Effect of mutations in the pea genes <Emphasis Type="Italic">Sym33</Emphasis> and <Emphasis Type="Italic">Sym40</Emphasis>

Two symbiotic pea (Pisum sativum L.) mutants SGEFix(-)-1 (sym40) and SGEFix(-)-2 (sym33) with abnormalities in infection thread formation in symbiotic root nodules were characterised with respect to dynamics of arbuscule development at 15 degrees C and 24 degrees C. Mutation of sym33 decreased mycorrhiza colonisation at both temperatures and delayed arbuscule development at 15 degrees C, whereas mutation of sym40 accelerated mycorrhiza colonisation and arbuscule senescence at 24 degrees C. The differences between the mutants and the wild-type were more pronounced at 24 degrees C, a temperature close to the optimum for pea growth. The results demonstrate that both pea genes are important in the control of arbuscular mycorrhiza development and can be considered necessary for the tripartite symbiosis in pea.     

UV-B radiation affects flavonoids and fungal colonisation in <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> and <Emphasis Type="Italic">F. tataricum</Emphasis>

In the present study, we have evaluated the effects of increased UV-B radiation that simulates 17% ozone depletion, on fungal colonisation and concentrations of rutin, catechin and quercetin in common buckwheat (Fagopyrum esculentum) and tartary buckwheat (Fagopyrum tataricum). Induced root growth and reduced shoot:root ratios were seen in both of these buckwheat species after enhanced UV-B radiation. There was specific induction of shoot quercetin concentrations in UV-B-treated common buckwheat, whereas there were no specific responses for flavonoid metabolism in tartary buckwheat. Root colonisation with arbuscular mycorrhizal fungi significantly reduced catechin concentrations in common buckwheat roots, and induced rutin concentrations in tartary buckwheat, but did not affect shoot concentrations of the measured phenolics. Specific UV-B-related reductions in the density of microsclerotia were observed in tartary buckwheat, indicating a mechanism that potentially affects fungus-plant interactions. The data support the hypothesis that responses to enhanced UV-B radiation can be influenced by the plant pre-adaptation properties and related changes in flavonoid metabolism.     

<Emphasis Type="Italic">Rhizophagus irregularis</Emphasis> as an elicitor of rosmarinic acid and antioxidant production by transformed roots of <Emphasis Type="Italic">Ocimum basilicum</Emphasis> in an in vitro co-culture system

Arbuscular mycorrhiza is a symbiotic association formed between plant roots and soil borne fungi that alter and at times improve the production of secondary metabolites. Detailed information is available on mycorrhizal development and its influence on plants grown under various edapho-climatic conditions, however, very little is known about their influence on transformed roots that are rich reserves of secondary metabolites. This raises the question of how mycorrhizal colonization progresses in transformed roots grown in vitro and whether the mycorrhizal fungus presence influences the production of secondary metabolites. To fully understand mycorrhizal ontogenesis and its effect on root morphology, root biomass, total phenolics, rosmarinic acid, caffeic acid and antioxidant production under in vitro conditions, a co-culture was developed between three Agrobacterium rhizogenes-derived, elite-transformed root lines of Ocimum basilicum and Rhizophagus irregularis. We found that mycorrhizal ontogenesis in transformed roots was similar to mycorrhizal roots obtained from an in planta system. Mycorrhizal establishment was also found to be transformed root line-specific. Colonization of transformed roots increased the concentration of rosmarinic acid, caffeic acid and antioxidant production while no effect was observed on root morphological traits and biomass. Enhancement of total phenolics and rosmarinic acid in the three mycorrhizal transformed root lines was found to be transformed root line-specific and age dependent. We reveal the potential of R. irregularis as a biotic elicitor in vitro and propose its incorporation into commercial in vitro secondary metabolite production via transformed roots.     

