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1.  Assess suitability of hydroaeroponic culture to establish tripartite symbiosis between different AMF species, beans, and rhizobia 
BMC Plant Biology  2009;9:73.
Background
Like other species of the Phaseoleae tribe, common bean (Phaseolus vulgaris L.) has the potential to establish symbiosis with rhizobia and to fix the atmospheric dinitrogen (N2) for its N nutrition. Common bean has also the potential to establish symbiosis with arbuscular mycorrhizal fungi (AMF) that improves the uptake of low mobile nutrients such as phosphorus, from the soil. Both rhizobial and mycorrhizal symbioses can act synergistically in benefits on plant.
Results
The tripartite symbiosis of common bean with rhizobia and arbuscular mycorrhizal fungi (AMF) was assessed in hydroaeroponic culture with common bean (Phaseolus vulgaris L.), by comparing the effects of three fungi spp. on growth, nodulation and mycorrhization of the roots under sufficient versus deficient P supplies, after transfer from initial sand culture. Although Glomus intraradices Schenck & Smith colonized intensely the roots of common bean in both sand and hydroaeroponic cultures, Gigaspora rosea Nicolson & Schenck only established well under sand culture conditions, and no root-colonization was found with Acaulospora mellea Spain & Schenck under either culture conditions. Interestingly, mycorrhization by Glomus was also obtained by contact with mycorrhized Stylosanthes guianensis (Aubl.) sw in sand culture under deficient P before transfer into hydroaeroponic culture. The effect of bean genotype on both rhizobial and mycorrhizal symbioses with Glomus was subsequently assessed with the common bean recombinant inbreed line 7, 28, 83, 115 and 147, and the cultivar Flamingo. Significant differences among colonization and nodulation of the roots and growth among genotypes were found.
Conclusion
The hydroaeroponic culture is a valuable tool for further scrutinizing the physiological interactions and nutrient partitioning within the tripartite symbiosis.
doi:10.1186/1471-2229-9-73
PMCID: PMC2713235  PMID: 19534785
2.  Enhancement of the efficacy of a carbamate nematicide against the potato cyst nematode, Globodera pallida, through mycorrhization in commercial potato fields 
Journal of Nematology  2010;42(1):22-32.
Two experiments were conducted over 2 years in commercial potato fields in Shropshire, UK, to evaluate the compatibility of the nematicide aldicarb with commercial inocula of arbuscular mycorrhizal fungi (AMF) in the control of the potato cyst nematode Globodera pallida. The AMF used were Vaminoc (mixed-AMF inoculum), Glomus intraradices (BioRize BB-E) and G. mosseae (isolate BEG 12). In the absence of AMF, the in-soil hatch of G. pallida increased 30% (P < 0.01) from wk-2 to wk-4 after planting. Inoculation of physiologically-aged potato (cv. Golden Wonder) tubers with AMF eliminated this delay in G. pallida hatch by stimulating a mean increase of 32% (P < 0.01) in hatch within 2 wk after planting. In the aldicarb-treated plots in Experiment 1, G. pallida multiplication rate was 38% lower (P < 0.05) in roots of AMF-inoculated than noninoculated plants, but in Experiment 2, this effect was slightly lower (P = 0.07). In these plots, the single AMF inocula showed also a weak trend (P = 0.10) towards greater tuber yields relative to their noninoculated counterparts. Mycorrhization therefore appears to enhance the efficacy of carbamate nematicides against G. pallida and consequently more research is proposed to validate these findings and fully explore the potential of this model.
PMCID: PMC3380507  PMID: 22736833
integrated pest management; potato cyst nematode; Globodera pallida; arbuscular mycorrhizal fungi; Glomus spp.; interaction; aldicarb; hatch; multiplication; Solanum tuberosum
3.  Impact of Land Use Intensity on the Species Diversity of Arbuscular Mycorrhizal Fungi in Agroecosystems of Central Europe 
The impact of land use intensity on the diversity of arbuscular mycorrhizal fungi (AMF) was investigated at eight sites in the “three-country corner” of France, Germany, and Switzerland. Three sites were low-input, species-rich grasslands. Two sites represented low- to moderate-input farming with a 7-year crop rotation, and three sites represented high-input continuous maize monocropping. Representative soil samples were taken, and the AMF spores present were morphologically identified and counted. The same soil samples also served as inocula for “AMF trap cultures” with Plantago lanceolata, Trifolium pratense, and Lolium perenne. These trap cultures were established in pots in a greenhouse, and AMF root colonization and spore formation were monitored over 8 months. For the field samples, the numbers of AMF spores and species were highest in the grasslands, lower in the low- and moderate-input arable lands, and lowest in the lands with intensive continuous maize monocropping. Some AMF species occurred at all sites (“generalists”); most of them were prevalent in the intensively managed arable lands. Many other species, particularly those forming sporocarps, appeared to be specialists for grasslands. Only a few species were specialized on the arable lands with crop rotation, and only one species was restricted to the high-input maize sites. In the trap culture experiment, the rate of root colonization by AMF was highest with inocula from the permanent grasslands and lowest with those from the high-input monocropping sites. In contrast, AMF spore formation was slowest with the former inocula and fastest with the latter inocula. In conclusion, the increased land use intensity was correlated with a decrease in AMF species richness and with a preferential selection of species that colonized roots slowly but formed spores rapidly.
