Phosphate transporters of Medicago truncatula and arbuscular mycorrhizal fungi

Most vascular plants acquire phosphate from their environment either directly, via the roots, or indirectly, via a symbiotic interaction with arbuscular mycorrhizal (AM) fungi. The symbiosis develops in the plant roots where the fungi colonize the cortex of the root to obtain carbon from the plant host, while assisting the plant with acquisition of phosphate and other mineral nutrients from the soil solution. As a first step toward understanding the molecular basis of the symbiosis and phosphate utilization, we have cloned and characterized phosphate transporter genes from the AM fungi Glomus versiforme and Glomus intraradices, and from the roots of a host plant, Medicago truncatula. Expression analyses and localization studies indicate that each of these transporters has a role in phosphate uptake from the soil solution.     

Effect of two AMF life strategies on the tripartite symbiosis with Bradyrhizobium japonicum and soybean

This study is the first in assessing the effect of soil disturbance on the contribution of arbuscular mycorrhizal fungi (AMF) with different life-history strategies to the tripartite symbiosis with soybeans and Bradyrhizobium japonicum (Kirchner) Jordan. We hypothesized that Gigaspora margarita Becker and Hall would be more affected by soil disturbance than Glomus clarum Nicol. and Schenck, and consequently, the tripartite symbiosis would develop more rapidly and lead to greater N₂ fixation in the presence of the latter. Soil pasteurization allowed the establishment of treatments with individual AMF species and soil disturbance enabled the development of contrasting root colonization potentials. In contrast, the colonization potential of B. japonicum was kept the same in all treatments. Soil disturbance significantly reduced root colonization by both AMF, with Gi. margarita being considerably more affected than G. clarum. Furthermore, the tripartite symbiosis progressed faster with G. clarum, and at 10 days after plant emergence, there was 30% more nodules when G. clarum was present compared to that when the bacterial symbiont alone was present. At flowering, the absence of soil disturbance stimulated N₂ fixation by 17% in mycorrhizal plants. However, this response was similar for both AMF.     

Cytophotometric investigation of DNA and RNA content in nuclei of active Strasburger cells in Pinus nigra var. austriaca (Hoess) Badoux

The nuclei of active, sieve cell-associated Strasburger cells in the secondary phloem of Pinus nigra var. austriaca (Hoess) Badoux have been studied for their structure and DNA and RNA content. No difference in size compared to those of ordinary ray cells was found. The nuclear surface is often increased by an ameboid or lobed shape. The amount of highly decondensed chromatin is greatly increased. Cytophotometric measurements of DNA content of both Feulgen and gallocyanine chromalum-stained nuclei showed normal DNA levels and proved absence of endomitotic polyploidization. RNA content, however, was significantly increased as compared to nuclei of young Strasburger cells and of ordinary ray parenchyma cells.Abbreviations StC₁ Strasburger cells in contact with young and immature sieve cells - StC₂ Srasburger cells in contact with mature and functionally active sieve cells - StC₃ dead Strasburger cells - eRPC pRPC erect and procumbent ray parenohyma cells, respectively - GCCA gallocyanine chromalum - T transmission - A absorbance Dedicated to Professor Dr. Wilhelm Halbsguth, Kiel, on the occation of his 65th birthday     

Impact of antifungals producing rhizobacteria on the performance of Vigna radiata in the presence of arbuscular mycorrhizal fungi

Plant growth-promoting rhizobacteria (PGPR) that produce antifungal metabolites are potential threats for the arbuscular mycorrhizal (AM) fungi known for their beneficial symbiosis with plants that is crucially important for low-input sustainable agriculture. To address this issue, we used a compartmented container system where test plants, Vigna radiata, could only reach a separate nutrient-rich compartment indirectly via the hyphae of AM fungi associated with their roots. In this system, where plants depended on nutrient uptake via AM symbiosis, we explored the impact of various PGPR. Plants were inoculated with or without a consortium of four species of AM fungi (Glomus coronatum, Glomus etunicatum, Glomus constrictum, and Glomus intraradices), and one or more of the following PGPR strains: phenazine producing (P⁺) and phenazine-less mutant (P⁻), diacetylphloroglucinol (DAPG) producing (G⁺) and DAPG-less mutant (G⁻) strains of Pseudomonas fluorescens, and an unknown antifungal metabolite-producing Alcaligenes faecalis strain, SLHRE425 (D). PGPR exerted only a small if any effect on the performance of AM symbiosis. G⁺ enhanced AM root colonization and had positive effects on shoot growth and nitrogen content when added alone, but not in combination with P⁺. D negatively influenced AM root colonization, but did not affect nutrient acquisition. Principal component analysis of all treatments indicated correlation between root weight, shoot weight, and nutrient uptake by AM fungus. The results indicate that antifungal metabolites producing PGPR do not necessarily interfere with AM symbiosis and may even promote it thus carefully chosen combinations of such bioinoculants could lead to better plant growth.     

