Effect of diazotrophic bacteria isolated from a mycelium of arbuscular mycorrhizal fungi on colonization of maize roots by Glomus fistulosum

The inoculation of mycorrhizal maize plants with three isolates of microaerophilic diazotrophic bacteria obtained from the mycelium of arbuscular mycorrhizal fungi associated with three grasses (Arrhenatherum elatius - bacterial isolate ARR, Agropyrum repens - isolate AGR and Poa annua - isolate POA) caused no increase in nitrogen content in plant biomass. The inoculation with bacterial isolate ARR resulted in the decreased plant growth. Bacterial isolate AGR decreased the percentage of the root length colonized by arbuscular mycorrhizal fungus Glomus fistulosum. The inoculation with both mycorrhizal fungus and isolate POA increased significantly the concentration of phosphorus in plant shoots compared to uninoculated control. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of spermine on proliferation of hyphae ofGlomus fistulosum, an arbuscular mycorrhizal fungus, in maize roots

Effects of two oligoamines, putrescine and spermine, on proliferation of intraradical hyphae in surface disinfected root segments were studied under axenic conditions in vitro. No significant effects of putrescine were observed. Spermine significantly stimulated hyphal growth at a concentration of about 1.5 mumol/L. High concentration (> 150 mumol/L) caused a strong inhibition of hyphal growth and of the percentage of root segments bearing proliferating hyphae. DL-alpha-difluoromethylornithine, a metabolic inhibitor of polyamine synthesis, caused a significant inhibition of proliferation of the hyphae only in the presence of 2 mumol/L spermine.     

Effect of phenanthrene and Rhodotorula glutinis on arbuscular mycorrhizal fungus colonization of maize roots

The effect of the polycyclic aromatic hydrocarbon (PAH) phenanthrene and the yeast Rhodotorula glutinis on the arbuscular mycorrhizal fungus (AMF) Glomus geosporum colonizing maize roots, was studied. During a 90-day experiment, the highest G. geosporum colonization values were found in control plants. Mycorrhiza root length, measured both on the basis of percentage of root colonization and on the activity of succinate dehydrogenase, showed similar patterns in different phenanthrene treatments. The presence of phenanthrene in the substrate reduced G. geosporum intraradical colonization. The presence of R. glutinis did not enhance AMF colonization in the presence of phenanthrene. The biomass of the external mycelium estimated on the basis of the fatty acid 16:1 omega 5 concentration showed a progressive increase through time, and the amounts of this fatty acid differed among treated and untreated substrates. However, this increase was found to be lowest in the phenanthrene and Rhodotorula treatment at 60 days. There was less phenanthrene accumulation in roots of maize inoculated with AMF and the yeast than in roots inoculated only with AMF. A similar pattern was observed in the phenanthrene content of G. geosporum spores collected after 90 days.     

Identification of genes differentially expressed in extraradical mycelium and ectomycorrhizal roots during Paxillus involutus-Betula pendula ectomycorrhizal symbiosis

The development of ectomycorrhizal symbiosis leads to drastic changes in gene expression in both partners. However, little is known about the spatial regulation of symbiosis-regulated genes. Using cDNA array profiling, we compared the levels of expression of fungal genes corresponding to approximately 1,200 expressed sequenced tags in the ectomycorrhizal root tips (ECM) and the connected extraradical mycelium (EM) for the Paxillus involutus-Betula pendula ectomycorrhizal association grown on peat in a microcosm system. Sixty-five unique genes were found to be differentially expressed in these two fungal compartments. In ECM, a gene coding for a putative phosphatidylserine decarboxylase (Psd) was up-regulated by 24-fold, while genes coding for urea (Dur3) and spermine (Tpo3) transporters were up-regulated 4.1- and 6.2-fold in EM. Moreover, urea was the major nitrogen compound found in EM by gas chromatography-mass spectrometry analysis. These results suggest that (i) there is a spatial difference in the patterns of fungal gene expression between ECM and EM, (ii) urea and polyamine transporters could facilitate the translocation of nitrogen compounds within the EM network, and (iii) fungal Psd may contribute to membrane remodeling during ectomycorrhiza formation.     

Variation in N2 fixation,N and P contents of mycorrhizalVigna unguiculata in relation to the progressive development of extraradical hyphae ofGlomus mosseae

Summary Vigna unguiculata cv. 58–185 grown in a sterile Dek soil was inoculated withRhizobium sp. orRhizobium sp. plusGlomus mosseae. Response of the host plant to the treatments was estimated by periodic measurements of shoot and nodule dry weights, N₂ fixation (C₂H₂ reduction activity) and N and P contents up to the 50th day of the growth cycle. It was only 45 days after planting that shoot dry weight of dually inoculated plants differed significantly from that of plants inoculated withRhizobium sp. alone. Nodule dry weight and N₂ fixation of dually inoculated plants were significantly higher than those of plants inoculated withRhizobium sp. alone from day 20 after planting, but there was no significant difference in N content (%). During the first 20 days, shoot P content (%) of both sets of plants decreased progressively, P content of dually inoculated plants being lower than that of the others. Later, P content of dually inoculated plants increased rapidly whereas P content of the other plants remained constant. Increase in nodule dry weight, N₂ fixation and P content of dually inoculated plants corresponded to the onset of the development of the extra-radical hyphae ofGlomus mosseae. In the rhizosphere.