doi:10.1128/AEM.69.5.2816-2824.2003
PMCID: PMC154529  PMID: 12732553
4.  Diversity of Arbuscular Mycorrhizal Fungus Populations in Heavy-Metal-Contaminated Soils 
High concentrations of heavy metals have been shown to adversely affect the size, diversity, and activity of microbial populations in soil. The aim of this work was to determine how the diversity of arbuscular mycorrhizal (AM) fungi is affected by the addition of sewage-amended sludge containing heavy metals in a long-term experiment. Due to the reduced number of indigenous AM fungal (AMF) propagules in the experimental soils, several host plants with different life cycles were used to multiply indigenous fungi. Six AMF ecotypes were found in the experimental soils, showing consistent differences with regard to their tolerance to the presence of heavy metals. AMF ecotypes ranged from very sensitive to the presence of metals to relatively tolerant to high rates of heavy metals in soil. Total AMF spore numbers decreased with increasing amounts of heavy metals in the soil. However, species richness and diversity as measured by the Shannon-Wiener index increased in soils receiving intermediate rates of sludge contamination but decreased in soils receiving the highest rate of heavy-metal-contaminated sludge. Relative densities of most AMF species were also significantly influenced by soil treatments. Host plant species exerted a selective influence on AMF population size and diversity. We conclude based on the results of this study that size and diversity of AMF populations were modified in metal-polluted soils, even in those with metal concentrations that were below the upper limits accepted by the European Union for agricultural soils.
PMCID: PMC91085  PMID: 9925606
5.  Assessment of arbuscular mycorrhizal fungi on the phytoremediation potential of Ipomoea aquatica on cadmium uptake 
3 Biotech  2012;2(3):193-198.
The phytoremedial potential of Ipomoea aquatica and role of arbuscular mycorrhizal fungi (AMF) during Cadmium uptake was studied under two different soils i.e., soil inoculated with and without AMF. The plants were treated with different concentrations of Cd(NO)3 starting from 0, 5, 10, 25, 50, and 100 ppm in three replicate design in soil with and without AMF inoculation. Results showed that AMF enhanced accumulation of cadmium in plant tissues at all concentrations. Plants in AMF exhibited tolerance for Cd up to 100 mg/l and accumulated 88.07% in its tissues with no visual symptoms of toxicity, whereas those in non-AMF showed marked growth reduction at the same concentration with a metal accumulation of 73.2%. A significant variation of antioxidant enzymes under different environments evaluated the defense pathways of plants during uptake of Cd. Physiological changes and nutrient uptake showed that plants inoculated in AMF were more unwavering during stress conditions. The study established that phytoremedial potential of I. aquatica depends on rhizospheric conditions which enhanced Cd uptake. Finally, it was established that AMF was able to maintain an efficient symbiosis with I. aquatica in soil moderately contaminated by Cd, viable due to relation between fungus and plant.
doi:10.1007/s13205-012-0046-8
PMCID: PMC3433885
Phytoremediation; Cadmium; AMF; Ipomoea aquatica; Antioxidant enzymes
6.  Arbuscular mycorrhizal fungi alter plant allometry and biomass–density relationships 
Annals of Botany  2010;107(3):407-413.
Background and Aims
Plant biomass–density relationships during self-thinning are determined mainly by allometry. Both allometry and biomass–density relationship have been shown to vary with abiotic conditions, but the effects of biotic interactions have not been investigated. Arbuscular mycorrhizal fungi (AMF) can promote plant growth and affect plant form. Here experiments were carried out to test whether AMF affect plant allometry and the self-thinning trajectory.
Methods
Two experiments were conducted on Medicago sativa L., a leguminous species known to be highly dependent on mycorrhiza. Two mycorrhizal levels were obtained by applying benomyl (low AMF) or not (high AMF). Experiment 1 investigated the effects of AMF on plant growth in the absence of competition. Experiment 2 was a factorial design with two mycorrhizal levels and two plant densities (6000 and 17 500 seeds m−2). Shoot biomass, root biomass and canopy radius were measured 30, 60, 90 and 120 d after sowing. The allometric relationships among these aspects of size were estimated by standardized major axis regression on log-transformed data.
Key Results
Shoot biomass in the absence of competition was lower under low AMF treatment. In self-thinning populations, the slope of the log (mean shoot biomass) vs. log density relationship was significantly steeper for the high AMF treatment (slope = –1·480) than for the low AMF treatment (–1·133). The canopy radius–biomass allometric exponents were not significantly affected by AMF level, but the root–shoot allometric exponent was higher in the low AMF treatment. With a high level of AMF, the biomass–density exponent can be predicted from the above-ground allometric model of self-thinning, while this was not the case when AMF were reduced by fungicide.
