Recombination in Glomus intraradices, a supposed ancient asexual arbuscular mycorrhizal fungus

Background: Arbuscular mycorrhizal fungi (AMF) are important symbionts of most plant species, promoting plant diversity and productivity. This symbiosis is thought to have contributed to the early colonisation of land by plants. Morphological stasis over 400 million years and the lack of an observed sexual stage in any member of the phylum Glomeromycota led to the controversial suggestion of AMF being ancients asexuals. Evidence for recombination in AMF is contradictory. Results: We addressed the question of recombination in the AMF Glomus intraradices by sequencing 11 polymorphic nuclear loci in 40 morphologically identical isolates from one field. Phylogenetic relationships among genotypes showed a reticulate network pattern providing a rationale to test for recombination. Five statistical tests predicted multiple recombinant regions in the genome of a core set of isolates. In contrast, five clonal lineages had fixed a large number of differences. Conclusion: Our data show that AMF from one field have undergone recombination but that clonal lineages coexist. This finding has important consequences for understanding AMF evolution, co-evolution of AMF and plants and highlights the potential for commercially introduced AMF inoculum recombining with existing local populations. Finally, our results reconcile seemingly contradictory studies on whether AMF are clonal or form recombining populations.

Identity and combinations of arbuscular mycorrhizal fungal isolates influence plant resistance and insect preference

1. Accumulating evidence indicates that plant resistance against above-ground herbivores can be affected by the presence of arbuscular mycorrhizal fungi (AMF) in association with the host plant. Little is known, however, about how AMF composition can influence herbivore choice to feed on a particular plant. 2. Unravelling the preference-performance hypothesis in a multitrophic context is needed to expand our knowledge of complex multitrophic interactions in natural systems. If given mycorrhizal fungal genotypes increase attractiveness for a herbivore (reduced plant resistance), then the benefits of increased unpalatability provided by the mycorrhizal fungi (increased plant resistance) might be outweighed by the increased herbivore recruitment. 3. This was addressed by designing three experiments to test the effects of different AMF genotypes, inoculated either alone or in combination, to measure intraspecific AMF effects on plant resistance and insect herbivore preference. Using strawberry (Fragaria vesca L.) plants that were colonised by eight different combinations of Rhizophagus irregularis isolates, we measured effects on plant growth, insect growth and survival, as well as feeding preferences of a generalist herbivore caterpillar (Spodoptera littoralis Boisduval). 4. Overall, it was found that: (i) AMF influenced plant resistance in an AMF genotype-specific manner; (ii) some AMF inoculations decreased insect performance; (iii) insects preferentially chose to feed more on leaves originating from non-mycorrhizal plants; but also that (iv) in a whole plant bioassay, insects preferentially chose the biggest plant, regardless of their mycorrhizal status. 5. Therefore, AMF-mediated trade-offs between growth and resistance against herbivores have been shown. Such trade-offs, particularly driven by plant attractiveness to herbivores, buffer the positive effects of the mycorrhizal symbiosis on enhanced plant growth.

Glyphosate drift affects arbuscular mycorrhizal association in coffee

Mycorrhizal association promotes better survival and nutrition of colonized seedling on field, and consequently, increasing of productivity. However, the weed management can interfere on this association, due to incorrect use of glyphosate. This work has assessed the effects of glyphosate drift on the growth and nutrition of arabica coffee plants (Catuaí Vermelho - IAC 99) colonized with arbuscular mycorrhizal fungi (AMF). The experiment was conducted in 2 x 5 factorial scheme, and included inoculated and non-inoculated plants, and five glyphosate subdoses (0.0, 57.6, 115.2, 230.4, and 460.8 g ha-1 of glyphosate), in randomized blocks with five replication. The inoculation was carried during the greenhouse phase of seedlings production with a mixture of Rhizophagus clarus and Gigaspora margarita, and after to transplanting, when the plants had seven pairs of leaves, glyphosate subdoses were applied. The product caused intoxication in up to 60% of non-inoculated and 45% on inoculated plants, when the highest dose of 460.8 g a.e. ha-1 was applied. A negative effect was noted on the growth and phosphorus content of coffee plants, this effect increased depending on glyphosate subdose, but regardless of inoculation. Glyphosate drift reduces the growth and nutrition of plants colonized by species of AMF and native fungi, negatively affecting root colonization of plants treated.

