monoxenic in vitro-produced Glomus intraradices spores. The isolated spores were produced using a two-compartment in vitro growth system previously described. A spore suspension was used as inoculum and was compared to the inoculum potential of endomycorrhizal root segments of pot-grown leek (Allium porrum L.) plants. The leeks were grown in a controlled environment and two types of sterilized growth media were tested: calcined montmorillonite clay and a soil mix. Root colonization progressed faster in the soil mix than in the clay. However, in this medium, after an initial delay, root colonization from in vitro-produced spores was essentially the same as that observed with the root-segment inoculum, reaching 44% and 58% respectively, after 16 weeks. Leek roots colonized by the monoxenically-produced spores harbored only the studied AMF fungi while the roots colonized from the root segments were substantially contaminated by other fungi. Accepted: 25 December 1998     

In situ analysis of anastomosis in representative genera of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) form obligate symbiotic associations with plants. As a result, the role of hyphal interactions in the establishment and maintenance of common mycorrhizal networks is poorly understood because of constraints on methods for in situ analysis. We designed a rhizohyphatron that allows the examination of intact mycelia growing from whole mycorrhizal plants. Plants preinoculated with spores were cultivated in a compartment with a connecting tube from which hyphae extend through a fine nylon mesh onto agar-coated slides. Species selected from each of the five AMF genera were used to assess and characterize the anastomosis behavior in the rhizohyphatron. Hyphal networks of Paraglomus occultum, Ambispora leptoticha, Scutellospora heterogama, and Gigaspora gigantea growing on the agar-coated slides showed no evidence of hyphal fusion. In contrast, anastomosis occurred in the hyphal networks of Glomus clarum and Glomus intraradices at an average frequency of less than 15% for both species. The rhizohyphatron developed in this study will provide knowledge of the biology and genetics of self/non-self recognition in AMF and help to better understand Glomeromycotan life history strategies.     

Influence of liming,inoculum level and inoculum placement on root colonization of subterranean clover

Arbuscular mycorrhizal (AM) fungi differ in their response to soil pH. Thus, change in soil pH may influence the relative abundance of mycorrhizal fungi inside roots. Root colonization by two AM fungi was studied in relation to addition of lime (CaCO3), quantity of inoculum and inoculum placement. Addition of CaCO3 to an acid soil decreased the colonization of roots by Acaulospora laevis but increased colonization by Glomus invermaium when both fungi were present. In acid soil (pH 4.7), almost all roots were colonized by A. laevis, while G. invermaium was dominant when soil pH was increased to pH 7.3. This occurred regardless of whether the inoculum was banded or mixed throughout the soil. There was no effect of CaCO3 on the relative abundance of fungi inside roots at intermediate rates of CaCO3 application (pH 5.3-6.3) when both fungi were inoculated together. In this experiment, both fungi colonized roots at all levels of CaCO3 when inoculated alone, except for A. laevis at the highest level of CaCO3. We conclude that soil pH affects the competitive ability of these two AM fungi during mycorrhiza formation primarily by affecting hyphae growth in soil and thus the relative abundance of hyphae at the root surface and subsequently inside the root.     

Assessment of Natural Mycorrhizal Potential in a Desertified Semiarid Ecosystem

A survey of the natural mycorrhizal potential has been carried out in a representative area of a desertified semiarid ecosystem in the southeast of Spain. Many indigenous plants from the field site were mycorrhizal, including the dominant Anthyllis cytisoides, which had high levels of colonization by arbuscular mycorrhizal fungi (AMF). Low numbers of AMF spores were present in the soil, although a range of species, including Scutellospora calospora, Glomus coronatum, Glomus constrictum, and several Acaulospora species, was represented. Soil infectivities, as determined by a soil dilution method, were similar for most plants tested but were significantly lower for Anthyllis cytisoides. Nevertheless, when a less disruptive method to determine soil infectivity was used, the importance of the mycelial network in maintaining the infectivity of soil under perennial shrubs, such as Anthyllis cytisoides, was highlighted. Seasonal variations in the mycorrhizal infectivity showed that it was higher towards the end of the summer period than in midwinter. In screening trials in a greenhouse, the indigenous AMF did not significantly improve the growth of plants compared with that of noninoculated controls. Augmentation of the soil with an inoculum of Glomus intraradices resulted in improved growth of Anthyllis cytisoides in both sterile and nonsterile conditions, in contrast to results obtained following inoculation with Glomus mosseae or another Glomus sp. Our findings suggest that the indigenous inoculum levels of AMF are inadequate to support an extensive revegetation program in the absence of an additional mycorrhizal inoculum.     

Diversity and structure of AMF communities as affected by tillage in a temperate soil

Arbuscular mycorrhizal fungi (AMF) were studied in differently tilled soils from a long-term field experiment in Switzerland. Diversity and structure of AMF communities were surveyed either directly on spores isolated from the field soil or on spores isolated from trap cultures, planted with different host plants. Single-spore cultures were established from the AMF spores obtained from trap cultures. Identification of the AMF was made by observation of spore morphology and confirmed by sequencing of ITS rDNA. At least 17 recognised AMF species were identified in samples from field and/or trap cultures, belonging to five genera of AMF--Glomus, Gigaspora, Scutellospora, Acaulospora, and Entrophospora. Tillage had a significant influence on the sporulation of some species and non- Glomus AMF tended to be more abundant in the no-tilled soil. The community structure of AMF in the field soil was significantly affected by tillage treatment. However, no significant differences in AMF diversity were detected among different soil tillage treatments. AMF community composition in trap cultures was affected much more by the species of the trap plant than by the original tillage treatment of the field soil. The use of trap cultures for fungal diversity estimation in comparison with direct observation of field samples is discussed.