Arbuscular mycorrhizal potential and mycorrhizal fungi diversity associated with Agave potatorum Zucc. in Oaxaca, Mexico

The status of the arbuscular mycorrhizal association in wild Agave potatorum Zucc. was studied at three semiarid sites over a 1-year period of rhizospheric soil sampling. Root colonization present at all sites and at all times of year, ranged from 20 to 83 %. The extraradical mycelia length was estimated to be from 2.64 to 5.22 m g^?1 of dry soil. Spore number ranged from 20 to 192 in 100 g of soil. The number of viable mycorrhizal propagules ranged from 500 to 2,640 in 100 g of soil. Twenty species of arbuscular mycorrhizal fungi (AMF) were identified, the family Glomeracea comprising the greatest number of species (45 %). Two other fungi families Acaulosporaceae and Gigasporaceae made up 35 and 15 %, respectively. High alpha diversity and low beta diversity of AMF were found in this study. Arbuscular mycorrhizal fungi species richness in A. potatorum is high and the associated fungi appear to be an important component in semiarid ecosystems in this region of Mexico. Arbuscular mycorrhizal fungi species with small spores might be better adapted to the local environment.     

Patterns of arbuscular mycorrhiza down the profile of a heavy textured soil do not reflect associated colonization potential

Colonization of roots by arbuscular mycorrhizal (AM) fungi in several annual crops in two consecutive seasons was compared with, in the second season, the density of fungal propagules in the soil with the use of a bioassay. Root density decreased down the soil profile in both years in all crops, and a high proportion of roots were mycorrhizal throughout the profile. AM colonization decreased down the profile in cotton and lablab in the second season only. The bioassay indicated that most propagules of AM fungi in soils under cotton were located near the surface, with virtually no propagules at 1 m. The absence of propagules at depth indicates a lack of mycelium deep in the soil, and suggests that mycorrhizas are primarily initiated in the surface soil and that the fungi colonize the root system mostly through secondary spread down the profile. The use of AM colonization in the field as an indicator of propagule density and symbiotic function should be qualified by an understanding of the depth in the soil from which roots were extracted.     

Fungi from the Lower Devonian Rhynie chert: My Coparasitism

Several examples of mycoparasitism are described from the Lower Devonian (Siegenian) Rhynie chert. These fungal interactions include thick-walled chlamydospores and vesicles in which epibiotic fungi are attached to the outer surface of the spore. Other fossil spores are characterized by mycoparasites developing between the layers of the spore wall or within the lumen. The presence of callosities extending from the inner surface of some fossil spores demonstrates that the hosts were alive when parasitized. This response by the mycohost is identical to that found in certain modem mycoparasitic symbioses involving vesicular arbuscular mycorrhizae that are parasitized by various aquatic fungi. The presence of mycoparasitism in a 400-million-year-old ecosystem underscores the potential significance of the fungal genome early in the evolution of other organisms.     

In vitro mycorrhization of the rubber tree Hevea brasiliensis Müll Arg

In vitro cultivation systems of arbuscular mycorrhizal fungi are useful tools to study the interaction between plants and their fungal symbiont, and also to develop new biotechnologies. Plantlets of the latex-producing species Hevea brasiliensis clone PB 260 were grown in a dense extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833 developed from a mycelium donor plant (Medicago truncatula A17). The factors indole-3-butyric acid (IBA), 2-morpholineoethanesulfonic acid monohydrate (MES) buffer, and carbon dioxide (CO₂) were tested on root development and colonization by the fungus. No colonization was observed in the presence of plantlets pre-treated with IBA. The highest levels of root colonization were obtained when plantlets were mycorrhized under a high CO₂ concentration (1,000 μmol?mol^?1) with MES (10 mM) added to the growth medium. Widespread root colonization (with presence of arbuscules, intraradical mycelium, and spores/vesicles) was predominantly observed in newly produced roots. Therefore, it appears essential to improve root initiation and growth for improving in vitro mycorrhization of H. brasiliensis. We demonstrated the potential of the “mycelium donor plant” in vitro culture system to produce colonized H. brasiliensis plantlets before their transfer to ex vitro conditions.     