Spatial structuring of arbuscular mycorrhizal communities in benchmark and modified temperate eucalypt woodlands

Arbuscular mycorrhizal fungi (AMF) are crucial to the functioning of the plant–soil system, but little is known about the spatial structuring of AMF communities across landscapes modified by agriculture. AMF community composition was characterized across four sites in the highly cleared south-western Australian wheatbelt that were originally dominated by forb-rich eucalypt woodlands. Environmentally induced spatial structuring in AMF composition was examined at four scales: the regional scale associated with location, the site scale associated with past management (benchmark woodlands with no agricultural management history, livestock grazing, recent revegetation), the patch scale associated with trees and canopy gaps, and the fine scale associated with the herbaceous plant species beneath which soils were sourced. Field-collected soils were cultured in trap pots; then, AMF composition was determined by identifying spores and through ITS1 sequencing. Structuring was strongest at site scales, where composition was strongly related to prior management and associated changes in soil phosphorus. The two fields were dominated by the genera Funneliformis and Paraglomus, with little convergence back to woodland composition after revegetation. The two benchmark woodlands were characterized by Ambispora gerdemannii and taxa from Gigasporaceae. Their AMF communities were strongly structured at patch scales associated with trees and gaps, in turn most strongly related to soil N. By contrast, there were few patterns at fine scales related to different herbaceous plant species, or at regional scales associated with the 175 km distance between benchmark woodlands. Important areas for future investigation are to identify the circumstances in which recolonization by woodland AMF may be limited by fungal propagule availability, reduced plant diversity and/or altered chemistry in agricultural soils.

Dual mycorrhizal associations of jarrah (Eucalyptus marginata) in a nurse-pot system

Jarrah (Eucalyptus marginata Donn ex Sm.) plants, like many other eucalypts, can form symbiotic associations with both arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi. To study this tripartite relationship we developed a novel nurse-pot system to allow us to investigate the extent and temporal colonisation dynamics of jarrah by two AM species (Rhizophagus irregularis (Błaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler comb. nov. and Scutellospora calospora Nicol. & Gerd.) and two putative ECM species (Austroboletus occidentalis Watling & N.M. Greg. and Scleroderma sp.) and their potential effects on jarrah growth and nutrition. Our nurse-pot system, using jarrah as both the nurse plant and test plant, was developed to establish extraradical hyphal networks of both AM and ECM fungi that act as single or dual inoculum for test plants. Mycorrhizal colonisation was described and quantified, and growth and nutritional effects measured and analysed. Mycorrhizal colonisation increased with time for the test seedlings exposed to hyphae networks from S. calospora and Scleroderma sp. The nurse-pot system was effective at initiating colonisation of functioning AM or (putative) ECM systems separately but the ECM symbiosis was inhibited where a dual AM + ECM inoculum (R. irregularis and Scleroderma sp.) was present. The presence of S. calospora, A. occidentalis and Scleroderma sp. individually significantly increased the shoot biomass of seedlings compared with non-mycorrhizal controls. The two AM isolates had different physiological effects on jarrah plants. S. calospora improved growth and micronutrient uptake of jarrah seedlings whereas no positive response was observed with R. irregularis. In addition, as an interesting observation, the non-responsive AM fungus R. irregularis suppressed the ECM symbiosis in dually inoculated plants where ECM structures, positive growth response and nutritional effects were absent. When inoculated individually, ECM isolates dominated the growth response and uptake of P and other nutrients in this dual symbiotic plant. Despite the positive growth response in the A. occidentalis treatment, ECM structures were not observed in either nurse or test seedlings. From the effects of A. occidentalis on jarrah we hypothesise that this fungus forms a functional mycorrhizal-type partnership even without forming archetypal structures in and on the root

Carbon trading for phosphorus gain: the balance between rhizosphere carboxylates and arbuscular mycorrhizal symbiosis in plant phosphorus acquisition

Two key plant adaptations for phosphorus (P) acquisition are carboxylate exudation into the rhizosphere and mycorrhizal symbioses. These target different soil P resources, presumably with different plant carbon costs. We examined the effect of inoculation with arbuscular mycorrhizal fungi (AMF) on amount of rhizosphere carboxylates and plant P uptake for 10 species of low-P adapted Kennedia grown for 23 weeks in low-P sand. Inoculation decreased carboxylates in some species (up to 50%), decreased plant dry weight (21%) and increased plant P content (23%). There was a positive logarithmic relationship between plant P content and the amount of rhizosphere citric acid for inoculated and uninoculated plants. Causality was indicated by experiments using sand where little citric acid was lost from the soil solution over 2 h and citric acid at low concentrations desorbed P into the soil solution. Senesced leaf P concentration was often low and P-resorption efficiencies reached >90%. In conclusion, we propose that mycorrhizally mediated resource partitioning occurred because inoculation reduced rhizosphere carboxylates, but increased plant P uptake. Hence, presumably, the proportion of plant P acquired from strongly sorbed sources decreased with inoculation, while the proportion from labile inorganic P increased. Implications for plant fitness under field conditions now require investigation.