Conclusions
AMF affected the importance of below-ground relative to above-ground interactions and changed root vs. shoot allocation. This changed allometric allocation of biomass and altered the self-thinning trajectory.
doi:10.1093/aob/mcq249
PMCID: PMC3043928  PMID: 21169608
Arbuscular mycorrhizal fungi; biomass–density relationship; canopy radius–biomass allometry; root–shoot biomass allometry; Medicago sativa; self-thinning
7.  Hypericin and pseudohypericin concentrations of a valuable medicinal plant Hypericum perforatum L. are enhanced by arbuscular mycorrhizal fungi 
Mycorrhiza  2011;22(2):149-156.
Hypericum perforatum L. (St. John’s-wort, Hypericaceae) is a valuable medicinal plant species cultivated for pharmaceutical purposes. Although the chemical composition and pharmacological activities of H. perforatum have been well studied, no data are available concerning the influence of arbuscular mycorrhizal fungi (AMF) on this important herb. A laboratory experiment was therefore conducted in order to test three AMF inocula on H. perforatum with a view to show whether AMF could influence plant vitality (biomass and photosynthetic activity) and the production of the most valuable secondary metabolites, namely anthraquinone derivatives (hypericin and pseudohypericin) as well as the prenylated phloroglucinol—hyperforin. The following treatments were prepared: (1) control—sterile soil without AMF inoculation, (2) Rhizophagus intraradices (syn. Glomus intraradices), (3) Funneliformis mosseae (syn. Glomus mosseae), and (4) an AMF Mix which contained: Funneliformis constrictum (syn. Glomus constrictum), Funneliformis geosporum (syn. Glomus geosporum), F. mosseae, and R. intraradices. The application of R. intraradices inoculum resulted in the highest mycorrhizal colonization, whereas the lowest values of mycorrhizal parameters were detected in the AMF Mix. There were no statistically significant differences in H. perforatum shoot mass in any of the treatments. However, we found AMF species specificity in the stimulation of H. perforatum photosynthetic activity and the production of secondary metabolites. Inoculation with the AMF Mix resulted in higher photosynthetic performance index (PItotal) values in comparison to all the other treatments. The plants inoculated with R. intraradices and the AMF Mix were characterized by a higher concentration of hypericin and pseudohypericin in the shoots. However, no differences in the content of these metabolites were detected after the application of F. mosseae. In the case of hyperforin, no significant differences were found between the control plants and those inoculated with any of the AMF applied. The enhanced content of anthraquinone derivatives and, at the same time, better plant vitality suggest that the improved production of these metabolites was a result of the positive effect of the applied AMF strains on H. perforatum. This could be due to improved mineral nutrition or to AMF-induced changes in the phytohormonal balance. Our results are promising from the biotechnological point of view, i.e. the future inoculation of H. perforatum with AMF in order to improve the quality of medicinal plant raw material obtained from cultivation.
doi:10.1007/s00572-011-0391-1
PMCID: PMC3261393  PMID: 21626142
AMF species specificity; Anthraquinone derivatives; Arbuscular mycorrhiza; Hyperforin; Photosynthetic performance index; St. John’s-wort
8.  A Common Garden Test of Host-Symbiont Specificity Supports a Dominant Role for Soil Type in Determining AMF Assemblage Structure in Collinsia sparsiflora 
PLoS ONE  2013;8(2):e55507.
Specialization in plant host-symbiont-soil interactions may help mediate plant adaptation to edaphic stress. Our previous field study showed ecological evidence for host-symbiont specificity between serpentine and non-serpentine adapted ecotypes of Collinsia sparsiflora and arbuscular mycorrrhizal fungi (AMF). To test for adapted plant ecotype-AMF specificity between C. sparsiflora ecotypes and field AMF taxa, we conducted an AMF common garden greenhouse experiment. We grew C. sparsiflora ecotypes individually in a common pool of serpentine and non-serpentine AMF then identified the root AMF by amplifying rDNA, cloning, and sequencing and compared common garden AMF associates to serpentine and non-serpentine AMF controls. Mixing of serpentine and non-serpentine AMF soil inoculum resulted in an intermediate soil classified as non-serpentine soil type. Within this common garden both host ecotypes associated with AMF assemblages that resembled those seen in a non-serpentine soil. ANOSIM analysis and MDS ordination showed that common garden AMF assemblages differed significantly from those in the serpentine-only controls (R = 0.643, P<0.001), but were similar the non-serpentine-only control AMF assemblages (R = 0.081, P<0.31). There was no evidence of adapted host ecotype-AMF specificity. Instead soil type accounted for most of the variation AM fungi association patterns, and some differences between field and greenhouse behavior of individual AM fungi were found.
doi:10.1371/journal.pone.0055507
PMCID: PMC3564749  PMID: 23393588
9.  Specific bottom–up effects of arbuscular mycorrhizal fungi across a plant–herbivore–parasitoid system 
Oecologia  2009;160(2):267-277.