Microwave-assisted technology for the clearing and staining of arbuscular mycorrhizal fungi in roots

The use of microwave irradiation as a source of energy to clear and stain intra-radical arbuscular mycorrhizal fungi propagules has been tested on a variety of indigenous and cultivated herbaceous plants. The aim of the study was to evaluate the efficiency of microwave irradiation on root softening, fungi tissue staining, and preservation of DNA integrity for subsequent molecular analyses. The proposed methodology has been adapted from the standard procedures used to detect and quantify mycorrhizal root colonization levels. Using a domestic microwave oven, tissue clearing and staining required together between 30 s and 1.5 min of microwave treatment to be completed, depending the diameter size of the roots. The well-performing chemical stains tested were acid fuchsin, trypan blue, and aniline blue. The acid fuchsin clearing and staining processes, as performed, were also demonstrated to preserve DNA integrity for further molecular analyses. Irradiation by microwaves has been used with success in our laboratory within the frame of several studies. It offers considerable time saving over traditional method, reducing processing times from several hours to a few minutes while decreasing considerably the amount of chemicals and energy required to perform analyses.     

Axenic culture and encapsulation of the intraradical forms of Glomus spp.

In recent years there have been many attempts to cultivate in vitro vesicular-arbuscular mycorrhizal (VAM) fungi which are obligate symbionts. Resting spores extracted from soils are often used as inoculum. Mycorrhizal root pieces are also used for inoculation but the role of intra-radical structures has not been clearly established. On agar medium vegetative mycelium was regenerated from individual intra-radical vesicles and from hyphae extracted by enzymatic maceration. After cell penetration, the mycelium probably accumulates substances which allow growth of VAM fungi in pure culture. When associated with tomato roots, this mycelium forms typical mycorrhizae. Encapsulation stabilized the biological properties of mycorrhizal roots and isolated vesicles. The immobilization also preserved the infectivity of the intra-radical hyphae and vesicles. After 25 years of exclusive utilization of resting spores as starting material for axenic and dual cultures of VAM fungi, it appears that intra-radical vesicles may be preferable propagules.D.-G. Strullu and C. Romand are with the Université d'Angers, Laboratoire de Phytonique, 2 Boulevard Lavoisier, 49045 Angers Cedex, France. C. Plenchette is with INRA, Station d'Agronomie, 17 rue Sully, 21034 Dijon Cedex, France.     

An unusual microfungus in a fungal spore from the Lower Devonian Rhynie chert

Abstract: A new fossil microfungus, Kryphiomyces catenulatus gen. et sp. nov., occurs as an endobiotic mycelial thallus in a large spore of a glomeromycotan fungus from the Lower Devonian Rhynie chert. The thallus consists of branched (?pseudo‐)septate hyphae with numerous catenulate swellings. Some hyphal tips produce spherical reproductive structures or propagules. Hyphal morphology in K. catenulatus is reminiscent of that in certain extant Hyphochytridiomycota, Chytridiomycota, and even Ascomycota, but specific diagnostic features that allow assignment of the fossil to modern groups are absent. The discovery of this interfungal association broadens our knowledge about the diversity of microfungi and their intricate associations in early continental ecosystems.     

Effect of crop rotation on native vesicular arbuscular mycorrhizal propagules in soil

The effect of crop rotation of native vesicular arbuscular mycorrhizal fungi was studied. Finger millet was grown as the first season crop in 15 plots. In the second season a mycorhizal host (cowpea) and a non-mycorrhizal host (mustard) were grown in 5 plots each, and the remaining 5 plots were left fallow. In the third season cowpea was grown in all the plots. Leaving the land fallow reduced the mycorrhizal propagules by 40% while growing a non-mycorrhizal host reduced it by 13%. Cowpea grown in the third season coincided with a slow build up of mycorrhizal propagules in soil. There was a slow build up of mycorrhizal propagules late in the season irrespective of the treatment in the preceding season.     

