Ulrich Hildebrandt

Selective Element Deposits in Maize Colonized by a Heavy Metal Tolerance Conferring Arbuscular Mycorrhizal Fungus

Summary The Glomus isolate Br1 from the zinc violet, Viola calaminaria (DC.) Lej., confers heavy metal tolerance to plants including maize, alfalfa, barley and others (see accompanying paper). In the present study, the bulk analysis of maize grown in two different heavy metal soils in greenhouse experiments indicated that roots and shoots contained considerably lower heavy metal concentrations when maize was colonized with the isolate Br1 compared with plants grown with a common Glomus strain or to non-colonized controls. Essential elements like K, P and Mg were enriched in roots in Br1 colonized maize. Since arbuscular mycorrhizal (AM) plants grew much faster until flower and seed formation and had an approximately 25-fold higher dry weight than the controls, a massive acquisition of essential elements has happened. Data from three different microbeam techniques indicated distinct differences in the cellular distribution of heavy metals and essential elements in AM colonized roots compared with the non-mycorrhizal controls. SIMS images showed a selective enrichment of Mg, Ca, Fe, Ni and Zn in the inner cortical cell region containing the fungal structures (arbuscules) and a lower concentration of the heavy metals Fe, Zn and Ni in the stele than in the cortex. EDXA measurements indicated a selective enrichment of Mg and K in the stele. The data from SIMS and LAMMA suggested Al to be more or less evenly distributed in the root cells. The present investigation appears to be the first comprehensive approach to map elemental distribution within root tissues in AM colonized and control maize by three different methods of microbeam analysis. Since the microbeam techniques had to be applied near the detection limit of the methods, the data obtained by the three different approaches were not always uniform. However, the combination of these three techniques showed that the growth of maize in the heavy metal soil was at least partly due to a selective immobilization of heavy metals within those root tissues containing the fungal cells. The measurements also indicated that AM fungi might cope with heavy metal toxicity for each metal individually.

The Zinc Violet and its Colonization by Arbuscular Mycorrhizal Fungi

Summary Among the plants growing on several heavy metal soils, the zinc violets (the yellow Viola calaminaria (DC.) Lej. s. str. of the Aachen/Liege area and the blue Viola guestphalica Nauenburg of Blankenrode/Paderborn) were consistently colonized by arbuscular mycorrhizal (AM) fungi. The degree of AM-colonization apparently correlated with the heavy metal content in soils as indicated by the composition of the plant community. Among diverse violets examined from various non-polluted areas, Viola lutea (DC.) Lej. and some other alpine violets showed high mycorrhizal colonizations of the roots. A specific Glomus Br1 isolate was obtained from the roots of the yellow zinc violet ( V. calaminaria s. str.) of the Breinigerberg area near Aachen. RFLP-analysis indicated the uniformity of this isolate. Incubation with Glomus Br1 allowed plants like maize, barley, alfalfa and zinc violets to grow until flower and seed formation in two different heavy metal soils supplemented with nutrient solutions in greenhouse experiments. Controls (sterilized heavy metal soils not inoculated with Glomus Br1 or yellow lupins as non-mycorrhizal plants) did not grow. The Glomus Br1 isolate from the zinc violet more efficiently supported growth of maize or alfalfa in heavy metal soils than a commonly used Glomus intraradices Schenck and Smith isolate. The potential applications of these findings are discussed.

The Developmental Pattern of Tomato Fruit Wax Accumulation and Its Impact on Cuticular Transpiration Barrier Properties: Effects of a Deficiency in a β-Ketoacyl-Coenzyme A Synthase (LeCER6)

Cuticular waxes play a pivotal role in limiting transpirational water loss across the primary plant surface. The astomatous fruits of the tomato (Lycopersicon esculentum) ‘MicroTom’ and its lecer6 mutant, defective in a β-ketoacyl-coenzyme A synthase, which is involved in very-long-chain fatty acid elongation, were analyzed with respect to cuticular wax load and composition. The developmental course of fruit ripening was followed. Both the ‘MicroTom’ wild type and lecer6 mutant showed similar patterns of quantitative wax accumulation, although exhibiting considerably different water permeances. With the exception of immature green fruits, the lecer6 mutant exhibited about 3- to 8-fold increased water loss per unit time and fruit surface area when compared to the wild type. This was not the case with immature green fruits. The differences in final cuticular barrier properties of tomato fruits in both lines were fully developed already in the mature green to early breaker stage of fruit development. When the qualitative chemical composition of fruit cuticular waxes during fruit ripening was investigated, the deficiency in a β-ketoacyl-coenzyme A synthase in the lecer6 mutant became discernible in the stage of mature green fruits mainly by a distinct decrease in the proportion of n-alkanes of chain lengths > C28 and a concomitant increase in cyclic triterpenoids. This shift in cuticular wax biosynthesis of the lecer6 mutant appears to be responsible for the simultaneously occurring increase of water permeance. Changes in cutin composition were also investigated as a function of developmental stage. This integrative functional approach demonstrates a direct relationship between cuticular transpiration barrier properties and distinct chemical modifications in cuticular wax composition during the course of tomato fruit development.

