Characterizing and handling different kinds of AM fungal spores in the rhizosphere

Abstract

Spores are important propagules as well as the most reliable species-distinguishing traits of arbuscular mycorrhizal (AM) fungi. During surveys of AM fungal communities, spore enumeration and spore identification are frequently conducted, but generally little attention is given to the age and viability of the spores. In this study, AM fungal spores in the rhizosphere were characterized as live or dead by vital staining and by performing a germination assay. A considerable proportion of the spores in the rhizosphere were dead despite their intact appearance. Furthermore, morphological and molecular analyses of spores to determine species identity revealed that both viable spores and dead spores with contents were identified. The accurate identification of spores at different developmental stages on the basis of morphology requires considerable experience. Our findings suggest that surveys of AM fungal communities based on spore enumeration and morphological and molecular identification are likely to be inaccurate, primarily because of the large proportion of dead spores in the rhizosphere. A viability check is recommended prior to spore molecular identification, and the use of trap cultures would give more reliable morphological identification results. We show that the abundance and activity of AM fungi in the rhizosphere can be determined by calculating the density of viable spores and the density of spores that could germinate. The adoption of these methods should provide a more reliable basis for further AM fungal community analysis.

Stimulation of Hyphal Ramification and Sporulation in Funneliformis mosseae by Root Extracts Is Host Phosphorous Status-Dependent

“…The effects of RE on AM fungal growth could be controversial, with some studies showing positive effects, such as the formation of highly branched structures [18,26,27,52], while others only found a slight effect on hyphal elongation [6,19,47]. In this study, we did not observe significant hyphal elongation; however, more branches formed in the presence of RE compared with the control.…”

“…The plantlets were cultured on a shaker (40 rpm) for three days (under the same cultivation conditions as those described above) before harvesting the RE by collecting the distilled-water solution in which the plantlets were growing. RE were adjusted to 5 or 50 mL/g fresh roots by adding sterile distilled water or concentrated using a rotary evaporator at a low temperature (below 35 • C) (1:5 and 1:50, respectively), and the pH was adjusted to 6.5 (around the middle of pH range of crude root exudates and extracts that we prepared; it is also the optimal pH for F. mosseae growth according to previous reports [45][46][47][48]). RE were then stored at 4 • C until ready for further use.…”

“…A single F. mosseae spore was placed on each disc. Petri dishes were sealed with parafilm and incubated at 25 • C (optimal temperature for F. mosseae growth according to previous reports [45][46][47]) in the dark for 10 d. Data collected from 10 dishes were combined as one sample and each treatment had five replicates.…”

Arbuscular Mycorrhizal Fungi from Argentinean Highland Puna Soils Unveiled by Propagule Multiplication

“…Some authors argue that TC does not reflect in situ reality [ 19 ]; others [ 20 ] state that TC should not be used to assess AMF diversity whenever the richness is less than 21 species per site, whereas the sole use of direct analysis of field samples should be avoided if the total AMF richness is below that value. However, it was demonstrated [ 21 ] both by morphological and molecular methods that a considerable proportion of spores in the rhizosphere were dead despite their intact appearance. The authors then stated that using TC would give more reliable results for AMF diversity studies.…”

The Tripartite Rhizobacteria-AM Fungal-Host Plant Relationship in Winter Wheat: Impact of Multi-Species Inoculation, Tillage Regime and Naturally Occurring Rhizobacteria Species

“…The AM fungal inoculum was obtained from wet sieving through a series of 40, 35 and 28 µm mesh sizes in succession. Collected spores were divided into morphology under stereomicroscope (Apex ® stereomicroscope, Wiltshire, UK) at a magnification of ×50 following the protocol described by Sun et al [ 28 ]. Spores identified as AM fungal were grown in a 250 mL conical flask of Czapek Dox broth with surface sterilised wheat roots attached to the side of the flask at 15 °C for 1 week.…”