Nitrogen-tixing and vesicular-arbuscular mycorrhizal symbioses in some tropical legume trees of tribe Mimoseae

Response to mineral fertilization and inoculation with rhizobia and/or arbuscular mycorrhiza fungi (AMF) of the Anadenanthera colubrina, Mimosa bimucronata and Parapiptadenia rigida (Leguminosae-Mimosoideae) native trees from Brazilian riparian forests, were studied in nursery conditions. Each species was submitted to seven treatments, varying nitrogen and phosphorous fertilization and inoculation with rhizobia (r), mycorrhiza (m) or both (rm): NP, P, P + r, P + rm, N, N + m and N + rm. Results showed that AMF inoculations did not enhance the mycorrhizal colonization, and P uptake was not sufficient to sustain good growth of plants. The level of P mineral added affected negatively the AMF colonization in A. colubrina and M. bimucronata, but not in P. rigida. Native fungi infected the three legume hosts. The absence of mineral N limited growth of A. colubrina and P. rigida, but in M. bimucronata the lack of N was corrected by biological nitrogen fixation. N mineral added inhibited the nodulation, although spontaneous nodulation had occurred in A. colubrina and M. bimucronata. Rhizobia inoculation enhanced the number of nodules, nitrogenase activity and leghemoglobin content of these two species. Thus, the extent of rhizobial and mycorrhizal symbiosis in these species under nursery conditions can affect growth and consequently the post-planting success. (C) 2004 Elsevier B.V. All rights reserved.

Nodulation, arbuscular mycorrhizal colonization and growth of some legumes native from Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Arbuscular mycorrhizal fungal communities and soil aggregation as affected by cultivation of various crops during the sugarcane fallow period

Management systems involving crop rotation, ground cover species and reduced soil tillage can improve the soil physical and biological properties and reduce degradation. The primary purpose of this study was to assess the effect of various crops grown during the sugarcane fallow period on the production of glomalin and arbuscular mycorrhizal fungi in two Latosols, as well as their influence on soil aggregation. The experiment was conducted on an eutroferric Red Latosol with high-clay texture (680 g clay kg-1) and an acric Red Latosol with clayey texture (440 g kg-1 clay) in Jaboticabal (São Paulo State, Brazil). A randomized block design involving five blocks and four crops [soybean (S), soybean/fallow/soybean (SFS), soybean/millet/soybean (SMS) and soybean/sunn hemp/soybean (SHS)] was used to this end. Soil samples for analysis were collected in June 2011. No significant differences in total glomalin production were detected between the soils after the different crops. However, total external mycelium length was greater in the soils under SMS and SHS. Also, there were differences in easily extractable glomalin, total glomalin and aggregate stability, which were all greater in the eutroferric Red Latosol than in the acric Red Latosol. None of the cover crops planted in the fallow period of sugarcane improved aggregate stability in either Latosol.

Phosphorus and Nitrogen Regulate Arbuscular Mycorrhizal Symbiosis in Petunia hybrida

Phosphorus and nitrogen are essential nutrient elements that are needed by plants in large amounts. The arbuscular mycorrhizal symbiosis between plants and soil fungi improves phosphorus and nitrogen acquisition under limiting conditions. On the other hand, these nutrients influence root colonization by mycorrhizal fungi and symbiotic functioning. This represents a feedback mechanism that allows plants to control the fungal symbiont depending on nutrient requirements and supply. Elevated phosphorus supply has previously been shown to exert strong inhibition of arbuscular mycorrhizal development. Here, we address to what extent inhibition by phosphorus is influenced by other nutritional pathways in the interaction between Petunia hybrida and R. irregularis. We show that phosphorus and nitrogen are the major nutritional determinants of the interaction. Interestingly, the symbiosis-promoting effect of nitrogen starvation dominantly overruled the suppressive effect of high phosphorus nutrition onto arbuscular mycorrhiza, suggesting that plants promote the symbiosis as long as they are limited by one of the two major nutrients. Our results also show that in a given pair of symbiotic partners (Petunia hybrida and R. irregularis), the entire range from mutually symbiotic to parasitic can be observed depending on the nutritional conditions. Taken together, these results reveal complex nutritional feedback mechanisms in the control of root colonization by arbuscular mycorrhizal fungi.