Arbuscular mycorrhizal colonization of halophytes in Central European salt marshes

Abstract Halophytes from both coastal and inland Central European salt marshes were examined for colonization by arbuscular mycorrhizal (AM) fungi. Plants from different families were strongly colonized but the degree of colonization varied with the individual plant and apparently during the vegetation period, too. Members of the typical non-mycorrhizal families like Armeria maritima of the Plumbaginaceae and Salicornia europaea of the Chenopodiaceae were found to be colonized, particularly in the drier salt marshes. High numbers of Glomus spores were found in the saline soils, especially those of the inland locations examined. Approximately 80% of these spores were from Glomus geosporum as shown by a typical restriction fragment length polymorphism (RFLP) pattern of the amplified internal transcribed spacer regions. The present study demonstrates that RFLP analysis is useful when screening habitats for the occurrence of mycorrhizal fungi which can be identified only with difficulty by morphological criteria.

Towards Growth of Arbuscular Mycorrhizal Fungi Independent of a Plant Host

ABSTRACT When surface-sterilized spores of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices Sy167 were germinated on agar plates in the slightly modified minimum mineral medium described by G. Bécard and J. A. Fortin (New Phytol. 108:211-218, 1988), slime-forming bacteria, identified as Paenibacillus validus, frequently grew up. These bacteria were able to support growth of the fungus on the agar plates. In the presence of P. validus, hyphae branched profusely and formed coiled structures. These were much more densely packed than the so-called arbuscule-like structures which are formed by AMF grown in coculture with carrot roots transformed with T-DNA from Agrobacterium rhizogenes. The presence of P. validus alone also enabled G. intraradices to form new spores, mainly at the densely packed hyphal coils. The new spores were not as abundant as and were smaller than those formed by AMF in the monoxenic culture with carrot root tissues, but they also contained lipid droplets and a large number of nuclei. In these experiments P. validus could not be replaced by bacteria such as Escherichia coli K-12 or Azospirillum brasilense Sp7. Although no conditions under which the daughter spores regerminate and colonize plants have been found yet, and no factor(s) from P. validus which stimulates fungal growth has been identified, the present findings might be a significant step forward toward growth of AMF independent of any plant host.

The arbuscular mycorrhizal fungus Glomus geosporum in European saline, sodic and gypsum soils

Abstract. Plants of saline and sodic soils of the Hungarian steppe and of gypsum rock in the German Harz mountains, thus soils of high ionic strength and electric conductivity, were examined for their colonization by arbuscular mycorrhizal fungi (AMF). Roots of several plants of the saline and sodic soils such as Artemisiamaritima, Astertripolium or Plantagomaritima are strongly colonized and show typical AMF structures (arbuscules, vesicles) whereas others like the members of the Chenopodiaceae, Salicorniaeuropaea, Suaedamaritima or Camphorosmaannua, are not. The vegetation of the gypsum rock is totally different, but several plants are also strongly colonized there. The number of spores in samples from the saline and sodic soils examined is rather variable, but high on average, although with an apparent low species diversity. Spore numbers in the soil adjacent to the roots of plants often, but not always, correlate with the degree of AMF colonization of the plants. As in German salt marshes [Hildebrandt et al. (2001)], the dominant AMF in the Hungarian saline and sodic soils is Glomusgeosporum. All these isolates provided nearly identical restriction fragment length polymorphism (RFLP) patterns of the internal transcribed spacer (ITS) region of spore DNA amplified by polymerase chain reaction (PCR). Cloning and sequencing of several PCR products of the ITS regions indicated that ecotypes of the G.geosporum/Glomuscaledonium clade might exist at the different habitats. A phylogenetic dendrogram constructed from the ITS or 5.8S rDNA sequences was nearly identical to the one published for 18S rDNA data (Schwarzott et al. 2001). It is tempting to speculate that specific ecotypes may be particularly adapted to the peculiar saline or sodic conditions in such soils. They could have an enormous potential in conferring salt resistance to plants.

Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt marshes.