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Resumen Se cultivóVigna unguiculata cv. 58–185 en un suelo estéril tipo Dek, se inoculó conRhizobium sp. o conRhizobium sp. másGlomus mosseae. La respuesta de la planta huésped a los tratamientos se estudió midiendo periodicamente el peso seco de la parte aerea y de los nódulos, la fijación de N (actividad reductora de C₂H₂) y los contenidos de N y P hasta el 50° día del ciclo de crecimiento. La diferencia entre el peso seco de la parte aerea de las plantas con doble inoculación y aquellas inoculadas conRhizobium sp. unicamente, no fue significativa hasta 45 días despúés de la siembra. A los 20 días de la siembra tanto el peso seco de los nódulos como la fijación de nitrógeno de las plantas con doble inoculación eran significativamente superiores a los valores obtenidos para las plantas con soloRhizobium sp., aunque no se observaron diferencias en el contenido en N (%). Durante los primeros 20 días del ciclo el contenido en P (%) de ambos grupos de plantas disminuyó progresivamente, siendo los valores obtenidos por las plantas con doble inoculación inferiores a los de las demás. Más tarde el contenido en P de las plantas con doble inoculación aumentó rapidamente manteniéndose constante el de las demás. El incremento en el peso seco de los nódulos, en la fijación de N y en el contenido en P de las plantas con doble inoculación se correspondió con el inicio del desarrollo de las hifas extraradiculares deGlomus mosseae.

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Résumé On a inoculéV. unguiculata poussant dans un sol Dek stérile avecRhizobium etRhizobium plusGlomus mosseae. On a recherché la réponse de la plante-hôte à ces deux traitements en estimant périodiquement les poids des nodules et des parties aériennes de la plante, la fixation d'azote (activité réductrice de C₂H₂), les teneurs en N et P jusqu'au 50^e jour du cycle de végétation. C'est seulement au 45^e jour après la plantation que le poids sec des parties aériennes des plantes inoculées avec deux symbiotes (plantes doublement inoculées) diffère significativement de celui des plantes inoculées avec Rhizobium seul. Le poids sec des nodules et la fixation N₂ des plantes doublement inoculées sont significativement plus élevés que ceux des plantes inoculées avecRhizobium seul au 20^e jour après la plantation mais il n'y a pas de différence significative pour la teneur en N (%). Pendant les 20 premiers jours, la teneur en P (%) des parties aériennes des deux catégories de plantes décroit progressivement; la teneur en P des plantes doublement inoculées est plus faible que celle des plantes inoculées seulement avecRhizobium. Plus tard, la teneur en P des plantes doublement inoculées augmente rapidement tandis que celle des autres plantes reste constante. L'accroissement du poids sec des nodules, de la fixation d'azote et de la teneur en P observé chez les plantes doublement inoculées correspond au démarrage du développement des hyphes extra-radicales deGlomus mosseae dans la rhizosphère.

     

Effect ofAgrobacterium radiobacter on vesicular-arbuscular-mycorrhizal infection and external mycelium of maize

Agrobacterium radiobacter influenced the development of mycorrhizal infection, length of external mycelium and metabolic activity of the mycelium in a hydroponic culture system, with maize as a host plant. Infection caused byGlomus fasciculatum, and metabolic activity of the external mycelium ofG. fasciculatum andG. etunicatum, was stimulated by bacterial inoculation. The results underline the importance of the soil saprophytic microflora for development and activity of the extraradical phase of vesicular-arbuscular mycorrhizal fungi.     

Cadmium immobilization in the rhizosphere of arbuscular mycorrhizal plants by the fungal extraradical mycelium

The aim of the study was to assess how the extraradical mycelium (ERM) of arbuscular mycorrhizal (AM) fungi contributes to Cd immobilization in the rhizosphere. Substrates prepared by cultivation of AM and non-mycorrhizal tobacco (Nicotiana tabacum L.) in quartz sand in two experiments were amended with Cd in a range of concentrations and Cd immobilization was assessed as Cd toxicity using root growth tests. Split-root plants, inoculated at one part of the root system, and hyphal compartments colonized by ERM only were used to separate the effects of ERM from plant-mediated effects of mycorrhiza and from the effects of roots. AM decreased Cd toxicity in the substrates obtained by 12 weeks of cultivation (Experiment 1), while the effect was less clear after 8 weeks (Experiment 2). No indication was found for an involvement of plant-mediated effects; in contrast, the effect of ERM could be clearly demonstrated. Lower Cd toxicity in the substrates colonized by ERM was related to ERM-induced alkalinization, but not directly to ERM density. It is concluded that the ERM of AM fungi may enhance Cd immobilisation in soil not only due to its high Cd sorption capacity but also by its activity.     