Responses of mycorrhizal and non-mycorrhizal<Emphasis Type="Italic"> Erica cinerea</Emphasis> and<Emphasis Type="Italic"> Vaccinium macrocarpon</Emphasis> to<Emphasis Type="Italic"> Glomus mosseae</Emphasis>

An investigation was carried out on the mycorrhizal colonisation, growth and nutrition of two members of the Ericaceae in close proximity to an arbuscular mycorrhizal (AM) association. This was undertaken by separating mycorrhizal (EM) and non-mycorrhizal (NEM) Erica cinerea and Vaccinium macrocarpon from AM (inoculated by Glomus mosseae) and non-mycorrhizal (NAM) Plantago lanceolata using a 30 µm nylon mesh in a sand culture/pot system. Ericoid mycorrhizal colonisation by Hymenoscyphus ericae on root systems of E. cinerea and V. macrocarpon was in the range 14–22% and 58–69%, respectively. The presence of AM P. lanceolata had no effect on the ericoid mycorrhizal colonisation of E. cinerea and V. macrocarpon. NEM E. cinerea showed reductions in shoot biomass and shoot nitrogen concentrations after exposure to AM P. lanceolata after incubations of 6 and 9 weeks but there were no differences in dry mass, length, and nitrogen and phosphorus concentrations of the root systems between the treatment combinations. Reductions were also found, after incubations of 6 and 9 weeks, in shoot dry mass, leaf area and shoot nitrogen concentrations of NEM V. macrocarpon in the presence of AM P. lanceolata but no changes occurred in the length and dry mass of the root systems. There were no differences in maximum photosynthesis in V. macrocarpon between treatment combinations but NEM V. macrocarpon in the presence of AM P. lanceolata had the lowest transpiration rates and stomatal conductance and the highest nitrogen- and phosphorus-use efficiencies compared with the other treatment combinations. These results are discussed in relation to the type of interaction found in these compatible and incompatible mycorrhizal associations.     

The crucial role of roots in increased cadmium-tolerance and Cd-accumulation in the pea mutant <Emphasis Type="Italic">SGECd</Emphasis><Superscript><Emphasis Type="Italic">t</Emphasis></Superscript>

Elucidation of mechanisms underlying plant tolerance to cadmium, a widespread toxic soil pollutant, and accumulation of Cd in plants are urgent tasks. For this purposes, the pea (Pisum sativum L.) mutant SGECd^t (obtained by treatment of the laboratory pea line SGE with ethylmethane sulfonate) was reciprocally grafted with the parental line SGE, and four scion/rootstock combinations were obtained: SGE/SGE, SGECd^t/SGECd^t, SGE/SGECd^t, and SGECd^t/SGE. They were grown in hydroponics in the presence of 1 μM CdCl₂ for 30 d. The SGE and SGECd^t scions on the SGECd^t rootstock had a higher root and shoot biomass and an elevated root and shoot Cd content compared with the grafts having SGE rootstock. Only the grafts with the SGE rootstock showed chlorosis and roots demonstrating symptoms of Cd toxicity. The content of nutrient elements in roots (Fe, K, Mg, Mn, Na, P, and Zn) was higher in the grafts having the SGECd^t rootstock, and three elements, namely Ca, Fe, and Mn, were efficiently transported by the SGECd^t root to the shoot of these grafts. The content of other measured elements (K, Mg, Na, P, and Zn) was similar in the root and shoot in all the grafts. Then, the non-grafted plants were grown in the presence of Cd and subjected to deficit or excess concentrations of Ca, Fe, or Mn. Exclusion of these elements from the nutrient solution retained or increased differences between SGE and SGECd^t in growth response to Cd toxicity, whereas excess of Ca, Fe, or Mn decreased or eliminated such differences. The obtained results assign a principal role of roots to realizing the increased Cd-tolerance and Cdaccumulation in the SGECd^t mutant. Efficient translocation of Ca, Fe, and Mn from roots to shoots appeared to counteract Cd toxicity, although Cd was actively taken up by roots and accumulated in shoots.     