Arbuscular mycorrhizal propagules in soils from a tropical forest and an abandoned cornfield in Quintana Roo, Mexico: visual comparison of most-probable-number estimates

The present study was aimed at comparing the number of arbuscular mycorrhizal fungi (AMF) propagules found in soil from a mature tropical forest and that found in an abandoned cornfield in Noh-Bec Quintana Roo, Mexico, during three seasons. Agricultural practices can dramatically reduce the availability and viability of AMF propagules, and in this way delay the regeneration of tropical forests in abandoned agricultural areas. In addition, rainfall seasonality, which characterizes deciduous tropical forests, may strongly influence AMF propagules density. To compare AMF propagule numbers between sites and seasons (summer rainy, winter rainy and dry season), a “most probable number” (MPN) bioassay was conducted under greenhouse conditions employing Sorgum vulgare L. as host plant. Results showed an average value of 3.5 ± 0.41 propagules in 50 ml of soil for the mature forest while the abandoned cornfield had 15.4 ± 5.03 propagules in 50 ml of soil. Likelihood analysis showed no statistical differences in MPN of propagules between seasons within each site, or between sites, except for the summer rainy season for which soil from the abandoned cornfield had eight times as many propagules compared to soil from the mature forest site for this season. Propagules of arbuscular mycorrhizal fungi remained viable throughout the sampling seasons at both sites. Abandoned areas resulting from traditional slash and burn agriculture practices involving maize did not show a lower number of AMF propagules, which should allow the establishment of mycotrophic plants thus maintaining the AMF inoculum potential in these soils.     

FUNGI FROM THE LOWER DEVONIAN RHYNIE CHERT: CHYTRIDIOMYCETES

Several different chytridiomycetes are described from the Lower Devonian (Siegenian) Rhynie chert. Included are both eucarpic and apparently holocarpic forms that occur in Palaeonitella, Aglaophyton, Lyonophyton, Horneophyton, and clusters of algal cells, as well as in the surrounding chert matrix. Holocarpic types consist of endobiotic sporangia, each characterized by one discharge tube. Sporangia can be traced from the thallus stage to the discharge of zoospores. Monocentric and polycentric eucarpic chytrids are associated with the miospores of Aglaophyton and various thick-walled fungal spores. In these forms the sporangia are variable in size and shape ranging up to 30 μm. Most appear to be inoperculate and there is evidence that the sporangium ruptured on the distal surface. Some contain zoospores with flagella. One operculate eucarpic form had parasitized the cellular gametophyte emerging from the proximal surface of an Aglaophyton spore. Several of the Rhynie chert chytrids are comparable with a number of extant forms (e.g., Olpidiaceae and Spizellomycetaceae), while others possess features that encompass several groups. These fossil fungi are discussed in the context of their interactions with other organisms in this Lower Devonian freshwater paleoecosystem.     

Correlation of abundance of arbuscular mycorrhizal fungi, bacteria and saprophytic microfungi with soil carbon, nitrogen and phsophorus

A significant positive correlation between the concentration of CFU of soil saprophytic microfungi and total soil carbon content, organic matter (oxidizable carbon) and available phosphorus was observed in field collected soil samples. Concentration of CFU of culturable bacteria correlated negatively with soil organic matter. Specific length of hyphae of arbuscular mycorrhizal fungi in the soil correlated only with soil respiration rate. The results indicate that arbuscular mycorrhizal fungi are associated with soil microsites rich in some easily mineralizable fraction of soil, organic matter rather than with total or oxidizable organic carbon.     

13C incorporation into signature fatty acids as an assay for carbon allocation in arbuscular mycorrhiza

The ubiquitous arbuscular mycorrhizal fungi consume significant amounts of plant assimilated C, but this C flow has been difficult to quantify. The neutral lipid fatty acid 16:1omega5 is a quantitative signature for most arbuscular mycorrhizal fungi in roots and soil. We measured carbon transfer from four plant species to the arbuscular mycorrhizal fungus Glomus intraradices by estimating (13)C enrichment of 16:1omega5 and compared it with (13)C enrichment of total root and mycelial C. Carbon allocation to mycelia was detected within 1 day in monoxenic arbuscular mycorrhizal root cultures labeled with [(13)C]glucose. The (13)C enrichment of neutral lipid fatty acid 16:1omega5 extracted from roots increased from 0.14% 1 day after labeling to 2.2% 7 days after labeling. The colonized roots usually were more enriched for (13)C in the arbuscular mycorrhizal fungal neutral lipid fatty acid 16:1omega5 than for the root specific neutral lipid fatty acid 18:2omega6,9. We labeled plant assimilates by using (13)CO(2) in whole-plant experiments. The extraradical mycelium often was more enriched for (13)C than was the intraradical mycelium, suggesting rapid translocation of carbon to and more active growth by the extraradical mycelium. Since there was a good correlation between (13)C enrichment in neutral lipid fatty acid 16:1omega5 and total (13)C in extraradical mycelia in different systems (r(2) = 0.94), we propose that the total amount of labeled C in intraradical and extraradical mycelium can be calculated from the (13)C enrichment of 16:1omega5. The method described enables evaluation of C flow from plants to arbuscular mycorrhizal fungi to be made without extraction, purification and identification of fungal mycelia.     