Forage radish and cereal rye cover crop effects on mycorrhizal fungus colonization of maize roots

Forage radish (Raphanus sativus L. var. longipinnatus) is being used by increasing numbers of farmers as a winter cover crop in the Mid-Atlantic USA. It is a non-host to arbuscular mycorrhizal fungi (AMF) and releases anti-fungal isothiocyanates (ITCs) upon decomposition in the winter. Field experiments were conducted to determine the effect of forage radish and cereal rye (Secale cereale L.) cover crops on arbuscular mycorrhizal fungus colonization of and P acquisition by a subsequent maize (Zea mays L.) silage crop. Cover crop treatments included forage radish, rye, a mix of forage radish and rye, and no cover crop. Mycorrhizal fungus colonization of maize roots at the V4 stage following forage radish cover crops was not significantly different from that in the no cover crop treatment. In 3 out of 6 site-years, a rye cover crop increased AMF colonization of V4 stage maize roots compared to no cover crop. These findings suggest that forage radish cover crops do not have a negative effect on AMF colonization of subsequent crops.     

Molecular identification of the edible ectomycorrhizal fungus Lactarius deliciosus in the symbiotic and extraradical mycelium stages

Specific rDNA ITS amplifications, microsatellite-primed PCR and ITS-SSCP analysis were applied to identify and characterize pre-selected isolates of the edible ectomycorrhizal fungus Lactarius deliciosus in different stages of the life cycle. Sampling was performed from pure cultures, mycorrhizas and soil from experimental plots established with nursery-inoculated pine seedlings. A newly-designed reverse primer (LDITS2R) combined with the universal forward ITS1 allowed to perform specific amplifications of L. deliciosus from all the samples. Microsatellite-primed PCR using the (GTG)5 oligonucleotide as a primer showed clear polymorphisms among the different L. deliciosus isolates. The patterns of mycorrhiza samples showed additional bands corresponding to the plant DNA. Single strand conformation polymorphism (SSCP) analysis of the specific rDNA ITS fragment amplified from 18 L. deliciosus isolates showed nine clearly different patterns. Mycorrhiza and soil samples showed coincident patterns with their respective fungal isolates. Specific rDNA ITS amplifications had not been previously used for SSCP analysis of ectomycorrhizas and extraradical mycelium. This relatively simple and inexpensive technique allows tracking L. deliciosus isolates in different stages of the fungus development. Specific ITS-SSCP analysis is promising in studies of the persistence of inoculated L. deliciosus isolates and their competitiveness with native ectomycorrhizal fungi, especially at the extraradical mycelium stage.     

Extraction of extraradical arbuscular mycorrhizal mycelium from compartments filled with soil and glass beads

This study presents a novel method for the extraction and quantification of extraradical mycelium (ERM) of arbuscular mycorrhizal fungi (AMF) from a substrate that simulates soil better than previously used artificial growth media. Fungal compartments were constructed from small net pots with a latticed wall and filled with a mixture of glass beads and 40 m wet sieved soil. The net pots were surrounded by a 30-m mesh membrane through which hyphae but not roots could grow. They were inserted into soil where a Glomus intraradices (BEG 110) colonized potato plant was growing. The ERM that had grown out from roots through the membrane was successfully collected and quantified after harvest by washing out the soil/glass bead mixture through a sieve with a mesh width of 40 m. Concentrations of P, Zn, Cu and Mn in the AMF ERM were analysed.     

Rhizosphere disturbance influences fungal colonization and community development on dead fine roots

Little is known about the community dynamics of fungi on decomposing fine roots, despite the importance of fine roots as a source of carbon to detrital systems in forests. We examined fungal communities on dead roots in a sugar-maple dominated northern hardwood forest to test the hypothesis that community development is sensitive to rhizosphere disruption. We generated cohorts of dead fine roots in root windows and disturbed the rhizosphere microbial community in half of the windows by moving roots into sieved bulk soil. We sampled root fragments repeatedly over time and cultured fungi from these fragments to explore temporal patterns of fungal species composition. Disturbing the root rhizosphere prior to initiating decomposition changed the dominant fungal taxa, the distribution of dominant species within the community, and the temporal development in the culturable fungal community. Dominance in control roots shifted from Neonectria in early decay to Umbelopsis in later decay. Disturbance roots were more evenly dominated over time by Trichoderma, Neonectria, another species of Umbelopsis, and Pochonia. Our results suggest that species interactions are important in the ecology of fine root decay fungi, with the rhizosphere community of the living root influencing development of the decay community.     

