Michael Kaldorf

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.

Combining nested PCR and restriction digest of the internal transcribed spacer region to characterize arbuscular mycorrhizal fungi on roots from the field

Identification of arbuscular mycorrhizal fungi (AMF) on roots is almost impossible with morphological methods and, due to the presence of contaminating fungi, it is also difficult with molecular biological techniques. To allow broad investigation of the population structure of AMF in the field, we have established a new method to selectively amplify the internal transcribed spacer (ITS) region of most AMF with a unique primer set. Based on available sequences of the rDNA, one primer pair specific for AMF and a few other fungal groups was designed and combined in a nested PCR with the already established primer pair ITS5/ITS4. Amplification from contaminating organisms was reduced by an AluI restriction after the first reaction of the nested PCR. The method was assessed at five different field sites representing different types of habitats. Members of all major groups within the Glomeromycota (except Archaeosporaceae) were detected at the different sites. Gigasporaceae also proved detectable with the method based on cultivated strains.

Characterization and spatial distribution of ectomycorrhizas colonizing aspen clones released in an experimental field

Ectomycorrhizas (EM) from aspen clones released on an experimental field were characterized by morphotyping, restriction analysis and internal transcribed spacer (ITS) sequencing. In addition, their community structure and spatial distribution was analyzed. Among the 23 observed morphotypes, six mycobionts dominated, forming roughly 90% of all ectomycorrhizas: Cenococcum geophilum, Laccaria sp., Phialocephala fortinii, two different Thelephoraceae, and one member of the Pezizales. The three most common morphotypes had an even spatial distribution, reflecting the high degree of homogeneity of the experimental field. The distribution of three other morphotypes was correlated with the distances to the spruce forest and deciduous trees bordering the experimental field. These two patterns allowed two invasion strategies of ectomycorrhizal fungi (EMF) to be recognized, the success of which depends on adaptation of the EMF to local ecological conditions.

Indole-3-butyric acid (IBA) is enhanced in young maize (Zea mays L.) roots colonized with the arbuscular mycorrhizal fungus Glomus intraradices

Abstract Inoculation of maize ( Zea mays L.) roots with the mycorrhizal fungus Glomus intraradices resulted in an increase of indole-3-butyric acid (IBA) during early stages of infection compared to control roots. The increase in IBA was accompanied by an increase of IBA synthetase activity, but the enzyme activity was also enhanced at later stages of infection. No IBA was detected in spores of Glomus , whereas small amounts of indole-3-acetic acid (IAA) were found. The endogenous IBA concentration is not important for colonization of roots with the fungus, since two other maize varieties with lower IBA content had the same infection rate as the variety with higher IBA content. The increase of IBA in AM-colonized roots was also confirmed in the variety ‘Alize’.

Diversity of the ectomycorrhiza community at a uranium mining heap

Ectomycorrhiza (EM) community structure was analyzed at one bare heap site (BHS), one heap site with organic cover (HS-OH) and one reference site (RS) in the former uranium mining area near Ronneburg (Thuringia, Germany). Twenty-three EM morphotypes were distinguished, and 14 of them were additionally characterized by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and internal transcribed spacer (ITS) sequence analysis. Colonization of birch by the different morphotypes was quantified, and the EM diversity at the different sites was investigated. Compared to RS, total EM colonization was reduced by 6% (P=0.851) at HS-OH and by 58% (P<0.001) at BHS. Likewise, EM diversity was reduced by 16% (P=0.229) at HS-OH and 52% (P<0.001) at BHS. The Sørensen similarity between EM samples from RS was nearly independent from the sampling date, whereas at HS-OH and especially BHS, the Sørensen similarity decreased with increasing time between the samplings. All EM fungal species dominating at the two heap sites were also present at RS. Thus, fungi with high tolerance against uranium and other stress factors at the heap sites (e.g. heavy metals, nutrient limitation, drought) were selected among all EM fungi of the area. Highly adapted fungi with a distribution restricted to the contaminated soils were not detected.

Chitinase activities, scab resistance, mycorrhization rates and biomass of own-rooted and grafted transgenic apple

This study investigated the impact of constitutively expressed Trichoderma atroviride genes encoding exochitinase nag70 or endochitinase ech42 in transgenic lines of the apple cultivar Pinova on the symbiosis with arbuscular mycorrhizal fungi (AMF). We compared the exo- and endochitinase activities of leaves and roots from non-transgenic Pinova and the transgenic lines T386 and T389. Local and systemic effects were examined using own-rooted trees and trees grafted onto rootstock M9. Scab susceptibility was also assessed in own-rooted and grafted trees. AMF root colonization was assessed microscopically in the roots of apple trees cultivated in pots with artificial substrate and inoculated with the AMF Glomus intraradices and Glomus mosseae. Own-rooted transgenic lines had significantly higher chitinase activities in their leaves and roots compared to non-transgenic Pinova. Both of the own-rooted transgenic lines showed significantly fewer symptoms of scab infection as well as significantly lower root colonization by AMF. Biomass production was significantly reduced in both own-rooted transgenic lines. Rootstock M9 influenced chitinase activities in the leaves of grafted scions. When grafted onto M9, the leaf chitinase activities of non-transgenic Pinova (M9/Pinova) and transgenic lines (M9/T386 and M9/T389) were not as different as when grown on their own roots. M9/T386 and M9/T389 were only temporarily less infected by scab than M9/Pinova. M9/T386 and M9/T389 did not differ significantly from M9/Pinova in their root chitinase activities, AMF root colonization and biomass.

Physiologische und molekularbiologische Ansätze zur Analyse der Funktion von Indol-3-buttersäure in der Entwicklung arbuskulärer Mykorrhizen

Inoculation of maize with the AM fungus Glomus intraradices resulted in a distinct root phenotype with a significant increase in the percentage of lateral roots during early stages of colonization. Auxins are known to play a role in different developmental processes including fine root formation and are therefore suitable candidates for the regulation of AM symbiosis. The increase in lateral root development coincided with an increase in the levels of the auxin indole-3-butyric acid (fA). The phenotype of mycorrhizal maize roots could be mimicked by exogenously applied IBA to non-mycorrhizal roots. Addition of trifluoro-IBA (IFIBA), an inhibitor of IBA-induced root growth and lateral root induction, simultaneously with IBA resulted in a phenotype resembling that of untreated controls. In roots treated with TFIBA the inoculation with AM-fungi did not increase the formation of fine roots. The IFIBA-treatment also reduced endogenous free IBA and the percentage of colonization in mycorrhizal roots. Using activity screening and cDNA-AFLP analysis, we have started to isolate the gene for IBA synthetase and other genes differentially induced by IBA and AM.