Fungal colonization of the rooting system of the early land plant Asteroxylon mackiei from the 407‐Myr‐old Rhynie Chert (Scotland,UK)

Associations between plants and fungi were an important and varied feature of early terrestrial ecosystems, but in most instances their biological functions remain poorly understood. We document a new species of fungus colonizing the rooting system of the early lycopod Asteroxylon mackiei, based on exceptionally well‐preserved fossils from the Rhynie Chert. We investigated historical petrographic thin sections using standard optical microscopy and confocal laser scanning microscopy. P alaeozoosporites renaultii gen. nov., sp. nov. colonized the inner cortex of the plant rooting system. The fungus had an aseptate thallus with isotomous or sympodial branching. The mycelium bore distinctive porate, globose to elongated structures that we interpret as zoosporangia and resting sporangia. Doubts remain over the precise systematic affinity of P. renaultii, which closely resembles chytrids. Whereas most of the Rhynie Chert plants developed symbiotic associations of the mycorrhizal type, it seems that this was not the case for Asteroxylon mackiei, which possessed the most evolved rooting system among the Rhynie Chert plants. We argue that the new root‐borne fungus was probably parasitic. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 201–213.     

Germination shields in Scutellospora (Glomeromycota: Diversisporales, Gigasporaceae) from the 400 million-year-old Rhynie chert

Glomeromycotan spores from the Lower Devonian Rhynie chert provide the first evidence for germination shields in fossil fungi and demonstrate that this complex mode of germination was in place in some fungi at least 400 million years ago. Moreover, they represent the first direct marker relative to the precise systematic position of an Early Devonian endomycorrhizal fungus. In extant fungi, germination shields occur exclusively in the genus Scutellospora (Glomeromycota: Diversisporales, Gigasporaceae). These structures are regarded as a derived feature within the phylum Glomeromycota, and hence their presence in the Rhynie chert suggests that major diversification within this group of fungi occurred before the Early Devonian.Taxonomical novelties Scutellosporites Dotzler, M. Krings, T.N. Taylor and Agerer Scutellosporites devonicus Dotzler, M. Krings, T.N. Taylor and AgererStürmer 1998     

Fungal endophytes in a 400-million-yr-old land plant: infection pathways, spatial distribution, and host responses

The Early Devonian Rhynie chert has been critical in documenting early land plant-fungal interactions. However, complex associations involving several fungi that enter into qualitatively different relationships with a single host plant and even interact with one another have not yet been detailed. Here, we studied petrographic thin sections of the Rhynie chert plant Nothia aphylla. Three fungal endophytes (co)occur in prostrate axes of this plant: narrow hyphae producing clusters of small spores; large spherical spores/zoosporangia; and wide aseptate hyphae that form intercellular vesicles in the cortex. Host responses on attack include bulging of infected rhizoids, formation of encasement layers around intracellular hyphae, and separation of infected from uninfected tissues by secondarily thickened cell walls. A complex simultaneous interaction of N. aphylla with three endophytic fungi was discovered. The host responses indicate that some of the mechanisms causing host responses in extant plants were in place 400 million yr ago. Anatomical and life history features of N. aphylla suggest that this plant may have been particularly susceptible to colonization by fungi.     

丛枝菌根真菌菌丝体吸附重金属的潜力及特征

应用玻璃珠分室培养系统获得丛枝菌根真菌材料,研究了离体真菌菌丝体对pH缓冲体系中Zn、Cd和Mn等金属离子的吸附特征。试验结果表明,真菌菌丝体对各金属离子吸附能力差异显著,对Cd最强,Zn次之,Mn最弱。试验条件下,菌体可分别吸附相当于自身干物重1.6%的Mn、2.8%的Zn和13.3%的Cdo吸附于菌丝体的Cd2+绝大部分可以被Ca2+交换吸附。另外研究了宿主植物根系对Cd的吸附作用,证实菌根真菌侵染改变了根系的吸附特性,相对于非菌根根系,菌根的CEC较高,对Cd的吸附能力较强。试验结果为重金属污染条件下丛枝菌根强化根系的屏障作用提供了直接证据。     