Measurement of the extraradical mycelium of a vesicular-arbuscular mycorrhizal fungus in soil by chitin determination

Summary Development of a vesicular-arbuscular mycorrhizal (VAM) fungus in association with soybean was determined in a greenhouse soil mix by chitin assay. Samples were sieved to eliminate hexosamine-containing contaminants. This preparation reduced the interference caused by extraneous soil substances and permitted quantitative measurement of extraradical VAM fungal mycelium in the soil mix by colorimetric assay. Recovery of added chitin, used as an internal standard, was greater in the soil mix than in an inert medium indicating that some hexosamine was stabalized from chemical degradation by other soil components.     

Plugging into the network: belowground connections between germlings and extraradical mycelium of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs; nevertheless their spores can germinate in the absence of host plants. Such inconsistent behavior is balanced by diverse survival strategies. The ability of AM fungal hyphae to fuse might represent a fundamental survival strategy because germlings could plug into compatible mycorrhizal networks, thus gaining access to plant-derived carbon before asymbiotic growth arrest. An in vivo experimental system was used to grow extraradical mycelium produced by Glomus mosseae colonizing three different plant species and germlings of the same isolate. After symbiotic and asymbiotic mycelia came into contact we showed that germling hyphae fused with symbiotic network hyphae and established protoplasm connections with nuclei occurring in fusion bridges. The frequency of anastomoses between germling and symbiotic hyphae was 4.9-23.9%. Prefusion and postfusion incompatible responses, with protoplasm withdrawal in interacting hyphae, were evident in some hyphal contacts. Given the multigenomic nature of AMF, the mingling of germling nuclei with those of the mycorrhizal network through perfect fusions might represent a means for the maintenance of genetic diversity in the absence of sexual recombination.     

Effect of propagators and inhibitors on the ultraweak luminescence from maize roots

This study has investigated the kinetics and mechanism of ultraweak luminescence in maize roots. Mannitol induced the second maximum and enhanced the main maximum of the relative intensity of luminescence from the roots. Hydroquinone and quinone enhanced the relative intensity of the luminescence. Catalase enhanced the maximum of the luminescence and changed the kinetics of the light emission. The effect of catalase on the kinetics was abolished by superoxide dismutase. Ascorbate in the presence of catalase reduced the luminescence maximum, but did not alter the kinetics. In the presence of catalase only, or in the combination with superoxide dismutase, or ascorbate, the luminescence intensity in the stationary phase was significantly lower compared to the control. The results support the participation of superoxide-radical, singlet oxygen, electron transfer and the role of peroxidase in the reactions generating ultraweak luminescence in the roots. Ascorbate, catalase and superoxide dismutase have a protective role in the luminescent reactions.     

Differential morphogenesis of the extraradical mycelium of an arbuscular mycorrhizal fungus grown monoxenically on spatially heterogeneous culture media

A new in vitro experimental system was developed to study the morphogenesis of discrete regions of a single extraradical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, growing simultaneously in six different agar-based media. The media were (i) unamended water agar (WA), (ii) WA+PO(4)(3-) (PO(4)(3-)), (iii) WA+NO(3)(-) (NO(3)(-)), (iv) WA+NH(4)(+) (NH(4)(+)), (v) WA+NH(4)(+)+MES (NH(4)(+)+MES) and (vi) minimal medium (M, complete nutrients). Each medium was amended with the pH indicator bromocresol purple. The extraradical mycelium of the fungus showed between-treatment differences in morphogenesis, architecture, formation of branched absorbing structures (BAS) and sporulation. Extraradical hyphae that developed in WA or PO(4)(3-) compartments exhibited an economic development pattern, in which runner hyphae radially extended the external colony. Extraradical hyphal growth in the NO(3)(-) compartments was characterized by increased formation of runner hyphae, BAS and spores and an alkalinization of the medium. In the two NH(4)(+)-amended media (NH(4)(+), NH(4)(+)+MES), sporulation was suppressed and considerable morphological changes were noted. These results show the plasticity of G. intraradices that lets it efficiently exploit an heterogeneous substrate.     

Effect of white light on the growth of aseptically cultured maize roots

The effect of white light on elongation of aseptically grown maize (Zea mays L. cv. LG 11) root segments is influenced by the modifications of the chemical nature of the culture medium occurring during autoclaving. Light was found to be either stimulatory or inhibitory to root growth when the medium was, respectively, sterilised by autoclaving or by filtration. It was then postulated that sucrose hydrolysis occurring during the autoclaving was involved. A gas chromatographic analysis of the culture media gives accurate qualitative and quantitative evidence that such a reaction does indeed occur.