The majority of plants are involved in symbioses with arbuscular mycorrhizal fungi (AMF), and these associations are known to have a strong influence on the performance of both plants and insect herbivores. Little is known about the impact of AMF on complex trophic chains, although such effects are conceivable. In a greenhouse study we examined the effects of two AMF species, Glomus intraradices and G. mosseae on trophic interactions between the grass Phleum pratense, the aphid Rhopalosiphum padi, and the parasitic wasp Aphidius rhopalosiphi. Inoculation with AMF in our study system generally enhanced plant biomass (+5.2%) and decreased aphid population growth (−47%), but there were no fungal species-specific effects. When plants were infested with G. intraradices, the rate of parasitism in aphids increased by 140% relative to the G. mosseae and control treatment. When plants were associated with AMF, the developmental time of the parasitoids decreased by 4.3% and weight at eclosion increased by 23.8%. There were no clear effects of AMF on the concentration of nitrogen and phosphorus in plant foliage. Our study demonstrates that the effects of AMF go beyond a simple amelioration of the plants’ nutritional status and involve rather more complex species-specific cascading effects of AMF in the food chain that have a strong impact not only on the performance of plants but also on higher trophic levels, such as herbivores and parasitoids.
doi:10.1007/s00442-009-1294-0
PMCID: PMC2757589  PMID: 19219458
Aphidius rhopalosiphi; Insect herbivory; Multitrophic interactions; Parasitoid performance; Rhopalosiphum padi
10.  A Fungal Symbiont of Plant-Roots Modulates Mycotoxin Gene Expression in the Pathogen Fusarium sambucinum 
PLoS ONE  2011;6(3):e17990.
Fusarium trichothecenes are fungal toxins that cause disease on infected plants and, more importantly, health problems for humans and animals that consume infected fruits or vegetables. Unfortunately, there are few methods for controlling mycotoxin production by fungal pathogens. In this study, we isolated and characterized sixteen Fusarium strains from naturally infected potato plants in the field. Pathogenicity tests were carried out in the greenhouse to evaluate the virulence of the strains on potato plants as well as their trichothecene production capacity, and the most aggressive strain was selected for further studies. This strain, identified as F. sambucinum, was used to determine if trichothecene gene expression was affected by the symbiotic Arbuscular mycorrhizal fungus (AMF) Glomus irregulare. AMF form symbioses with plant roots, in particular by improving their mineral nutrient uptake and protecting plants against soil-borne pathogens. We found that that G. irregulare significantly inhibits F. sambucinum growth. We also found, using RT-PCR assays to assess the relative expression of trichothecene genes, that in the presence of the AMF G. irregulare, F. sambucinum genes TRI5 and TRI6 were up-regulated, while TRI4, TRI13 and TRI101 were down-regulated. We conclude that AMF can modulate mycotoxin gene expression by a plant fungal pathogen. This previously undescribed effect may be an important mechanism for biological control and has fascinating implications for advancing our knowledge of plant-microbe interactions and controlling plant pathogens.
doi:10.1371/journal.pone.0017990
PMCID: PMC3063790  PMID: 21455305
11.  Interactions of Heterodera glycines, Macrophomina phaseolina, and Mycorrhizal Fungi on Soybean in Kansas 
Journal of Nematology  1994;26(4S):675-682.
The impact of naturally occurring arbuscular mycorrhizal fungi on soybean growth and their interaction with Heterodera glycines were evaluated in nematode-infested and uninfested fields in Kansas. Ten soybean cultivars from Maturity Groups III-V with differential susceptibility to H. glycines were treated with the fungicide benomyl to suppress colonization by naturally occurring mycorrhizal fungi and compared with untreated control plots. In H. glycines-infested soil, susceptible cultivars exhibited 39% lower yields, 28% lower colonization by mycorrhizal fungi, and an eightfold increase in colonization by the charcoal rot fungus, Macrophomina phaseolina, compared with resistant cultivars. In the absence of the nematode, susceptible cultivars exhibited 10% lower yields than resistant cultivars, root colonization of resistant vs. susceptible soybean by mycorrhizal fungi varied with sampling date, and there were no differences in colonization by M. phaseolina between resistant and susceptible cultivars. Benomyl application resulted in 19% greater root growth and 9% higher seed yields in H. glycines-infested soil, but did not affect soybean growth and yield in the absence of the nematode. Colonization of soybean roots by mycorrhizal fungi was negatively correlated with H. glycines population densities due to nematode antagonism to the mycorrhizal fungi rather than suppression of nematode populations. Soybean yields were a function of the pathogenic effects of H. glycines and M. phaseolina, and, to a lesser degree, the stimulatory effects of mycorrhizal fungi.
PMCID: PMC2619575  PMID: 19279947
arbuscular mycorrhizal fungi; charcoal rot; endomycorrhizae; Glycine max; Heterodera glycines; interaction; Macrophomina phaseolina; nematode; soybean; soybean cyst nematode; vesicular-arbuscular mycorrhizal fungi
12.  Nonlegumes, Legumes, and Root Nodules Harbor Different Arbuscular Mycorrhizal Fungal Communities 
Applied and Environmental Microbiology  2004;70(10):6240-6246.