Intracellular pH in Arbuscular Mycorrhizal Fungi1 : A Symbiotic Physiological Marker

A method was developed to perform real-time analysis of cytosolic pH of arbuscular mycorrhizal fungi in culture using dye and ratiometric measurements (490/450 nm excitations). The study was mainly performed using photometric analysis, although some data were confirmed using image analysis. The use of nigericin allowed an in vivo calibration. Experimental parameters such as loading time and concentration of the dye were determined so that pH measurements could be made for a steady-state period on viable cells. A characteristic pH profile was observed along hyphae. For Gigaspora margarita, the pH of the tip (0–2 μm) was typically 6.7, increased sharply to 7.0 behind this region (9.5 μm), and decreased over the next 250 μm to a constant value of 6.6. A similar pattern was obtained for Glomus intraradices. The pH profile of G. margarita germ tubes was higher when cultured in the presence of carrot (Daucus carota) hairy roots (nonmycorrhizal). Similarly, extraradical hyphae of G. intraradices had a higher apical pH than the germ tubes. The use of a paper layer to prevent the mycorrhizal roots from being in direct contact with the medium selected hyphae with an even higher cytosolic pH. Results suggest that this method could be useful as a bioassay for studying signal perception and/or H+ cotransport of nutrients by arbuscular mycorrhizal hyphae.

Plant Genes Related to Gibberellin Biosynthesis and Signaling Are Differentially Regulated during the Early Stages of AM Fungal Interactions

Dear Editor, Phytohormones are essential regulators of plant development, but their role in the signaling processes between plants and fungi during arbuscular mycorrhizal (AM) establishment is far from being understood (Ludwig-Müller, 2010). AM colonization leads to extensive effects on host metabolism, as revealed by transcriptome studies of AM plants (Hogekamp et al., 2011). Some genes have been specified as an AM core set, since they are mycorrhizal-responsive, irrespective of the identity of the plant, of the fungus, and of the investigated organ. These data support the idea that, on colonization, plants activate a wide reprogramming of their major regulatory networks and argue that mobile factors of fungal or plant origin are involved in such generalized metabolic changes. In this context, hormones may be good candidates (Bonfante and Genre, 2010). However, the emerging picture of the interaction between phytohormones and AMs is very patchy, and information on gibberellin (GA) involvement is still more limited (García-Garrido et al., 2010). The role of GA during nodulation is instead known to control the nodulation signaling pathway (Ferguson et al., 2011).

The arbuscular mycorrhizal fungus Glomus intraradices is haploid and has a small genome size in the lower limit of eukaryotes.

The genome size, complexity, and ploidy of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was determined using flow cytometry, reassociation kinetics, and genomic reconstruction. Nuclei of G. intraradices from in vitro culture, were analyzed by flow cytometry. The estimated average length of DNA per nucleus was 14.07+/-3.52 Mb. Reassociation kinetics on G. intraradices DNA indicated a haploid genome size of approximately 16.54 Mb, comprising 88.36% single copy DNA, 1.59% repetitive DNA, and 10.05% fold-back DNA. To determine ploidy, the DNA content per nucleus measured by flow cytometry was compared with the genome estimate of reassociation kinetics. G. intraradices was found to have a DNA index (DNA per nucleus per haploid genome size) of approximately 0.9, indicating that it is haploid. Genomic DNA of G. intraradices was also analyzed by genomic reconstruction using four genes (Malate synthase, RecA, Rad32, and Hsp88). Because we used flow cytometry and reassociation kinetics to reveal the genome size of G. intraradices and show that it is haploid, then a similar value for genome size should be found when using genomic reconstruction as long as the genes studied are single copy. The average genome size estimate was 15.74+/-1.69 Mb indicating that these four genes are single copy per haploid genome and per nucleus of G. intraradices. Our results show that the genome size of G. intraradices is much smaller than estimates of other AMF and that the unusually high within-spore genetic variation that is seen in this fungus cannot be due to high ploidy.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

? The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. ? We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. ? We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. ? Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.