Identification of a mycorrhizal fungus in the roots of achlorophyllous Sciaphila tosaensis Makino (Triuridaceae)

The identity of a mycorrhizal fungus in the roots of achlorophyllous Sciaphila tosaensis was investigated by DNA analysis and examination of the morphology of the association. The morphological features of the mycorrhizal fungus, i. e. aseptate hyphal coils, vesicles, arbuscule-like branching, and degenerate coils were similar to those previously reported for other achlorophyllous plants. Spore-like propagules identified asa glomalean fungus were propagated from root pieces of S. tosaensis in pot culture using alfalfa as the host trap plant. A PCR product was obtained from colonized root of S. tosaensis using the taxon-specific primers, VANS1 and VAGLO. Sequence analysis of the DNA fragment showed it to be almost identical to other Glomus species. Although it has been reported many times that the morphology of mycorrhizal fungi in achlorophyllous plants is quite similar to that of arbuscular mycorrhizal fungi, this is the first report of the isolation and identification of such a fungus itself.     

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.     

Maintenance and preservation of ectomycorrhizal and arbuscular mycorrhizal fungi

Short- to long-term preservation of mycorrhizal fungi is essential for their in-depth study and, in the case of culture collections, for safeguarding their biodiversity. Many different maintenance/preservation methods have been developed in the last decades, from soil- and substrate-based maintenance to preservation methods that reduce (e.g., storage under water) or arrest (e.g., cryopreservation) growth and metabolism; all have advantages and disadvantages. In this review, the principal methods developed so far for ectomycorrhizal and arbuscular mycorrhizal fungi are reported and described given their distinct biology/ecology/evolutionary history. Factors that are the most important for their storage are presented and a protocol proposed which is applicable, although not generalizable, for the long-term preservation at ultra-low temperature of a large panel of these organisms. For ECM fungi, isolates should be grown on membranes or directly in cryovials until the late stationary growth phase. The recommended cryopreservation conditions are: a cryoprotectant of 10 % glycerol, applied 1–2 h prior to cryopreservation, a slow cooling rate (1 °C min^?1) until storage below ?130 °C, and fast thawing by direct plunging in a water bath at 35–37 °C. For AMF, propagules (i.e., spores/colonized root pieces) isolated from cultures in the late or stationary phase of growth should be used and incorporated in a carrier (i.e., soil or alginate beads), preferably dried, before cryopreservation. For in vitro-cultured isolates, 0.5 M trehalose should be used as cryoprotectant, while isolates produced in vivo can be preserved in dried soil without cryoprotectant. A fast cryopreservation cooling rate should be used (direct immersion in liquid nitrogen or freezing at temperatures below ?130 °C), as well as fast thawing by direct immersion in a water bath at 35 °C.     

Propagules of arbuscular mycorrhizal fungi in a secondary dry forest of Oaxaca, Mexico

Plant cover loss due to changes in land use promotes a decrease in spore diversity of arbuscular mycorrhizal fungi (AMF), viable mycelium and, therefore, in AMF colonization, this has an influence in community diversity and, as a consequence, in its recovery. To evaluate different AMF propagules, nine plots in a tropical dry forest with secondary vegetation were selected: 0, 1, 7, 10, 14, 18, 22, 25, and 27 years after abandonment in Nizanda, Oaxaca, Mexico. The secondary vegetation with different stages of development is a consequence of slash and burn agriculture, and posterior abandonment. Soil samples (six per plot) were collected and percentage of AMF field colonization, extrarradical mycelium, viable spore density, infectivity and most probable number (MPN) ofAMF propagules were quantified through a bioassay. Means for field colonization ranged between 40% and 70%, mean of total mycelium length was 15.7 +/- 1.88 mg(-1) dry soil, with significant differences between plots; however, more than 40% of extracted mycelium was not viable, between 60 and 456 spores in 100 g of dry soil were recorded, but more than 64% showed some kind of damage. Infectivity values fluctuated between 20% and 50%, while MPN showed a mean value of 85.42 +/- 44.17 propagules (100 g dry soil). We conclude that secondary communities generated by elimination of vegetation with agricultural purposes in a dry forest in Nizanda do not show elimination of propagules, probably as a consequence of the low input agriculture practices in this area, which may encourage natural regeneration.