The occurrence of arbuscular mycorrhizal fungi (AMF) was assessed by both morphological and molecular criteria in two salt marshes: (i) a NaCl site of the island Terschelling, Atlantic Coast, the Netherlands and (ii) a K(2)CO(3) marsh at Schreyahn, Northern Germany. The overall biodiversity of AMF, based on sequence analysis, was comparably low in roots at both sites. However, the morphological spore analyses from soil samples of both sites exhibited a higher AMF biodiversity. Glomus geosporum was the only fungus of the Glomerales that was detected both as spores in soil samples and in roots of the AMF-colonized salt plants Aster tripolium and Puccinellia sp. at both saline sites and on all sampling dates (one exception). In roots, sequences of Glomus intraradices prevailed, but this fungus could not be identified unambiguously from DNA of soil spores. Likewise, Glomus sp. uncultured, only deposited as sequence in the database, was widely detected by DNA sequencing in root samples. All attempts to obtain the corresponding sequences from spores isolated from soil samples failed consistently. A small sized Archaeospora sp. was detected, either/or by morphological and molecular analyses, in roots or soil spores, in dead AMF spores or orobatid mites. The study noted inconsistencies between morphological characterization and identification by DNA sequencing of the 5.8S rDNA-ITS2 region or part of the 18S rDNA gene. The distribution of AMF unlikely followed the salt gradient at both sites, in contrast to the zone formation of plant species. Zygotes of the alga Vaucheria erythrospora (Xanthophyceae) were retrieved and should not be misidentified with AMF spores.

Regulation of AmtR-controlled gene expression in Corynebacterium glutamicum: mechanism and characterization of the AmtR regulon

AmtR, the master regulator of nitrogen control in Corynebacterium glutamicum, represses transcription of a number of genes during nitrogen surplus. Repression is released by an interaction of AmtR with signal transduction protein GlnK. As shown by pull‐down assays and gel retardation experiments, only adenylylated GlnK, which is present in the cells during nitrogen limitation, is able to bind to AmtR.

Very‐long‐chain aldehydes promote in vitro prepenetration processes of Blumeria graminis in a dose‐ and chain length‐dependent manner

Surface properties of aerial plant organs have been shown to affect the interaction of fungal plant pathogens and their hosts. Conidial germination and differentiation - the so-called prepenetration processes - of the barley powdery mildew fungus (Blumeria graminis f. sp. hordei) are known to be triggered by n-hexacosanal (C(26)-aldehyde), a minor constituent of barley leaf wax. In order to analyze the differentiation-inducing capabilities of typical aldehyde wax constituents on conidia of wheat and barley powdery mildew, synthetic even-numbered very-long-chain aldehydes (C(22)-C(30)) were assayed, applying an in vitro system based on Formvar(®)/n-hexacosane-coated glass slides. n-Hexacosanal was the most effective aldehyde tested. Germination and differentiation rates of powdery mildew conidia increased with increasing concentrations of very-long-chain aldehydes. Relative to n-hexacosanal, the other aldehyde compounds showed a gradual decrease in germination- and differentiation-inducing capabilities with both decreasing and increasing chain length. In addition to n-hexacosanal, several other ubiquitous very-long-chain aldehyde wax constituents were capable of effectively stimulating B. graminis prepenetration processes in a dose- and chain length-dependent manner. Other wax constituents, such as n-alkanes, primary alcohols (with the exception of n-hexacosanol), fatty acids and alkyl esters, did not affect fungal prepenetration.

Two sides of a leaf blade: Blumeria graminis needs chemical cues in cuticular waxes of Lolium perenne for germination and differentiation.

Plant surface characteristics were repeatedly shown to play a pivotal role in plant–pathogen interactions. The abaxial leaf surface of perennial ryegrass (Lolium perenne) is extremely glossy and wettable compared to the glaucous and more hydrophobic adaxial surface. Earlier investigations have demonstrated that the abaxial leaf surface was rarely infected by powdery mildew (Blumeria graminis), even when the adaxial surface was densely colonized. This led to the assumption that components of the abaxial epicuticular leaf wax might contribute to the observed impairment of growth and development of B. graminis conidia on abaxial surfaces of L. perenne. To re-assess this hypothesis, we analyzed abundance and chemical composition of L. perenne ab- and adaxial epicuticular wax fractions. While the adaxial epicuticular waxes were dominated by primary alcohols and esters, the abaxial fraction was mainly composed of n-alkanes and aldehydes. However, the major germination and differentiation inducing compound, the C26-aldehyde n-hexacosanal, was not present in the abaxial epicuticular waxes. Spiking of isolated abaxial epicuticular Lolium waxes with synthetically produced n-hexacosanal allowed reconstituting germination and differentiation rates of B. graminis in an in vitro germination assay using wax-coated glass slides. Hence, the absence of the C26-aldehyde from the abaxial surface in combination with a distinctly reduced surface hydrophobicity appears to be primarily responsible for the failure of normal germling development of B. graminis on the abaxial leaf surfaces of L. perenne.