Medicago truncatula gene responses specific to arbuscular mycorrhiza interactions with different species and genera of Glomeromycota.

Plant genes exhibiting common responses to different arbuscular mycorrhizal (AM) fungi and not induced under other biological conditions have been sought for to identify specific markers for monitoring the AM symbiosis. A subset of 14 candidate Medicago truncatula genes was identified as being potentially mycorrhiza responsive in previous cDNA microarray analyses and exclusive to cDNA libraries derived from mycorrhizal root tissues. Transcriptional activity of the selected plant genes was compared during root interactions with seven AM fungi belonging to different species of Glomus, Acaulospora, Gigaspora, or Scutellospora, and under widely different biological conditions (mycorrhiza, phosphate fertilization, pathogenic/beneficial microbe interactions, incompatible plant genotype). Ten of the M. truncatula genes were commonly induced by all the tested AM fungal species, and all were activated by at least two fungi. Most of the plant genes were transcribed uniquely in mycorrhizal roots, and several were already active at the appressorium stage of fungal development. Novel data provide evidence that common recognition responses to phylogenetically different Glomeromycota exist in plants during events that are unique to mycorrhiza interactions. They indicate that plants should possess a mycorrhiza-specific genetic program which is comodulated by a broad spectrum of AM fungi.

Isolation and partial characterization of antagonistic peptides produced by Paenibacillus sp. strain B2 isolated from the sorghum mycorrhizosphere.

Paenibacillus sp. strain B2, isolated from the mycorrhizosphere of sorghum colonized by Glomus mosseae, produces an antagonistic factor. This factor has a broad spectrum of activity against gram-positive and gram-negative bacteria and also against fungi. The antagonistic factor was isolated from the bacterial culture medium and purified by cation-exchange, reverse-phase, and size exclusion chromatography. The purified factor could be separated into three active compounds following characterization by amino acid analysis and by combined reverse-phase chromatography and mass spectrometry (liquid chromatography-mass spectrometry and mass spectrometry-mass spectrometry). The first compound had the same retention time as polymyxin B1, whereas the two other compounds were more hydrophobic. The molecular masses of the latter compounds are 1,184.7 and 1,202.7 Da, respectively, and their structure is similar to that of polymyxin B1, with a cyclic heptapeptide moiety attached to a tripeptide side chain and a fatty acyl residue. They both contain threonine, phenylalanine, leucine, and 2,4-diaminobutyric acid residues. The peptide with a molecular mass of 1,184.7 contains a 2,3-didehydrobutyrine residue with a molecular mass of 101 Da replacing a threonine at the A2 position of the polymyxin side chain. This modification could explain the broader range of antagonistic activity of this peptide compared to that of polymyxin B.

Proteomics as a tool to monitor plant-microbe endosymbioses in the rhizosphere.

In recent years, outstanding molecular approaches have been used to investigate genes and functions involved in plant-microbe endosymbioses. In this review, we outline the use of proteomic analysis, based on two-dimensional electrophoresis and mass spectrometry, to characterize symbiosis-related proteins. During the last decade, proteomics succeeded in identifying about 400 proteins associated with the development and functioning of both mycorrhizal and rhizobial symbioses. Further progress in prefractionation procedures is expected to allow the detection of symbiotic proteins showing low abundance or being present in certain cell compartments.

Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions.

Suppression subtractive hybridization (SSH), expression profiling and EST sequencing identified 12 plant genes and six fungal genes that are expressed in the arbuscular mycorrhizal symbiosis between Medicago truncatula and Glomus mosseae. All the plant genes and three of the fungal genes were up-regulated in symbiotic tissues. Expression of 15 of the genes is described for the first time in mycorrhizal roots and two are novel sequences. Six M. truncatula genes were also activated during appressorium formation at the root surface, suggesting a role in this early stage of mycorrhiza establishment, whilst the other six plant genes were only induced in the late stages of mycorrhization and could be involved in the development or functioning of the symbiosis. Phosphate fertilization had no significant influence on expression of any of the plant genes. Expression profiling of G. mosseae genes indicated that two of them may be associated with appressorium development on roots and one with arbuscule formation or function. The other three fungal genes were expressed throughout the life-cycle of G. mosseae.

Common gene expression in Medicago truncatula roots in response to Pseudomonas fluorescens colonization, mycorrhiza development and nodulation.

• Beneficial rhizosphere microorganisms may share similar molecular steps during root colonization. To test this hypothesis, we compared Medicago truncatula Gaertn. gene expression in roots colonized, or not colonized, by Glomus mosseae BEG12, Pseudomonas fluorescens C7R12 or Sinorhizobium meliloti 2011. • Pseudomonas fluorescens C7R12 formed colonies on the surface of M. truncatula roots and colonized root tissues intercellularly and intracellularly in a way similar to that previously described for other plants. • Semiquantitative reverse transcriptase polymerase chain reaction of a set of 12 mycorrhiza upregulated M. truncatula genes revealed different expression profiles in roots 3 weeks after inoculation with P. fluorescens or S. meliloti. Pseudomonas fluorescens colonization activated seven of the plant genes while nodulated root systems showed increased expression in only three genes and five appeared to be downregulated. • This first report of similar gene induction by a fluorescent pseudomonad and a mycorrhizal fungus in roots supports the hypothesis that some plant cell programmes may be shared during root colonization by these beneficial microorganisms. Less similarity existed in expression of the gene set with nodulation by S. meliloti.

Purification and partial amino acid sequencing of a mycorrhiza-related chitinase isoform from Glomus mosseae-inoculated roots of Pisum sativum L.

Colonization of Pisum sativum L. cv. Frisson roots with the arbuscular mycorrhizal fungus Glomus mosseae leads to the induction of four acidic symbiosis-related chitinase (SR-chi) isoforms (EC 3.2.1.14). These isoforms were characterized as 30-kDa proteins with isoelectric points ranging between 5.2 and 5.85. One of these SR-chis was purified by affinity and anion-exchange chromatographies, and isoelectric focusing electrophoresis. The sequences of four internal peptides were obtained. They showed high homology to a class-I chitinase isoform from pea shoots. Parts of the conserved regions of class-I chitinases were found in this SR-chi. This result strongly supports the argument that this SR-chi isoform is of plant origin. The functional role of the SR-chis in arbuscular mycorrhizal symbiosis is discussed.

25S rDNA-based molecular monitoring of glomalean fungi in sewage sludge-treated field plots.

Recycling of sewage wastes in agriculture is likely to affect the biological activity of soils through contamination of ecosystems by pathogens and metallic or organic micropollutants. The impact of sewage sludge spreading under field conditions on arbuscular mycorrhiza (AM) formation by a community of glomalean fungi was evaluated using a nested polymerase chain reaction (PCR) and discriminating primers based on 25S rDNA polymorphisms to detect different fungal species within root systems. Medicago truncatula was grown in soil of field plots amended or not with a composted sewage sludge, spiked or not with organic or metallic micropollutants. Overall AM development in roots decreased with sewage sludge application, and the relative abundance of five AM fungal morphotypes in root fragments was modified by the input of composted sludges. Sewage sludge spiked or not with organic pollutants had a generally positive effect on the relative diversity of AM fungal populations in planta, whereas after spreading of the sludge spiked with metallic pollutants, no variation was observed in the abundance of different species.

Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco.

In arbuscular mycorrhizas, H+-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson et al. 1995, Can J Bot 73: S526-S532). The proton-pumping H+-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H+-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H+-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of beta-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H+-ATPase activity in the periarbuscular membrane results from selective induction of two H+-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface.

Is a fully established arbuscular mycorrhizal symbiosis required for a bioprotection of Pisum sativum roots against Aphanomyces euteiches?

Bioprotection of pea roots against Aphanomyces euteiches by the arbuscular mycorrhizal fungus G. mosseae was demonstrated to depend on a fully established symbiosis. This was related with induction of mycorrrhiza-related chitinolytic enzymes. Possible mechanisms implicated in bioprotection are discussed.

Effects on growth and comparison of root tissue colonization patterns of Eucalyptus viminalis by pathogenic and nonpathogenic strains of Fusarium oxysporum.

Soilborne pathogens, especially Fusarium oxysporum, are responsible for damping-off and root necrosis in Eucalyptus nurseries. New technologies are increasingly considering strategies for plant disease control other than chemical fungicides. Among these, natural fungal antagonists, which are colonizers of the root cortex, are potential biocontrol agents. An in vitro system was used: (1) to test the pathogenic effects of F. oxysporum strain Foeu1 which was recovered from a forest nursery soil; (2) to explore the potential of the nonpathogenic F. oxysporum strain Fo47, which is known for its efficiency in biological control, to suppress damping-off of Eucalyptus seedlings; (3) to compare the patterns of root colonization and host response to invasion by the two Fusarium strains inoculated separately in a time-course study. Root inoculation of E. viminalis with F. oxysporum strain Foeu1 caused damping-off in young seedlings in vitro, whilst disease symptoms were not visible in plants inoculated with F. oxysporum strain Fo47 or when both strains (Foeu1 + Fo47) were inoculated simultaneously. Each strain showed similarities in patterns of root tissue colonization, and in the processes of root penetration and initial colonization. