The role of in vitro cultivation on asymbiotic trait variation in a single species of arbuscular mycorrhizal fungus

Cultivating arbuscular mycorrhizal (AM) fungi in vitro is an efficient way to produce material for industry and research. However, such artificial growing conditions may impose selective pressure on fungi grown in vitro over many generations. We hypothesized that isolates subjected to long term propagation in vitro may develop increasingly ruderal traits. We proposed a predictive framework for the effect of in vitro cultivation on asymbiotic AM fungal traits. Using photomicrography and image processing, we analyzed morphology and growth traits for 14 isolates representing an in vitro cultivation gradient from 0 to >80 generations in vitro. We investigated the range of trait variation among asymbiotic growth of arbuscular mycorrhizal (AM) fungus isolates (Rhizoglomus irregulare). Spore dormancy was strongly associated with in vitro cultivation. We observed extremely high levels of inter-isolate variation for most fungal traits, but this was not related to time in vitro. Our results indicate that intra-specific diversity may have a strong ecological role in AM fungal communities.     

Putative sites for nutrient uptake in arbuscular mycorrhizal fungi

Nutrition of the arbuscular mycorrhiza (AM) is addressed from a fungal point of view. Intraradical and extraradical structures proposed as preferential sites for nutrient acquisition in arbuscular mycorrhizal (AM) fungi are considered, and their main features compared. This comparison includes the formation and function of branched structures (either intra- or extraradical) as putative nutrient uptake sites with unique morphological and physiological features in the AM fungal colony. The morphology and functioning of these structures are further affected by intra- or extraradical environmental factors. A model is presented which portrays the intrinsic developmental and physiological duality of the AM fungus. This revised version was published online in June 2006 with corrections to the Cover Date.     

The arbuscular mycorrhizal fungus Rhizophagus intraradices and other microbial groups affect plant species in a copper-contaminated post-mining soil

Background and aimArbuscular mycorrhizal fungi (AMF) have an important role in plant-microbe interactions. But, there are few studies in which the combined effect of AMF with a stress factor, such as the presence of a metal, on plant species were assessed. This study investigated the effect of arbuscular mycorrhizal (AM) fungus Rhizophagus intraradices and other soil microbial groups in the presence of copper on three plant species in a microcosm experiment.MethodsTwo grass species Poa compressa and Festuca rubra and one herb species Centaurea jacea were selected as model plants in a pot-design test in which soils were artificially contaminated with copper. Treatments were bacteria (control), saprophytic fungi, protists, and a combined treatment of saprophytic fungi and protists, all in the presence or absence of the AM fungal species. After sixty days, plants were harvested and the biomass of grass and herb species and microbial respiration were measured.ResultsThe results showed almost equal above- and belowground plant biomass and microbial respiration in the treatments in the presence or absence of R. intraradices. The herb species C. jecea responded significantly to the soil inoculation with AM fungus, while grass species showed inconsistent patterns. Significant effect of AMF and copper and their interactions was observed on plant biomass when comparing contaminated vs. non-contaminated soils.ConclusionStrong effect of AMF on the biomass of herb species and slight changes in plant growth with the presence of this fungal species in copper-spiked test soils indicates the importance of mycorrhizal fungi compared to other soil microorganisms in our experimental microcosms.     

Changes in arbuscular mycorrhizal fungal communities during invasion by an exotic invasive plant

Exotic invasive plants can show strong plant–soil feedback responses, but little is known about time scales for significant changes in soil microbial communities to occur after invasion. Previous work has suggested that plant invasions can modify arbuscular mycorrhizal (AM) fungal community structure. However, there is a lack of understanding about how long it takes for these changes to develop. To test this we investigated temporal changes in AM fungal communities colonising the invasive plant Vincetoxicum rossicum (Apocynaceae). We hypothesised that AM fungal community structure would change in a particular direction during the invasion process. We collected soil from two sites with a long history of invasion by this plant, with each site having paired invaded and uninvaded plots. Soil from these plots was used in a glasshouse experiment to characterise AM fungal community structure in the roots of V. rossicum at different times throughout a simulated growing season. AM fungal community structure differed between invaded and uninvaded plots. However, contrasting with our hypothesis, AM fungal communities colonising V. rossicum growing in soil from uninvaded plots did not change towards those in plants growing in previously invaded soil. Our data suggest that changes to AM fungal communities in the presence of V. rossicum require longer than the first growing season after establishment to develop.     