Legumes are an important plant functional group since they can form a tripartite symbiosis with nitrogen-fixing Rhizobium bacteria and phosphorus-acquiring arbuscular mycorrhizal fungi (AMF). However, not much is known about AMF community composition in legumes and their root nodules. In this study, we analyzed the AMF community composition in the roots of three nonlegumes and in the roots and root nodules of three legumes growing in a natural dune grassland. We amplified a portion of the small-subunit ribosomal DNA and analyzed it by using restriction fragment length polymorphism and direct sequencing. We found differences in AMF communities between legumes and nonlegumes and between legume roots and root nodules. Different plant species also contained different AMF communities, with different AMF diversity. One AMF sequence type was much more abundant in legumes than in nonlegumes (39 and 13%, respectively). Root nodules contained characteristic AMF communities that were different from those in legume roots, even though the communities were similar in nodules from different legume species. One AMF sequence type was found almost exclusively in root nodules. Legumes and root nodules have relatively high nitrogen concentrations and high phosphorus demands. Accordingly, the presence of legume- and nodule-related AMF can be explained by the specific nutritional requirements of legumes or by host-specific interactions among legumes, root nodules, and AMF. In summary, we found that AMF communities vary between plant functional groups (legumes and nonlegumes), between plant species, and between parts of a root system (roots and root nodules).
doi:10.1128/AEM.70.10.6240-6246.2004
PMCID: PMC522056  PMID: 15466571
13.  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.
PMCID: PMC1388798  PMID: 16535273
14.  Positive Feedback between Mycorrhizal Fungi and Plants Influences Plant Invasion Success and Resistance to Invasion 
PLoS ONE  2010;5(8):e12380.
Negative or positive feedback between arbuscular mycorrhizal fungi (AMF) and host plants can contribute to plant species interactions, but how this feedback affects plant invasion or resistance to invasion is not well known. Here we tested how alterations in AMF community induced by an invasive plant species generate feedback to the invasive plant itself and affect subsequent interactions between the invasive species and its native neighbors. We first examined the effects of the invasive forb Solidago canadensis L. on AMF communities comprising five different AMF species. We then examined the effects of the altered AMF community on mutualisms formed with the native legume forb species Kummerowia striata (Thunb.) Schindl. and on the interaction between the invasive and native plants. The host preferences of the five AMF were also assessed to test whether the AMF form preferred mutualistic relations with the invasive and/or the native species. We found that S. canadensis altered AMF spore composition by increasing one AMF species (Glomus geosporum) while reducing Glomus mosseae, which is the dominant species in the field. The host preference test showed that S. canadensis had promoted the abundance of AMF species (G. geosporum) that most promoted its own growth. As a consequence, the altered AMF community enhanced the competitiveness of invasive S. canadensis at the expense of K. striata. Our results demonstrate that the invasive S. canadensis alters soil AMF community composition because of fungal-host preference. This change in the composition of the AMF community generates positive feedback to the invasive S. canadensis itself and decreases AM associations with native K. striata, thereby making the native K. striata less dominant.
doi:10.1371/journal.pone.0012380
PMCID: PMC2927435  PMID: 20808770
15.  How does phosphate status influence the development of the arbuscular mycorrhizal symbiosis? 
Plant Signaling & Behavior  2011;6(9):1300-1304.
Most terrestrial plant roots form mutualistic symbiosis with soil-borne arbuscular mycorrhizal fungi (AMF), a characteristic feature of which is nutrient exchange between the two symbiotic partners. Phosphate (Pi) is the main benefit the host plants acquired from the AMF. It has long been a common realization that high Pi supply could suppress the AMF development. However, the direct molecular regulatory mechanisms underlying this plant directed suppression are lacking. Here we reviewed the recent work providing the evidences that high Pi supply induces transcriptional alteration, leading to the inhibition of AMF development at different stages of AM symbiosis, and gave our view on potential cross-talk among Pi starvation, AM as well as phytohormone signaling.
doi:10.4161/psb.6.9.16365
PMCID: PMC3258057  PMID: 22019636
phosphate; arbuscular mycorrhiza; symbiosis; signaling; cross-talk; phytohormone
16.  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.
doi:10.1186/1471-2148-9-13
PMCID: PMC2630297  PMID: 19146661
17.  Genetic analysis of the interaction between Allium species and arbuscular mycorrhizal fungi 
The response of Alliumcepa, A. roylei, A. fistulosum, and the hybrid A. fistulosum × A. roylei to the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was studied. The genetic basis for response to AMF was analyzed in a tri-hybrid A. cepa × (A. roylei × A. fistulosum) population. Plant response to mycorrhizal symbiosis was expressed as relative mycorrhizal responsiveness (R′) and absolute responsiveness (R). In addition, the average performance (AP) of genotypes under mycorrhizal and non-mycorrhizal conditions was determined. Experiments were executed in 2 years, and comprised clonally propagated plants of each genotype grown in sterile soil, inoculated with G. intraradices or non-inoculated. Results were significantly correlated between both years. Biomass of non-mycorrhizal and mycorrhizal plants was significantly positively correlated. R′ was negatively correlated with biomass of non-mycorrhizal plants and hence unsuitable as a breeding criterion. R and AP were positively correlated with biomass of mycorrhizal and non-mycorrhizal plants. QTLs contributing to mycorrhizal response were located on a linkage map of the A. roylei × A. fistulosum parental genotype. Two QTLs from A. roylei were detected on chromosomes 2 and 3 for R, AP, and biomass of mycorrhizal plants. A QTL from A. fistulosum was detected on linkage group 9 for AP (but not R), biomass of mycorrhizal and non-mycorrhizal plants, and the number of stem-borne roots. Co-segregating QTLs for plant biomass, R and AP indicate that selection for plant biomass also selects for enhanced R and AP. Moreover, our findings suggest that modern onion breeding did not select against the response to AMF, as was suggested before for other cultivated species. Positive correlation between high number of roots, biomass and large response to AMF in close relatives of onion opens prospects to combine these traits for the development of more robust onion cultivars.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-010-1501-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s00122-010-1501-8
PMCID: PMC3043257  PMID: 21222096
18.  Arbuscular mycorrhizal fungi reduce effects of physiological integration in Trifolium repens 
Annals of Botany  2009;104(2):335-344.