Potential role of D-<Emphasis Type="Italic">myo</Emphasis>-inositol-3-phosphate synthase and 14-3-3 genes in the crosstalk between <Emphasis Type="Italic">Zea mays</Emphasis> and <Emphasis Type="Italic">Rhizophagus intraradices</Emphasis> under drought stress

Arbuscular mycorrhizal (AM) symbiosis is known to stimulate plant drought tolerance. However, the mechanisms underlying the synergistic responses of the symbiotic partners to drought stress are largely unknown. A split-root experiment was designed to investigate the molecular interactions between a host plant and an AM fungus (AMF) under drought stress. In the two-compartment cultivation system, an entire or only a half root system of a maize plant was inoculated with an AMF, Rhizophagus intraradices, in the presence of localized or systemic drought treatment. Plant physiological parameters including growth, water status, and phosphorus concentration, and the expression of drought tolerance-related genes in both roots and R. intraradices were recorded. Although mycorrhizal inoculation in either one or both compartments systemically decreased abscisic acid (ABA) content in the whole root system subjected to systemic or local drought stress, we observed local and/or systemic AM effects on root physiological traits and the expression of functional genes in both roots and R. intraradices. Interestingly, the simultaneous increase in the expression of plant genes encoding D-myo-inositol-3-phosphate synthase (IPS) and 14-3-3-like protein GF14 (14-3GF), which were responsible for ABA signal transduction, was found to be involved in the activation of 14-3-3 protein and aquaporins (GintAQPF1 and GintAQPF2) in R. intraradices. These findings suggest that coexpression of IPS and 14-3GF is responsible for the crosstalk between maize and R. intraradices under drought stress, and potentially induces the synergistic actions of the symbiotic partners in enhancing plant drought tolerance.     

<Emphasis Type="Italic">Gr</Emphasis> and <Emphasis Type="Italic">hp</Emphasis>-<Emphasis Type="Italic">1</Emphasis> tomato mutants unveil unprecedented interactions between arbuscular mycorrhizal symbiosis and fruit ripening

Main conclusion

Systemic responses to an arbuscular mycorrhizal fungus reveal opposite phenological patterns in two tomato ripening mutants depending whether ethylene or light reception is involved. The availability of tomato ripening mutants has revealed many aspects of the genetics behind fleshy fruit ripening, plant hormones and light signal reception. Since previous analyses revealed that arbuscular mycorrhizal symbiosis influences tomato berry ripening, we wanted to test the hypothesis that an interplay might occur between root symbiosis and fruit ripening. With this aim, we screened seven tomato mutants affected in the ripening process for their responsiveness to the arbuscular mycorrhizal fungus Funneliformis mosseae. Following their phenological responses we selected two mutants for a deeper analysis: Green ripe (Gr), deficient in fruit ethylene perception and high-pigment-1 (hp-1), displaying enhanced light signal perception throughout the plant. We investigated the putative interactions between ripening processes, mycorrhizal establishment and systemic effects using biochemical and gene expression tools. Our experiments showed that both mutants, notwithstanding a normal mycorrhizal phenotype at root level, exhibit altered arbuscule functionality. Furthermore, in contrast to wild type, mycorrhization did not lead to a higher phosphate concentration in berries of both mutants. These results suggest that the mutations considered interfere with arbuscular mycorrhiza inducing systemic changes in plant phenology and fruits metabolism. We hypothesize a cross talk mechanism between AM and ripening processes that involves genes related to ethylene and light signaling.

     

Impact of <Emphasis Type="Italic">Heterobasidion</Emphasis> root-rot on fine root morphology and associated fungi in <Emphasis Type="Italic">Picea abies</Emphasis> stands on peat soils

We examined differences in fine root morphology, mycorrhizal colonisation and root-inhabiting fungal communities between Picea abies individuals infected by Heterobasidion root-rot compared with healthy individuals in four stands on peat soils in Latvia. We hypothesised that decreased tree vitality and alteration in supply of photosynthates belowground due to root-rot infection might lead to changes in fungal communities of tree roots. Plots were established in places where trees were infected and in places where they were healthy. Within each stand, five replicate soil cores with roots were taken to 20 cm depth in each root-rot infected and uninfected plot. Root morphological parameters, mycorrhizal colonisation and associated fungal communities, and soil chemical properties were analysed. In three stands root morphological parameters and in all stands root mycorrhizal colonisation were similar between root-rot infected and uninfected plots. In one stand, there were significant differences in root morphological parameters between root-rot infected versus uninfected plots, but these were likely due to significant differences in soil chemical properties between the plots. Sequencing of the internal transcribed spacer of fungal nuclear rDNA from ectomycorrhizal (ECM) root morphotypes of P. abies revealed the presence of 42 fungal species, among which ECM basidiomycetes Tylospora asterophora (24.6 % of fine roots examined), Amphinema byssoides (14.5 %) and Russula sapinea (9.7 %) were most common. Within each stand, the richness of fungal species and the composition of fungal communities in root-rot infected versus uninfected plots were similar. In conclusion, Heterobasidion root-rot had little or no effect on fine root morphology, mycorrhizal colonisation and composition of fungal communities in fine roots of P. abies growing on peat soils.     