Host dependent differential spread of <Emphasis Type="Italic">Glomus intraradices</Emphasis> on various Ri T-DNA transformed roots <Emphasis Type="Italic">in vitro</Emphasis>

Carrots (Daucus carota) are used extensively for culturing arbuscular mycorrhizal fungi under the root organ culture system (ROC) in vitro. Four different cultivars of carrot and one of clover (Trifolium subterraneum) root cultures were used to investigate symbiotic events with Glomus intraradices when introduced to different host-transformed roots. The investigation was directed to study the state of mycorrhization, its pattern and variation, if any and, the differences in nutritional uptake of each AM-host symbiosis. The findings suggest the role of the host on the differential expression of G. intraradices with respect to colonization, spore production, intraradical and extramatrical spread of the fungus, nutritional kinetics of each host-fungus symbiosis, pattern of growth and differences in anatomical and morphological features of host roots.     

Evidence of DNA replication in an arbuscular mycorrhizal fungus in the absence of the host plant

Summary This study provides evidence thatGigaspora margarita replicates its nuclear DNA, even in the absence of a host plant. Three experimental approaches were used: (i) static cytofluorimetry to quantify the DNA content, (ii) pulse treatments with bromodeoxyuridine (BrdU), which is an analogue of thymidine, to reveal nuclei undergoing DNA synthesis, and (iii) ultrastructural observations to study changes in chromatin morphology during the fungal cell cycle. A slight second peak of approximately twice the value of a major peak was found by cytofluorimetry, showing that a small number of nuclei had entered in cycle during in vitro development. Nuclei which had incorporated BrdU were observed after pulses of 24 h; nuclei with condensed chromatin were also apparent at this time. The results demonstrate thatG. margarita has all the metabolic pathways needed to replicate its nuclear DNA even in the absence of the host, suggesting that more complex mechanisms inhibit the extended growth in vitro of arbuscular mycorrhizal fungi.Abbreviations AM-fungi arbuscular mycorrhizal fungi - A.U. arbitrary units - BrdU 5-bromo-2-deoxyuridine - DAPI 4,6-diamidino-2-phenylindole - UV ultraviolet light     

Peptidoglycan induces loss of a nuclear peptidoglycan recognition protein during host tissue development in a beneficial animal-bacterial symbiosis

Peptidoglycan recognition proteins (PGRPs) are mediators of innate immunity and recently have been implicated in developmental regulation. To explore the interplay between these two roles, we characterized a PGRP in the host squid Euprymna scolopes (EsPGRP1) during colonization by the mutualistic bacterium Vibrio fischeri . Previous research on the squid-vibrio symbiosis had shown that, upon colonization of deep epithelium-lined crypts of the host light organ, symbiont-derived peptidoglycan monomers induce apoptosis-mediated regression of remote epithelial fields involved in the inoculation process. In this study, immunofluorescence microscopy revealed that EsPGRP1 localizes to the nuclei of epithelial cells, and symbiont colonization induces the loss of EsPGRP1 from apoptotic nuclei. The loss of nuclear EsPGRP1 occurred prior to DNA cleavage and breakdown of the nuclear membrane, but followed chromatin condensation, suggesting that it occurs during late-stage apoptosis. Experiments with purified peptidoglycan monomers and with V. fischeri mutants defective in peptidoglycan-monomer release provided evidence that these molecules trigger nuclear loss of EsPGRP1 and apoptosis. The demonstration of a nuclear PGRP is unprecedented, and the dynamics of EsPGRP1 during apoptosis provide a striking example of a connection between microbial recognition and developmental responses in the establishment of symbiosis.     

