Martina Janoušková

Arbuscular mycorrhiza decreases cadmium phytoextraction by transgenic tobacco with inserted metallothionein

The effect of arbuscular mycorrhiza (AM) on the phytoextraction efficiency of transgenic tobacco with increased ability to tolerate and accumulate cadmium (Cd) was tested in a pot experiment. The tobacco plants bearing the yeast metallothionein CUP1 combined with a polyhistidine cluster were compared to non-transgenic tobacco of the same variety at four Cd concentrations in soil, non-inoculated or inoculated with two isolates of the AM fungus Glomus intraradices. Mycorrhizal inoculation improved the growth of both the transgenic and non-transgenic tobacco and decreased Cd concentrations in shoots and root to shoot translocation. Differences were found between the two AM fungal isolates: one isolate supported more efficient phosphorus uptake and plant growth in the soil without Cd addition, while the other isolate alleviated the inhibitory effect of cadmium on plant growth. The resulting effect of inoculation on Cd accumulation was dependent on Cd level in soil and differed between the more Cd tolerant transgenic plants and the less tolerant non-transgenic plants. Mycorrhiza mostly decreased the phytoextraction efficiency of transgenic plants while increased that of non-transgenic plants at Cd levels in soil inhibitory to tobacco growth. Mechanisms of the observed effects of inoculation on growth and Cd uptake are discussed as well as the possible implications of the results for the exploitation of AM in phytoextraction of heavy metals from contaminated soils.

Effects of Inoculum Additions in the Presence of a Preestablished Arbuscular Mycorrhizal Fungal Community

ABSTRACT Communities of arbuscular mycorrhizal fungi (AMF) are crucial for promoting plant productivity in most terrestrial systems, including anthropogenically managed ecosystems. Application of AMF inocula has therefore become a widespread practice. It is, however, pertinent to understand the mechanisms that govern AMF community composition and their performance in order to design successful manipulations. Here we assess whether the composition and plant growth-promotional effects of a synthetic AMF community can be altered by inoculum additions of the isolates forming the community. This was determined by following the effects of three AMF isolates, each inoculated in two propagule densities into a preestablished AMF community. Fungal abundance in roots and plant growth were evaluated in three sequential harvests. We found a transient positive response in AMF abundance to the intraspecific inoculation only in the competitively weakest isolate. The other two isolates responded negatively to intra- and interspecific inoculations, and in some cases plant growth was also reduced. Our results suggest that increasing the AMF density may lead to increased competition among fungi and a trade-off with their ability to promote plant productivity. This is a key ecological aspect to consider when introducing AMF into soils.

Cultivation of high-biomass crops on coal mine spoil banks: can microbial inoculation compensate for high doses of organic matter?

Two greenhouse experiments were focused on the application of arbuscular mycorrhizal fungi (AMF) and plant growth promoting rhizobacteria (PGPR) in planting of high-biomass crops on reclaimed spoil banks. In the first experiment, we tested the effects of different organic amendments on growth of alfalfa and on the introduced microorganisms. While growth of plants was supported in substrate with compost amendment, mycorrhizal colonization was suppressed. Lignocellulose papermill waste had no negative effects on AMF, but did not positively affect growth of plants. The mixture of these two amendments was found to be optimal in both respects, plant growth and mycorrhizal development. Decreasing doses of this mixture amendment were used in the second experiment, where the effects of microbial inoculation (assumed to compensate for reduced doses of organic matter) on growth of two high-biomass crops, hemp and reed canarygrass, were studied. Plant growth response to microbial inoculation was either positive or negative, depending on the dose of the applied amendment and plant species.

Extraradical mycelium of arbuscular mycorrhizal fungi radiating from large plants depresses the growth of nearby seedlings in a nutrient deficient substrate

The effect of arbuscular mycorrhiza (AM) on the interaction of large plants and seedlings in an early succession situation was investigated in a greenhouse experiment using compartmented rhizoboxes. Tripleurospermum inodorum, a highly mycorrhiza-responsive early coloniser of spoil banks, was cultivated either non-mycorrhizal or inoculated with AM fungi in the central compartment of the rhizoboxes. After two months, seedlings of T. inodorum or Sisymbrium loeselii, a non-host species colonising spoil banks simultaneously with T. inodorum, were planted in lateral compartments, which were colonised by the extraradical mycelium (ERM) of the pre-cultivated T. inodorum in the inoculated treatments. The experiment comprised the comparison of two AM fungal isolates and two substrates: spoil bank soil and a mixture of this soil with sand. As expected based on the low nutrient levels in the substrates, the pre-cultivated T. inodorum plants responded positively to mycorrhiza, the response being more pronounced in phosphorus uptake than in nitrogen uptake and growth. In contrast, the growth of the seedlings, both the host and the non-host species, was inhibited in the mycorrhizal treatments. Based on the phosphorus and nitrogen concentrations in the biomass of the experimental plants, this growth inhibition was attributed to nitrogen depletion in the lateral compartments by the ERM radiating from the central compartment. The results point to an important aspect of mycorrhizal effects on the coexistence of large plants and seedlings in nutrient deficient substrates.

Plant-fungus competition for nitrogen erases mycorrhizal growth benefits of Andropogon gerardii under limited nitrogen supply.

Abstract Considered to play an important role in plant mineral nutrition, arbuscular mycorrhizal (AM) symbiosis is a common relationship between the roots of a great majority of plant species and glomeromycotan fungi. Its effects on the plant host are highly context dependent, with the greatest benefits often observed in phosphorus (P)‐limited environments. Mycorrhizal contribution to plant nitrogen (N) nutrition is probably less important under most conditions. Moreover, inasmuch as both plant and fungi require substantial quantities of N for their growth, competition for N could potentially reduce net mycorrhizal benefits to the plant under conditions of limited N supply. Further compounded by increased belowground carbon (C) drain, the mycorrhizal costs could outweigh the benefits under severe N limitation. Using a field AM fungal community or a laboratory culture of Rhizophagus irregularis as mycorrhizal inoculants, we tested the contribution of mycorrhizal symbiosis to the growth, C allocation, and mineral nutrition of Andropogon gerardii growing in a nutrient‐poor substrate under variable N and P supplies. The plants unambiguously competed with the fungi for N when its supply was low, resulting in no or negative mycorrhizal growth and N‐uptake responses under such conditions. The field AM fungal communities manifested their potential to improve plant P nutrition only upon N fertilization, whereas the R. irregularis slightly yet significantly increased P uptake of its plant host (but not the hosts growth) even without N supply. Coincident with increasing levels of root colonization by the AM fungal structures, both inoculants invariably increased nutritional and growth benefits to the host with increasing N supply. This, in turn, resulted in relieving plant P deficiency, which was persistent in non‐mycorrhizal plants across the entire range of nutrient supplies.

Response to cadmium of Daucus carota hairy roots dual cultures with Glomus intraradices or Gigaspora margarita

Ri T-DNA-transformed carrot roots were cultivated in two experiments either non-inoculated or inoculated with the arbuscular mycorrhizal (AM) fungi Glomus intraradices or Gigaspora margarita. The influence of two concentrations of cadmium (Cd) in the medium (2 mg l−1, 4 mg l−1) on both root and mycelium growth was tested. Both parameters were estimated at 10-day intervals for 70 or 100 days for G. intraradices and Gi. margarita, respectively. In the first experiment, G. intraradices showed a rapid spread of extraradical mycelium (ERM) and reached average densities per treatment of about 90 cm cm−2 agar medium after 70 days. At the higher Cd level, the growth of ERM was delayed in comparison to the treatment without Cd addition. Root growth was inhibited by both Cd levels; the inhibition was, however, significantly lower in the treatments inoculated with G. intraradices compared to the non-inoculated control. In the second experiment, the ERM of Gi. margarita started to grow after a period of 50 days and reached average densities per treatment of only up to 27 cm cm−2 by the end of the cultivation. The growth of Gi. margarita mycelium was not inhibited by Cd. No differences in root growth were observed between the Gi. margarita inoculated and non-inoculated treatments. The inhibitory effect of Cd on root growth differed between the non-inoculated treatments in both experiments. The study has shown that the AM fungus Glomus intraradices can alleviate Cd-induced growth inhibition to carrot hairy roots. The potential and limits of the monoxenic system in studying the interaction between AM fungi and heavy metals are discussed.

Intraradical Dynamics of Two Coexisting Isolates of the Arbuscular Mycorrhizal Fungus Glomus intraradices Sensu Lato as Estimated by Real-Time PCR of Mitochondrial DNA

ABSTRACT Real-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates of Glomus intraradices sensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.

Duration and intensity of shade differentially affects mycorrhizal growth- and phosphorus uptake responses of Medicago truncatula

Plant and fungal partners in arbuscular mycorrhizal symbiosis trade mineral nutrients for carbon, with the outcome of this relationship for plant growth and nutrition being highly context-dependent and changing with the availability of resources as well as with the specific requirements of the different partners. Here we studied how the model legume Medicago truncatula, inoculated or not with a mycorrhizal fungus Rhizophagus irregularis, responded to a gradient of light intensities applied over different periods of time, in terms of growth, phosphorus nutrition and the levels of root colonization by the mycorrhizal fungus. Short-term (6 d) shading, depending on its intensity, resulted in a rapid decline of phosphorus uptake to the shoots of mycorrhizal plants and simultaneous accumulation of phosphorus in the roots (most likely in the fungal tissues), as compared to the non-mycorrhizal controls. There was, however, no significant change in the levels of mycorrhizal colonization of roots due to short-term shading. Long-term (38 d) shading, depending on its intensity, provoked a multitude of plant compensatory mechanisms, which were further boosted by the mycorrhizal symbiosis. Mycorrhizal growth- and phosphorus uptake benefits, however, vanished at 10% of the full light intensity applied over a long-term. Levels of root colonization by the mycorrhizal fungus were significantly reduced by long-term shading. Our results indicate that even short periods of shade could have important consequences for the functioning of mycorrhizal symbiosis in terms of phosphorus transfer between the fungus and the plants, without any apparent changes in root colonization parameters or mycorrhizal growth response, and call for more focused research on temporal dynamics of mycorrhizal functioning under changing environmental conditions.

Monitoring CO2 emissions to gain a dynamic view of carbon allocation to arbuscular mycorrhizal fungi

Quantification of carbon (C) fluxes in mycorrhizal plants is one of the important yet little explored tasks of mycorrhizal physiology and ecology. 13CO2 pulse-chase labelling experiments are increasingly being used to track the fate of C in these plant–microbial symbioses. Nevertheless, continuous monitoring of both the below- and aboveground CO2 emissions remains a challenge, although it is necessary to establish the full C budget of mycorrhizal plants. Here, a novel CO2 collection system is presented which allows assessment of gaseous CO2 emissions (including isotopic composition of their C) from both belowground and shoot compartments. This system then is used to quantify the allocation of recently fixed C in mycorrhizal versus nonmycorrhizal Medicago truncatula plants with comparable biomass and mineral nutrition. Using this system, we confirmed substantially greater belowground C drain in mycorrhizal versus nonmycorrhizal plants, with the belowground CO2 emissions showing large variation because of fluctuating environmental conditions in the glasshouse. Based on the assembled 13C budget, the C allocation to the mycorrhizal fungus was between 2.3% (increased 13C allocation to mycorrhizal substrate) and 2.9% (reduction of 13C allocation to mycorrhizal shoots) of the plant gross photosynthetic production. Although the C allocation to shoot respiration (measured during one night only) did not differ between the mycorrhizal and nonmycorrhizal plants under our experimental conditions, it presented a substantial part (∼10%) of the plant C budget, comparable to the amount of CO2 released belowground. These results advocate quantification of both above- and belowground CO2 emissions in future studies.

Plant Communities Rather than Soil Properties Structure Arbuscular Mycorrhizal Fungal Communities along Primary Succession on a Mine Spoil

Arbuscular mycorrhizal fungal (AMF) community assembly during primary succession has so far received little attention. It remains therefore unclear, which of the factors, driving AMF community composition, are important during ecosystem development. We addressed this question on a large spoil heap, which provides a mosaic of sites in different successional stages under different managements. We selected 24 sites of c. 12, 20, 30, or 50 years in age, including sites with spontaneously developing vegetation and sites reclaimed by alder plantations. On each site, we sampled twice a year roots of the perennial rhizomatous grass Calamagrostis epigejos (Poaceae) to determine AMF root colonization and diversity (using 454-sequencing), determined the soil chemical properties and composition of plant communities. AMF taxa richness was unaffected by site age, but AMF composition variation increased along the chronosequences. AMF communities were unaffected by soil chemistry, but related to the composition of neighboring plant communities of the sampled C. epigejos plants. In contrast, the plant communities of the sites were more distinctively structured than the AMF communities along the four successional stages. We conclude that AMF and plant community successions respond to different factors. AMF communities seem to be influenced by biotic rather than by abiotic factors and to diverge with successional age.