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

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

Interactions between a fluorescent pseudomonad,an arbuscular mycorrhizal fungus and a hypovirulent isolate of Rhizoctonia solani affect plant growth and root architecture of tomato plants

Abstract

Although Rhizoctonia solani is a cosmopolitan soilborne pathogen, the genus includes isolates with different pathogenicity ranging from high virulence to avirulence. The biocontrol strain Pseudomonas fluorescens P190r and the arbuscular mycorrhizal (AM) fungus Glomus mosseae BEG12 were inoculated alone or in combination in tomato plants infested by the mildly virulent pathogen R. solani #235. Plant growth as well as root morphometric and topological parameters were evaluated. The infection of R. solani was significantly reduced by all the combinations of the beneficial microorganisms. Root systems of R. solani‐infected plants were weakly developed but highly branched with a herring‐bone pattern, while those inoculated with the AM fungus, alone or in combination with the bacterial strain, were longer and more developed, and displayed a dichotomous pattern. The interactions among these three microorganisms affected plant growth and root architecture of tomato plants.     

Regulation of arbuscular mycorrhizal development by plant host and fungus species in alfalfa

Two cvs of alfalfa ( Medicago sativa L.), Gilboa and Moapa 69, were inoculated in glasshouse pots with three arbuscular mycorrhizal (AM) fungi to investigate the efficacy of mycorrhizas with respect to the extent of colonization and sporulation. Paspalum notatum Flugge also was inoculated to describe fungal parameters on a routine pot culture host. Percentage root length of P. notatum colonized by Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe, Glomus intraradices Schenck & Smith, and Gigaspora margarita Becker & Hall increased from 10 to 21 wk, and all fungi sporulated during that period. In alfalfa, only colonization by G. intraradices increased over that time period, and it was the only fungus to sporulate in association with alfalfa at 10 wk. Glomus mosseae did not sporulate after 16–21 wk despite having colonized 30–35% of the root length of both alfalfa cvs. In vitro experiments in which Ri T-DNA-transformed roots of alfalfa were inoculated with AM fungi showed normal mycorrhizal formation by G. intraradices and a hypersensitivity-like response to Gi. margarita . Colonized cells became necrotic, and HPLC analysis indicated increased concentrations of phenolics and isoflavonoids in these root segments. These data strongly support the existence of a degree of specificity between AM fungi and host that might rely on specific biochemical regulatory processes initiated in the host as a result of the attempts at colonization by the fungus.     

Extraradical mycelium network of arbuscular mycorrhizal fungi allows fast colonization of seedlings under in vitro conditions

Actively growing extraradical hyphae extending from mycorrhizal plants are an important source of inoculum in soils which has seldom been considered in vitro to inoculate young plantlets. Seedlings of Medicago truncatula were grown in vitro in the extraradical mycelium network extending from mycorrhizal plants. After 3, 6, 9, 12, and 15 days of contact with the mycelium, half of the seedlings were harvested and analyzed for root colonization. The other half was carefully transplanted in vitro on a suitable growth medium and mycelium growth and spore production were evaluated for 4 weeks. Seedlings were readily colonized after 3 days of contact with the mycelium. Starting from 6 days of contact, the newly colonized seedlings were able to reproduce the fungal life cycle, with the production of thousands of spores within 4 weeks. The fast mycorrhization process developed here opens the door to a broad range of in vitro studies for which either homogenous highly colonized seedlings or mass-produced in vitro inoculum is necessary. Liesbeth Voets and Ivan Enrique de la Providencia contributed equally to this work. MUCL is part of the Belgian Coordinated Collections of Micro-organisms (BCCM).     

Effects of arbuscular mycorrhizal fungi on the growth,nutrient uptake and glycyrrhizin production of licorice (<Emphasis Type="Italic">Glycyrrhiza uralensis</Emphasis> Fisch)

The growth of licorice in arid areas faces nutritional and environmental stresses. Arbuscular mycorrhizal (AM) fungi have been shown to increase the abilities of plants to develop. However, little is known regarding the role of AM fungi in licorice (Glycyrrhiza uralensis) growth. In the present study, by inoculation with two AM fungi, Glomus mosseae (Nicolson & Gerdemann) Gerd. & Trappe and Glomus veriforme (P. Karst.), the effects on licorice growth in sand were examined by measuring plant height, number of leaves, shoot and root fresh weight, and by analyzing morphological parameters of the root system in sand. The influence of the two microorganisms on the accumulation of mineral nutritions and bioactive components in licorice were also investigated. The results showed that mycorrhyzae were of the Arum-type and their colonization frequency (F %), colonization intensity (M %) and colonization intensity (m %) of AM fungi inoculation were found to be 80.0–84.6%, 49.4–60.0% and 58.4–71.9%, respectively. The inoculation significantly improved plant growth during early and late growth stages in comparison with the control. Moreover, inoculation of G. mosseae and G. versiforme, alone or in combination, improved plant phosphorus acquisition in the leaf over non-inoculation plants. In addition, mycorrhiza formation enhanced the glycyrrhizin concentration in roots, but resulted in a considerable reduction of the root oxidase activity. The results indicate that the inoculation with AM fungi could be a useful approach to increase the licorice pharmic quality.     

Phylogenetic trait conservatism and the evolution of functional trade-offs in arbuscular mycorrhizal fungi

The diversity of functional and life-history traits of organisms depends on adaptation as well as the legacy of shared ancestry. Although the evolution of traits in macro-organisms is well studied, relatively little is known about character evolution in micro-organisms. Here, we surveyed an ancient and ecologically important group of microbial plant symbionts, the arbuscular mycorrhizal (AM) fungi, and tested hypotheses about the evolution of functional and life-history traits. Variation in the extent of root and soil colonization by AM fungi is constrained to a few nodes basal to the most diverse groups within the phylum, with relatively little variation associated with recent divergences. We found no evidence for a trade-off in biomass allocated to root versus soil colonization in three published glasshouse experiments; rather these traits were positively correlated. Partial support was observed for correlated evolution between fungal colonization strategies and functional benefits of the symbiosis to host plants. The evolution of increased soil colonization was positively correlated with total plant biomass and shoot phosphorus content. Although the effect of AM fungi on infection by root pathogens was phylogenetically conserved, there was no evidence for correlated evolution between the extent of AM fungal root colonization and pathogen infection. Variability in colonization strategies evolved early in the diversification of AM fungi, and we propose that these strategies were influenced by functional interactions with host plants, resulting in an evolutionary stasis resembling trait conservatism.     

High incidence of arbuscular mycorrhizal fungi in rare and endangered wild grapevine

High levels of arbuscular mycorrhizal fungi colonisation of 90–100%, as well as high vesicular and arbuscular colonisation were found in the roots of the rare and endangered wild grapevine (Vitis vinifera subsp. sylvestris) along the Neretva River in southwestern Bosnia and Herzegovina, surpassing colonisation in region vineyads.     

Twenty years of research on community composition and species distribution of arbuscular mycorrhizal fungi in China: a review

The biodiversity and distribution of arbuscular mycorrhizal fungi (AMF) in different ecosystems and plant communities in China has received increasing interest over the past decades. This has led to a steady increase in the number of scientific papers published on this topic. Studies have surveyed AMF-colonizing rhizospheres of most families of angiosperms, bryophytes, pteridophytes, and gymnosperms. China has about 30,000 plant species (one eighth of the plant species worldwide). A total of 104 AMF species within nine genera, including 12 new species, have been reported in environments such as croplands, grasslands, forests, and numerous disturbed environments. In this paper, we review data published over the past 20 years on AMF community composition and species distribution, the mycorrhizal status of plants, AMF spore communities in different habitats, and germplasm collections in China. Possible future trends in the study of the biodiversity of AMF are also briefly discussed. In particular, the aim of our review is to make some of the recent work published in the Chinese literature accessible to a wider international audience.     

Effects of mowing on fungal endophytes and arbuscular mycorrhizal fungi in subalpine grasslands

In French subalpine grasslands, cessation of mowing promotes dominance of Festuca paniculata, which alters plant diversity and ecosystem functioning. One of the mechanisms underpinning such effects may be linked to simultaneous changes in the abundance of fungal symbionts such as endophytes and arbuscular mycorrhizal fungi. In field conditions, mowing reduced the abundance of the endophyte Neotyphodium sp. in leaves of F. paniculata by a factor of 6, and increased mycorrhizal densities by a factor of 15 in the soil. In greenhouse experiments, the mycorrhizal colonization of Trifolium pratense and Allium porrum increased 3- fold and 3.8- fold respectively in mown vs unmown grassland soil. Significantly reduced growth of the two host plants was also observed on soil from the unmown grassland. Such opposite effects of mowing on the two functional groups of fungal symbionts could suggest interactions between these two groups, which in turn could contribute to structuring plant communities in subalpine grasslands.     

A novel in vitro cultivation system to produce and isolate soluble factors released from hyphae of arbuscular mycorrhizal fungi

The Petri plate dual in vitro culture system for arbuscular mycorrhizal fungi (AMF) is ill-suited for the production and isolation of extraradically released soluble factors, thereby preventing the further characterization of such compounds. To overcome this technical bottleneck, we here describe a novel cultivation system using the standard AMF Glomus intraradices-carrot dual culture as a model. This new system is capable of producing soluble factors in copious amounts without any interference from host roots. The greatest advantages of this culture method include ease of handling and reusability of the culture vessel, thus making it a very cost effective system.     

The cultivation bias: different communities of arbuscular mycorrhizal fungi detected in roots from the field,from bait plants transplanted to the field,and from a greenhouse trap experiment

The community composition of arbuscular mycorrhizal fungi (AMF) was investigated in roots of four different plant species (Inula salicina, Medicago sativa, Origanum vulgare, and Bromus erectus) sampled in (1) a plant species-rich calcareous grassland, (2) a bait plant bioassay conducted directly in that grassland, and (3) a greenhouse trap experiment using soil and a transplanted whole plant from that grassland as inoculum. Roots were analyzed by AMF-specific nested polymerase chain reaction, restriction fragment length polymorphism screening, and sequence analyses of rDNA small subunit and internal transcribed spacer regions. The AMF sequences were analyzed phylogenetically and used to define monophyletic phylotypes. Overall, 16 phylotypes from several lineages of AMF were detected. The community composition was strongly influenced by the experimental approach, with additional influence of cultivation duration, substrate, and host plant species in some experiments. Some fungal phylotypes, e.g., GLOM-A3 (Glomus mosseae) and several members of Glomus group B, appeared predominantly in the greenhouse experiment or in bait plants. Thus, these phylotypes can be considered r strategists, rapidly colonizing uncolonized ruderal habitats in early successional stages of the fungal community. In the greenhouse experiment, for instance, G. mosseae was abundant after 3 months, but could not be detected anymore after 10 months. In contrast, other phylotypes as GLOM-A17 (G. badium) and GLOM-A16 were detected almost exclusively in roots sampled from plants naturally growing in the grassland or from bait plants exposed in the field, indicating that they preferentially occur in late successional stages of fungal communities and thus represent the K strategy. The only phylotype found with high frequency in all three experimental approaches was GLOM A-1 (G. intraradices), which is known to be a generalist. These results indicate that, in greenhouse trap experiments, it is difficult to establish a root-colonizing AMF community reflecting the diversity of these fungi in the field roots because fungal succession in such artificial systems may bias the results. However, the field bait plant approach might be a convenient way to study the influence of different environmental factors on AMF community composition directly under the field conditions. For a better understanding of the dynamics of AMF communities, it will be necessary to classify AMF phylotypes and species according to their life history strategies.     

西藏高原针茅草地土壤因子对丛枝菌根真菌物种多样性的影响

在对西藏高原北部针茅草地根围土壤中的丛枝菌根(AM)真菌种类分离鉴定基础上,研究了藏北针茅草地的土壤质地、pH、有机质和有效磷含量对AM真菌孢子密度、分离频度、相对多度、重要值、物种多样性指数和均匀度的影响.结果表明: 针茅草地根围土壤中共分离鉴定出AM真菌3属15种,其中,球囊霉属9种、无梗囊霉属6种、盾巨孢囊霉属1种.球囊霉属和无梗囊霉属为藏北针茅草地AM真菌的优势属;近明球囊霉和光壁无梗囊霉为藏北高寒草原针茅属植物根围AM真菌的优势种.不同质地土壤中AM真菌孢子密度、分离频度、相对多度和重要值均表现出球囊霉属＞无梗囊霉属＞盾巨孢囊霉属的趋势;土壤pH值对AM真菌种群组成无明显影响,球囊霉属和无梗囊霉属真菌分离频度、相对多度和重要值随土壤pH升高而增加,盾巨孢囊霉属则呈现相反趋势;不同土壤有机质含量范围内,AM真菌孢子密度等各项指标均呈球囊霉属＞无梗囊霉属＞盾巨孢囊霉属,而AM真菌属的分布没有明显规律;土壤有效磷含量对AM真菌种丰度和孢子密度影响较小.研究区域内AM真菌物种多样性指数和均匀度随着土壤有效磷含量升高而增加.     

Medicago species affect the community composition of arbuscular mycorrhizal fungi associated with roots

Sequencing of the 5' end of the large ribosomal subunit (LSU rDNA) and quantitative polymerase chain reaction (qPCR) were combined to assess the impact of four annual Medicago species (Medicago laciniata, Medicago murex, Medicago polymorpha and Medicago truncatula) on the genetic diversity of arbuscular mycorrhizal (AM) fungi, and on the relative abundance of representative AM fungal genotypes, in a silty-thin clay soil (Mas d'Imbert, France). Two hundred and forty-six Glomeromycete LSU rDNA sequences from the four plant species and the bulk soil were analysed. The high bootstrap values of the phylogenetic tree obtained allowed the delineation of 12 operational taxonomic units (OTUs), all belonging to Glomus. Specific primers targeting Glomeromycetes and major OTUs were applied to quantify their abundance by qPCR. Glomeromycetes and targeted OTUs were significantly more abundant in the root tissues than in the bulk soil, and the frequencies of three of them differed significantly in the root tissues of the different plant species. These differences indicate that, despite the absence of strict host specificity in mycorrhizal symbiosis, there was a preferential association between some AM fungal and plant genotypes.