Changes in polypeptide patterns in tobacco roots colonized by two Glomus species

Changes in gene expression were studied during the establishment of arbuscular mycorrhizal symbiosis in tobacco roots from an amphidiploid hybrid Nicotiana glutinosa x N. debneyi. Polypeptide patterns from control roots and from roots infected by Glomus mosseae or G. intraradices were resolved by two-dimensional polyacrylamide gel electrophoresis and followed in a time-course analysis. Arbuscular mycorrhizal infection led to significant modifications in polypeptide patterns with: (a) decreased amounts of some polypeptides, (b) increased accumulation of others, and (c) appearance of newly-induced polypeptides. Comparisons made during infection development by the two Glomus species demonstrated that protein modifications changed in relation to the mycorrhizal state of the tobacco roots.     

Azolla and other plant-cyanobacteria symbioses: Aspects of form and function

Nostoc, a genus of filamentous, heterocystous, cyanobacteria, is widely distributed in the free-living state. It is also the most common phycobiont in N₂-fixing lichens and occurs as the N₂-fixing symbiont in a small and diverse group of green plants. These include several bryophyte genera (e.g. Anthoceros and Blasia), a pteridophyte genus (Azolla; while the symbiont is referred to asAnabaena azollae, it may be aNostoc spp.), a division of gymnosperms (the 10 cycad genera) and one angiosperm genus (Gunnera). In Gunnera the Nostoc apparently penetrates into the cells of the host. In the other associations Nostoc is extracellular but specific morphological modifications and/or structures of the host plant organs create an environment which fosters interaction and metabolite interchange.The individual group of Nostoc-green plant symbioses other than Azolla are summarized in regard to the current understanding of their establishment, perpetuation, and host-symbiont interaction. This includes available information on recognition and specificity, mode(s) of infection if applicable, and a synopsis of morphological modifications of the partners. The symbiosis withAzolla is then addressed separately with a more indepth account of the foregoing areas. In addition, the concept ofAzolla harboring a dominant, obiligately symbiotic Nostoc which has not been cultured as well as minor symbionts capable of free-living growth, the distinction between re-constituting and simply re-establishing the symbiosis, and current approaches to improving the symbiosis and to authenticating the establishment of new associations are considered.     

Distinct chromatin organization in the germ line founder cell of the Caenorhabditis elegans embryo

Cells belonging to the germ lineage segregate physically and molecularly from their somatic neighbors during embryogenesis. While germ line‐specific chromatin modifications have been identified at later stages in the Caenorhabditis elegans nematode, none have been found in the single P₄ germ line founder cell that arises at the beginning of gastrulation. Using light and electron microscopy, we now report that the chromatin organization in the germ line founder cell of the early C. elegans embryo is distinct from that in the neighboring somatic cells. This unique organization is characterized by a greater chromatin compaction and an expansion of the interchromatin compartment. The ultrastructure of individual chromatin domains does not differ between germ line and somatic cells, pointing to a specific organization mainly at the level of the whole nucleus. We show that this higher order reorganization of chromatin is not a consequence of the P₄ nucleus being smaller than somatic nuclei or having initiated mitosis. Imaging of living embryos expressing fluorescent markers for both chromatin and P granules revealed that the appearance of a distinct chromatin organization in the P₄ cell occurs approximately 10 min after its birth and coincides with the aggregation of P granules around the nucleus, suggesting a possible link between these two events. The higher order reorganization of chromatin that is reported here occurs during the establishment of definitive germ cell identity. The changes we have observed could therefore be a prerequisite for the programming of chromatin totipotency.     

A method for direct soil extraction and PCR amplification of endomycorrhizal fungal DNA

 DNA from endomycorrhizal fungi was extracted directly from a weathered soil (alfisol) mixed with sand. Mycorrhizae were established in a greenhouse culture of Glomus clarum with Sudan grass (Sorghum vulgare var. sudanense) host plants. The extraction procedure included enzymatic digestion of cell walls, sodium dodecyl sulfate lysis of cells, polyvinylpolypyrrolidone absorption of organic compounds, and ethanol precipitation of the DNA. DNA in the extracts was amplified by the polymerase chain reaction using primers from the nuclear 17S rRNA sequence that were general to fungi or were specific to endomycorrhizae. Accepted: 17 July 1996     

Sucrose synthase levels do not limit or regulate carbon transfer in the arbuscular mycorrhizal symbiosis

Abstract

Sucrose synthase (SuSy) is the main sucrose breakdown enzyme in plant sink tissues, including nodules, and is a possible candidate for the diversion of plant carbon to arbuscular mycorrhizal (AM) fungi in roots. We tested the involvement of SuSy in AM symbiosis of Glomus intraradices and Pisum sativum (pea). We observed that peas deficient in the predominant root isoform of SuSy were colonized successfully by AM fungi similar to wild-type roots. SuSy protein levels did not increase in roots as AM symbiosis developed, although SuSy protein levels did increase in nodules as the rhizobium symbiosis developed. Our results lead us to conclude that, unlike nodule symbiosis, SuSy protein does not limit or regulate carbon transfer in the AM symbiosis.     

Stimulation of vesicular-arbuscular mycorrhiza by fungicides or rhizosphere bacteria

Stimulation of vesicular-arbuscular (VA) mycorrhizal fungi may secure the early establishment of symbiosis and benefit the host plant at an earlier stage of development. The application of Bacillus mycoides resulted in particular in the acceleration of early VA mycorrhiza formation. An increase in vigour of the symbiosis could be measured later in terms of increased sporulation of the mycorrhizal fungi after shoot removal. Natural sporulation during later mycorrhizal development was affected by combination of bacteria and just one mycorrhizal isolate. The stimulation of mycorrhizal development was shown to be non-specific with regard to host plant and the isolate of the VAM fungus. However, the effect could not be achieved in all combinations of soil types and host plants. Application of the systemic fungicides triadimefon and pyrazophos promoted VAM formation. Combinations of fungicide and bacterial treatments were not synergistic.     

The diversity of arbuscular mycorrhizas of selected Australian Fabaceae

Abstract

Members of the Australian native perennial Fabaceae have been little explored with regard to their root biology and the role played by arbuscular mycorrhizal (AM) fungi in their establishment, nutrition and long-term health. The ultimate goal of our research is to determine the dependency of native perennial legumes on their co-evolved AM fungi and conversely, the impact of AM fungal species in agricultural fields on the productivity of sown native perennial legume pastures. In this paper we investigate the colonisation morphology in roots and the AMF, identified by spores extracted from rhizosphere soil, from three replicate plots of each of the native legumes, Cullen australasicum, C. tenax and Lotus australis and the exotic legumes L. pedunculatus and Medicago sativa. The plants were grown in an agricultural field. The level and density of colonisation by AM fungi, and the frequency of intraradical and extraradical hyphae, arbuscules, intraradical spores and hyphal coils all differed between host plants and did not consistently differ between native and exotic species. However, there were strong similarities between species in the same genus. The three dominant species of AM fungi in rhizosphere soil also differed with host plant, but one fungus (Glomus mosseae) was always the most dominant. Sub-dominant AM species were the same between species in the same genus. No consistent differences in dominant spores were observed between the exotic and native Fabaceae species. Our results suggest that plant host influences the mycorrhizal community in the rhizosphere soil and that structural and functional differences in the symbiosis may occur at the plant genus level, not the species level or due to provenance.     

Resistance of pea roots to endomycorrhizal fungus or Rhizobium correlates with enhanced levels of endogenous salicylic acid

The analysis of SA accumulation in roots of plant symbiotic mutants revealed two independent phenomena associated with the inability of either the plant or the microsymbiont to form a compatible symbiosis. SA accumulation in roots of the wild type and symbiosis-resistant P2 (Nod^-, MYC^-) Pisum sativum genotypes was induced upon interaction with Glomus mosseae. The amplitude of this accumulation was higher in P2 plants and increased with time, an effect that was not observed in roots of the wild-type, an effect that was not observed in roots of the wild-type P. sativum genotype. Likewise, Rhizobium leguminosarum wild type or a mutant blocked in Nod factor biosynthesis induced SA accumulation in P2, whereas SA accumulation in roots of the wild-type plant was dependent on the inability of the bacterium to produce Nod factors. These results suggest that the sym30 gene, which is mutated in P2 plants, could be implicated in a common pathway that leads to the suppression of an SA-dependent defence mechanism in legume plants against Rhizobium and endomycorrhizal fungi, thus allowing establishment of symbiosis.     