Background and Aims
One of the special properties of clonal plants is the capacity for physiological integration, which can increase plant performance through mechanisms such as resource sharing and co-ordinated phenotypic plasticity when plants grow in microsites with contrasting resource availabilities. However, many clonal plants are colonized by arbuscular mycorrhizal fungi (AMF). Since AMF are likely to reduce contrasts in effective resource levels, they could also reduce these effects of clonal integration on plasticity and performance in heterogeneous environments.
Methods
To test this hypothesis, pairs of connected and disconnected ramets of the stoloniferous herb Trifolium repens were grown. One ramet in a pair was given high light and low nutrients while the other ramet was given high nutrients and low light. The pairs were inoculated with zero, one or five species of AMF.
Key Results
Pairs of ramets grown without AMF developed division of labour and benefited from resource sharing, as indicated by effects of connection on allocation to roots, accumulation of mass, and ramet production. Inoculation with five species of AMF significantly reduced these effects of connection, both by inhibiting them in ramets given high nutrients and inducing them in ramets given high light. Inoculation with one species of AMF also reduced some effects of connection, but generally to a lesser degree.
Conclusions
The results show that AMF can significantly modify the effects of clonal integration on the plasticity and performance of clonal plants in heterogeneous environments. In particular, AMF may partly replace the effects and benefits of clonal integration in low-nutrient habitats, possibly more so where species richness of AMF is high. This provides the first test of interaction between colonization by AMF and physiological integration in a clonal plant, and a new example of how biotic and abiotic factors could interact to determine the ecological importance of clonal growth.
doi:10.1093/aob/mcp130
PMCID: PMC2710896  PMID: 19493856
Arbuscular mycorrhizal fungi; biomass allocation; clonal plant; division of labour; environmental heterogeneity; light availability; nutrients; white clover
19.  Effect of Arbuscular Mycorrhizal Fungi on Plant Biomass and the Rhizosphere Microbial Community Structure of Mesquite Grown in Acidic Lead/Zinc Mine Tailings 
The Science of the total environment  2011;409(6):1009-1016.
Mine tailings in arid and semi-arid environments are barren of vegetation and subject to eolian dispersion and water erosion. Revegetation is a cost-effective strategy to reduce erosion processes and has wide public acceptance. A major cost of revegetation is the addition of amendments, such as compost, to allow plant establishment. In this paper we explore whether arbuscular mycorrhizal fungi (AMF) can help support plant growth in tailings at a reduced compost concentration. A greenhouse experiment was performed to determine the effects of three AMF inocula on biomass, shoot accumulation of heavy metals, and changes in the rhizosphere microbial community structure of the native plant Prosopis juliflora (mesquite). Plants were grown in an acidic lead/zinc mine tailings amended with 10% (w/w) compost amendment, which is slightly sub-optimal for plant growth in these tailings. After two months, AMF-inoculated plants showed increased dry biomass and root length (p < 0.05) and effective AMF colonization compared to controls grown in uninoculated compost-amended tailings. Mesquite shoot tissue lead and zinc concentrations did not exceed domestic animal toxicity limits regardless of whether AMF inoculation was used. The rhizosphere microbial community structure was assessed using denaturing gradient gel electrophoresis (DGGE) profiles of the small subunit RNA gene for bacteria and fungi. Canonical correspondence analysis (CCA) of DGGE profiles showed that the rhizosphere fungal community structure at the end of the experiment was significantly different from the community structure in the tailings, compost, and AMF inocula prior to planting. Further, CCA showed that AMF inoculation significantly influenced the development of both the fungal and bacterial rhizosphere community structures after two months. The changes observed in the rhizosphere microbial community structure may be either a direct effect of the AMF inocula, caused by changes in plant physiology induced by AMF, or a combination of both mechanisms.
doi:10.1016/j.scitotenv.2010.11.020
PMCID: PMC3030643  PMID: 21211826
Prosopis juliflora; phytostabilization; DGGE; mycorrhizal fungi; mine tailing
20.  Communities of Arbuscular Mycorrhizal Fungi Detected in Forest Soil Are Spatially Heterogeneous but Do Not Vary throughout the Growing Season 
PLoS ONE  2012;7(8):e41938.
Despite the important ecosystem role played by arbuscular mycorrhizal fungi (AMF), little is known about spatial and temporal variation in soil AMF communities. We used pyrosequencing to characterise AMF communities in soil samples (n = 44) from a natural forest ecosystem. Fungal taxa were identified by BLAST matching of reads against the MaarjAM database of AMF SSU rRNA gene diversity. Sub-sampling within our dataset and experimental shortening of a set of long reads indicated that our approaches to taxonomic identification and diversity analysis were robust to variations in pyrosequencing read length and numbers of reads per sample. Different forest plots (each 10×10 m and separated from one another by 30 m) contained significantly different soil AMF communities, and the pairwise similarity of communities decreased with distance up to 50 m. However, there were no significant changes in community composition between different time points in the growing season (May-September). Spatial structure in soil AMF communities may be related to the heterogeneous vegetation of the natural forest study system, while the temporal stability of communities suggests that AMF in soil represent a fairly constant local species pool from which mycorrhizae form and disband during the season.
doi:10.1371/journal.pone.0041938
PMCID: PMC3413688  PMID: 22879900
21.  Interactions of Vesicular-Arbuscular Mycorrhizal Fungi, Phosphorus, and Heterodera glycines on Soybean 
Journal of Nematology  1991;23(1):122-133.
Effects of vesicular-arbuscular mycorrhizal (VAM) fungi and soil phosphorus (P) fertility on parasitism of soybean cultivars Bragg and Wright by soybean cyst nematode (SCN) were investigated in field micropiot and greenhouse experiments. VAM fungi increased height of both cultivars and yield of Wright in microplot studies in 1986 and 1987. Conversely, yield of mycorrhizal and nonmycorrhizal plants of both cultivars was suppressed by SCN. Soil population densities of SCN were unaffected by VAM fungi in 1986 but were greater in microplots infested with VAM fungi than in control microplots in 1987. Growth of Wright soybean was stimulated by VAM fungi and suppressed by SCN in greenhouse experiments. The effect of VAM fungi on SCN varied with time. Numbers of SCN in roots and soil were decreased by VAM fungi by as much as 73% at the highest SCN inoculum level through 49 days after planting. Later, however, SCN numbers were usually comparable on mycorrhizal and nonmycorrhizal plants. Soil P fertility generally had no effect on SCN. Results of a split-root experiment indicated that VAM fungal suppression of SCN was not systemic.
PMCID: PMC2619123  PMID: 19283102
endomycorrhizae; Glycine max; Heterodera glycines; interaction; microplot; phosphorus; soybean; soybean cyst nematode; vesicular-arbuscnlar mycorrhizal fungi
22.  Fungal root endophyte associations of plants endemic to the Pamir Alay Mountains of Central Asia 
Symbiosis (Philadelphia, Pa.)  2011;54(3):139-149.
The fungal root endophyte associations of 16 species from 12 families of plants endemic to the Pamir Alay Mountains of Central Asia are presented. The plants and soil samples were collected in Zeravshan and Hissar ranges within the central Pamir Alay mountain system. Colonization by arbuscular mycorrhizal fungi (AMF) was found in 15 plant species; in 8 species it was of the Arum type and in 4 of the Paris type, while 3 taxa revealed intermediate arbuscular mycorrhiza (AM) morphology. AMF colonization was found to be absent only in Matthiola integrifolia, the representative of the Brassicaceae family. The AM status and morphology are reported for the first time for all the species analyzed and for the genera Asyneuma, Clementsia, and Eremostachys. Mycelia of dark septate endophytes (DSE) accompanied the AMF colonization in ten plant species. The frequency of DSE occurrence in the roots was low in all the plants, with the exception of Spiraea baldschuanica. However, in the case of both low and higher occurrence, the percentage of DSE root colonization was low. Moreover, the sporangia of Olpidium spp. were sporadically found inside the root epidermal cells of three plant species. Seven AMF species (Glomeromycota) found in the trap cultures established with soils surrounding roots of the plants being studied were reported for the first time from this region of Asia. Our results provide information that might well be of use to the conservation and restoration programmes of these valuable plant species. The potential application of beneficial root-inhabiting fungi in active plant protection projects of rare, endemic and endangered plants is discussed.
doi:10.1007/s13199-011-0137-z
PMCID: PMC3228954  PMID: 22207783
Arbuscular mycorrhiza (AM); Arbuscular mycorrhizal fungi (AMF) species diversity; Arum/Paris morphotypes; Conservation; Dark septate endophytes (DSE); Glomeromycota
23.  Arbuscular mycorrhizal fungi restore normal growth in a white poplar clone grown on heavy metal-contaminated soil, and this is associated with upregulation of foliar metallothionein and polyamine biosynthetic gene expression 
Annals of Botany  2010;106(5):791-802.
Background and Aims
It is increasingly evident that plant tolerance to stress is improved by mycorrhiza. Thus, suitable plant–fungus combinations may also contribute to the success of phytoremediation of heavy metal (HM)-polluted soil. Metallothioneins (MTs) and polyamines (PAs) are implicated in the response to HM stress in several plant species, but whether the response is modulated by arbuscular mycorrhizal fungi (AMF) remains to be clarified. The aim of the present study was to check whether colonization by AMF could modify growth, metal uptake/translocation, and MT and PA gene expression levels in white poplar cuttings grown on HM-contaminated soil, and to compare this with plants grown on non-contaminated soil.