Defoliation increases carbon limitation in ectomycorrhizal symbiosis of <Emphasis Type="Italic">Betula pubescens</Emphasis>

Boreal forest trees are highly dependent on root-colonizing mycorrhizal fungi. Since the maintenance of mycorrhizal symbiosis implies a significant carbon cost for the host plant, the loss of photosynthetic leaf area due to herbivory is expected to reduce the host investment in mycorrhizae. We tested this hypothesis in a common garden experiment by exposing ectomycorrhizal white birch (Betula pubescens Ehrh.) seedlings to simulated insect defoliation of 50 or 100% intensity during either the previous or the current summer or repeatedly during both seasons before harvest. The shoot and root growth of the seedlings were distinctly reduced by both 100% defoliation and repeated 50% defoliation, and they were more strongly affected by previous-year than current-year defoliation. The root to shoot ratio significantly decreased after 100% defoliation, indicating reduced proportional allocation to the roots. Ergosterol concentration (i.e. fungal biomass) in the fine roots decreased by 100% defoliation conducted either in the year of harvest or in both years. No such decrease occurred following the 100% defoliation conducted in the previous year, indicating the importance of current photosynthates for fungal symbionts. The trend was similar in the colonization percentage of thick-mantled mycorrhizae in the roots, the most marked decline occurring in the repeatedly defoliated seedlings. The present results thus support the prediction that the plant investment in ectomycorrhizae may decline as a response to foliage loss. Moreover, the colonization percentage of thick-mantled mycorrhizae correlated positively with the ratio of leaf to heterotrophic plant biomass in the defoliated birch seedlings, but not in the control ones. This tends to indicate a stronger carbon limitation of ectomycorrhizal colonization in defoliated seedlings.     

Mycorrhiza does not alter low temperature impact on <Emphasis Type="Italic">Gnaphalium norvegicum</Emphasis>

Extreme arctic-alpine vegetation has relatively low affinity to form mycorrhizal symbiosis. We asked whether the mycorrhizal growth benefit for the host plant is lower at low temperatures. We investigated the role of two root-associated fungi and temperature in growth, carbon–nitrogen relations and germination of an arctic-alpine herb. Seeds of Gnaphalium norvegicum were germinated at 8° or 15°C with or without arbuscular mycorrhizal (AM, Glomus claroideum) and dark septate endophytic (DSE, Phialocephala fortinii) inocula in a climate chamber. We found that germination percentage, shoot and root biomass, shoot N% and root AM colonization were lower at 8°C than at 15°C. P. fortinii inoculation had a positive impact on germination at both temperatures, whereas G. claroideum produced no effect. N% was lower in AM plants at both temperatures. Plant biomass and shoot N content were higher in AM plants than in control plants at 15°C, but not at 8°C. DSE inoculation tended also to have positive effects on plant biomass and N content at 15°C. At 15°C, rate of photosynthesis, photosynthetic nutrient use efficiency and specific leaf area were positively affected by G. claroideum, which suggests that G. claroideum formed a carbon sink and possibly enhanced the seedling water economy. The positive effects of P. fortinii were probably due to its saprotrophic function in the substrate because it did not colonize the roots. These results suggest that the effects of AM and DSE on plant growth are affected by temperature and that the mycorrhizal benefit for the host plant was lower at the lower temperature. Low saprotrophic activity and decreased mycorrhiza-mediated nutrient acquisition may thus constrain plant nutrient acquisition in cold environments. Decreased mycorrhizal benefit may be related to the comparatively low mycotrophy of cold environment vegetation.     