Arbuscular mycorrhizal symbiosis increases host plant acceptance and population growth rates of the two-spotted spider mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Most terrestrial plants live in symbiosis with arbuscular mycorrhizal (AM) fungi. Studies on the direct interaction between plants and mycorrhizal fungi are numerous whereas studies on the indirect interaction between such fungi and herbivores feeding on aboveground plant parts are scarce. We studied the impact of AM symbiosis on host plant choice and life history of an acarine surface piercing-sucking herbivore, the polyphagous two-spotted spider mite Tetranychus urticae. Experiments were performed on detached leaflets taken from common bean plants (Phaseolus vulgaris) colonized or not colonized by the AM fungus Glomus mosseae. T. urticae females were subjected to choice tests between leaves from mycorrhizal and non-mycorrhizal plants. Juvenile survival and development, adult female survival, oviposition rate and offspring sex ratio were measured in order to estimate the population growth parameters of T. urticae on either substrate. Moreover, we analyzed the macro- and micronutrient concentration of the aboveground plant parts. Adult T. urticae females preferentially resided and oviposited on mycorrhizal versus non-mycorrhizal leaflets. AM symbiosis significantly decreased embryonic development time and increased the overall oviposition rate as well as the proportion of female offspring produced during peak oviposition. Altogether, the improved life history parameters resulted in significant changes in net reproductive rate, intrinsic rate of increase, doubling time and finite rate of increase. Aboveground parts of colonized plants showed higher concentrations of P and K whereas Mn and Zn were both found at lower levels. This is the first study documenting the effect of AM symbiosis on the population growth rates of a herbivore, tracking the changes in life history characteristics throughout the life cycle. We discuss the AM-plant-herbivore interaction in relation to plant quality, herbivore feeding type and site and the evolutionary implications in a multi-trophic context.     

Interactions between Lotus japonicus genotypes and arbuscular mycorrhizal fungi

Abstract

Interactions between three genotypes (Ljsym 71-1, Ljsym 71-2 and Ljsym 72) of Lotus japoicus and one isolate from each of four species of arbuscular mycorrhizal fungi (Glomus sp. R-10, Glomus intraradices, Glomus etunicatum, and Gigaspora margarita) were investigated and compared with the wild-type ‘Gifu’ B-129. All the three genotypes showed no or defective internal colonization after inoculation with these AM fungi. In Ljsym72 mutant, the AM fungi produced deformed appressoria on the root surface, but failed to form any internal structures (internal hyphae, arbuscules and vesicles) except only in Glomus intraradices. The Ljsym71-1 and Ljsym71-2 mutants had more deformed appressoria and occasionally formed internal hyphae, arbuscules and vesicles, depending on AM fungi used. Wild-type ‘Gifu’ (nod⁺myc⁺) plants had typical colonization. The colonization of mutants by several fungi varied and provides a basis for studying recognition and compatibility between plants and mycorrhizal fungal species. These mutants also will be useful in studies of the genetics of the symbiosis between plant species and AM fungi.     

Effect of liming on spore germination,germ tube growth and root colonization by vesicular-arbuscular mycorrhizal fungi

Summary The effect of soil acidity on spore germination, germ tube growth and root colonization of vesicular-arbuscular mycorrhizal (VAM) fungi was examined using a Florida Ultisol. Soil samples were treated with 0, 4, 8 and 12 meq Ca/MgCO₃/100 g soil and each lime level received 0, 240, and 720 ppm P as superphosphate. Corn (Zea mays L.) was planted in the soil treatments, inoculated with eitherGlomus mosseae orGigaspora margarita spores and grown for 31 days. Acid soil inhibits mycorrhizal formation byG. mosseae through its strong fungistatic effect against the spores. The dolomitic lime increased mycorrhizal formation by both fungal species.G. margarita is much less sensitive to acidic conditions thanG. mosseae. Al ions are a very important component of the fungistatic property against the VAM symbiosis. VAM fungus adaptation may be important for plants growing on infertile acid soils if soil inoculation with these fungi is to contribute significantly to low-input technology for tropical agricultural systems.