Methods
In this greenhouse study, plants of a Populus alba clone were pre-inoculated, or not, with either Glomus mosseae or G. intraradices and then grown in pots containing either soil collected from a multimetal- (Cu and Zn) polluted site or non-polluted soil. The expression of MT and PA biosynthetic genes was analysed in leaves using quantitative reverse transcription–PCR. Free and conjugated foliar PA concentrations were determined in parallel.
Results
On polluted soil, AMF restored plant biomass despite higher Cu and Zn accumulation in plant organs, especially roots. Inoculation with the AMF caused an overall induction of PaMT1, PaMT2, PaMT3, PaSPDS1, PaSPDS2 and PaADC gene expression, together with increased free and conjugated PA levels, in plants grown on polluted soil, but not in those grown on non-polluted soil.
Conclusions
Mycorrhizal plants of P. alba clone AL35 exhibit increased capacity for stabilization of soil HMs, together with improved growth. Their enhanced stress tolerance may derive from the transcriptional upregulation of several stress-related genes, and the protective role of PAs.
doi:10.1093/aob/mcq170
PMCID: PMC2958786  PMID: 20810743
Arbuscular mycorrhizal fungi; contaminated soil; heavy metals; metallothioneins; polyamines; Populus alba; white poplar
24.  Arbuscular mycorrhizal fungal spores host bacteria that affect nutrient biodynamics and biocontrol of soil-borne plant pathogens 
Biology Open  2011;1(1):52-57.
Summary
The aim of this research was to isolate and characterize bacteria from spores of arbuscular mycorrhizal fungi (AMF). We designated these bacteria ‘probable endobacteria’ (PE). Three bacterial strains were isolated from approximately 500 spores of Gigaspora margarita (Becker and Hall) using a hypodermic needle (diameter, 200 μm). The bacteria were identified by morphological methods and on the basis of ribosomal gene sequences as Bacillus sp. (KTCIGM01), Bacillus thuringiensis (KTCIGM02), and Paenibacillus rhizospherae (KTCIGM03). We evaluated the effect of these probable endobacteria on antagonistic activity to the soil-borne plant pathogens (SBPPs) Fusarium oxysporum f. sp. lactucae MAFF 744088, Rosellinia necatrix, Rhizoctonia solani MAFF 237426, and Pythium ultimum NBRC 100123. We also tested whether these probable endobacteria affected phosphorus solubilization, ethylene production, nitrogenase activity (NA), and stimulation of AMF hyphal growth. In addition, fresh samples of spores and hyphae were photographed using an in situ scanning electron microscope (SEM) (Quanta 250FEG; FEI Co., Japan). Bacterial aggregates (BAs), structures similar to biofilms, could be detected on the surface of hyphae and spores. We demonstrate that using extraction with an ultrathin needle, it is possible to isolate AMF-associated bacterial species that are likely derived from inside the fungal spores.
doi:10.1242/bio.2011014
PMCID: PMC3507164  PMID: 23213368
arbuscular mycorrhizal fungi; bacteria; biocontrol; nutrient biodynamics
25.  Selectivity by host plants affects the distribution of arbuscular mycorrhizal fungi: evidence from ITS rDNA sequence metadata 
Background
Arbuscular mycorrhizal fungi (AMF) can form obligate symbioses with the vast majority of land plants, and AMF distribution patterns have received increasing attention from researchers. At the local scale, the distribution of AMF is well documented. Studies at large scales, however, are limited because intensive sampling is difficult. Here, we used ITS rDNA sequence metadata obtained from public databases to study the distribution of AMF at continental and global scales. We also used these sequence metadata to investigate whether host plant is the main factor that affects the distribution of AMF at large scales.
Results
We defined 305 ITS virtual taxa (ITS-VTs) among all sequences of the Glomeromycota by using a comprehensive maximum likelihood phylogenetic analysis. Each host taxonomic order averaged about 53% specific ITS-VTs, and approximately 60% of the ITS-VTs were host specific. Those ITS-VTs with wide host range showed wide geographic distribution. Most ITS-VTs occurred in only one type of host functional group. The distributions of most ITS-VTs were limited across ecosystem, across continent, across biogeographical realm, and across climatic zone. Non-metric multidimensional scaling analysis (NMDS) showed that AMF community composition differed among functional groups of hosts, and among ecosystem, continent, biogeographical realm, and climatic zone. The Mantel test showed that AMF community composition was significantly correlated with plant community composition among ecosystem, among continent, among biogeographical realm, and among climatic zone. The structural equation modeling (SEM) showed that the effects of ecosystem, continent, biogeographical realm, and climatic zone were mainly indirect on AMF distribution, but plant had strongly direct effects on AMF.
Conclusion
The distribution of AMF as indicated by ITS rDNA sequences showed a pattern of high endemism at large scales. This pattern indicates high specificity of AMF for host at different scales (plant taxonomic order and functional group) and high selectivity from host plants for AMF. The effects of ecosystemic, biogeographical, continental and climatic factors on AMF distribution might be mediated by host plants.
doi:10.1186/1471-2148-12-50
PMCID: PMC3395829  PMID: 22498355

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