<Emphasis Type="Italic">Bacillus methylotrophicus</Emphasis> M4-96 isolated from maize (<Emphasis Type="Italic">Zea mays</Emphasis>) rhizoplane increases growth and auxin content in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> via emission of volatiles

Plant growth-promoting rhizobacteria stimulate plant growth and development via different mechanisms. In this study, we characterized the effect of volatiles from Bacillus methylotrophicus M4-96 isolated from the maize rhizosphere on root and shoot development, and auxin homeostasis in Arabidopsis thaliana. Phytostimulation occurred after 4 days of interaction between M4-96 and Arabidopsis grown on opposite sides of divided Petri plates, as revealed by enhanced primary root growth, root branching, leaf formation, and shoot biomass accumulation. Analysis of indole-3-acetic acid content revealed two- and threefold higher accumulation in the shoot and root of bacterized seedlings, respectively, compared to uninoculated plants, which was correlated with increased expression of the auxin response marker DR5::GUS. The auxin transport inhibitor 1-naphthylphthalamic acid inhibited primary root growth and lateral root formation in axenically grown seedlings and antagonized the plant growth-promoting effects of M4-96. Analysis of bacterial volatile compounds revealed the presence of four classes of compounds, including ten ketones, eight alcohols, one aldehyde, and two hydrocarbons. However, the abundance of ketones and alcohols represented 88.73 and 8.05%, respectively, of all airborne signals detected, with acetoin being the main compound produced. Application of acetoin had a different effect from application of volatiles, suggesting that either the entire pool or acetoin acting in concert with another unidentified compound underlies the strong phytostimulatory response. Taken together, our results show that B. methylotrophicus M4-96 generates bioactive volatiles that increase the active auxin pool of plants, stimulate the growth and formation of new organs, and reprogram root morphogenesis.     

Interactions between indigenous arbuscular mycorrhizal fungi and <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis> in field-grown pea

This is the first reported study of the interactions between indigenous arbuscular mycorrhizal fungi (AMF) and Aphanomyces euteiches in pea under field conditions. A. euteiches was applied to the soil by adding oospores produced in vitro. Attempts were made to create a non-mycorrhizal control by incorporating carbendazim (Derosal Fl) in the topsoil before sowing. However, all carbendazim-treated plants showed approximately 20% root colonisation with AMF. Pea plants not treated with carbendazim showed a wide variation in AMF colonisation of 35-70% at the full flowering stage. In these control plots, root length infected with oospores of A. euteiches and colonisation by AMF were negatively correlated. Application of carbendazim increased the percent root length infected with oospores by 50-70%, depending on inoculum density of A. euteiches. Despite the lower levels of AMF colonisation in these treated plots, a negative correlation with oospore-containing root length was still observed. No correlation was found between AMF colonisation and disease severity, disease incidence or pathogen enzymatic activity (glucose-6-phosphate dehydrogenase). Thus, AMF do not seem to influence the vegetative stage of pathogen development during which cortical root rotting takes place, but rather the reproductive stage when oospores are produced. The results of this study underline the importance of field experiments for validating the significance of mycorrhizal fungi for plant health.     

Phosphorus efficiencies and responses of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) to arbuscular mycorrhizal fungi grown in highly calcareous soil

Two experiments were carried out to investigate phosphorus efficiencies and mycorrhizal responsiveness in an improved cultivar (Clipper) and a landrace (Sahara) of barley (Hordeum vulgare L.). In experiment 1, two pot sizes were used to evaluate the effect of soil volume on P uptake and mycorrhizal responsiveness. In experiment 2, a compartmented ("cross-pot") system was used to monitor (32)P delivery by external hyphae of arbuscular mycorrhizal fungi (AMF) to the host plant. Results showed that, irrespective of growth conditions, Sahara had much larger root biomass than Clipper and consequently substantially more P was allocated to roots in Sahara than in Clipper. Specific root length in Clipper was much longer than in Sahara. Increase in soil volume enhanced percentage root length colonised by AMF, plant growth and P uptake, and Sahara was more sensitive to changes in soil volume than Clipper. Pot size (soil volume) used to assess responsiveness to AMF by different plant species or genotypes with different root/shoot ratios might be a confounding factor. Clipper was more responsive to AMF than Sahara in terms of tissue P concentrations, which is partly related to their differences in root/shoot ratios. However, increases in SPU [specific P uptake, mg P (g root biomass)(-1)] caused by AMF were bigger in Clipper, suggesting that AMF played a larger role in P uptake. In accordance with the larger increase in SPU, Clipper took up more (32)P via AMF hyphae than Sahara. The compartmented system using radioactive P might be an alternative approach to directly investigate mycorrhizal responsiveness of different plant species or varieties than conventional pot experiments, provided that the same AM fungus is used.     

Chemical defense,mycorrhizal colonization and growth responses in <Emphasis Type="Italic">Plantago lanceolata</Emphasis> L.

Allelochemicals defend plants against herbivore and pathogen attack aboveground and belowground. Whether such plant defenses incur ecological costs by reducing benefits from plant mutualistic symbionts is largely unknown. We explored a potential trade-off between inherent plant chemical defense and belowground mutualism with arbuscular mycorrhizal fungi (AMF) in Plantago lanceolata L., using plant genotypes from lines selected for low and high constitutive levels of the iridoid glycosides (IG) aucubin and catalpol. As selection was based on IG concentrations in leaves, we first examined whether IG concentrations covaried in roots. Root and leaf IG concentrations were strongly positively correlated among genotypes, indicating genetic interdependence of leaf and root defense. We then found that root AMF arbuscule colonization was negatively correlated with root aucubin concentration. This negative correlation was observed both in plants grown with monocultures of Glomus intraradices and in plants colonized from whole-field soil inoculum. Overall, AMF did not affect total biomass of plants; an enhancement of initial shoot biomass was offset by a lower root biomass and reduced regrowth after defoliation. Although the precise effects of AMF on plant biomass varied among genotypes, plants with high IG levels and low AMF arbuscule colonization in roots did not produce less biomass than plants with low IG and high AMF arbuscule colonization. Therefore, although an apparent trade-off was observed between high root chemical defense and AMF arbuscule colonization, this did not negatively affect the growth responses of the plants to AMF. Interestingly, AMF induced an increase in root aucubin concentration in the high root IG genotype of P. lanceolata. We conclude that AMF does not necessarily stimulate plant growth, that direct plant defense by secondary metabolites does not necessarily reduce potential benefits from AMF, and that AMF can enhance concentrations of root chemical defenses, but that these responses are plant genotype-dependent.     

Effect of sucrose application,minerals, and irradiance on the <Emphasis Type="Italic">in vitro</Emphasis> growth of <Emphasis Type="Italic">Cistus incanus</Emphasis> seedlings and plantlets

To study the effect of sucrose on the sink-source relationship in in vitro-grown plants, Cistus incanus seedlings and plantlets were grown horizontally in a two-compartment Petri dish (split dish), with the root system in one compartment and the shoot in the other. Shoots and roots were exposed to different sucrose concentrations (0–30 g dm⁻³), two irradiance levels (25 and 160 μmol m⁻²s⁻¹) and the presence or absence of a minimum medium containing minerals and vitamins (M medium). Root and shoot biomass of the seedlings was enhanced by an increase in irradiance when the growth medium was not supplemented with sucrose indicating the role of photosynthesis in biomass production. When sucrose was added to either organ growth was enhanced as well. In the presence of sucrose in the root compartment, sucrose applied to the shoot compartment enhanced growth of both organs under low irradiance, while under high irradiance, sucrose had no further additive effect. In the absence of sucrose in the root compartment, the enhancement of root biomass by sucrose added to the shoot compartment was lower under high irradiance than under low irradiance. The response of Cistus plantlets to sucrose and irradiance differed from that of seedlings, probably reflecting a greater susceptibility of the plantlets to sucrose feedback inhibition on photosynthesis and biomass accumulation. The decrease in root and shoot growth when M medium was added to the shoot compartment and the relatively better growth of these organs when the roots were supplied with minerals and the shoot with sucrose, indicate that growth of the two organs in our experimental set-up was regulated by opposing fluxes of C and nutrients.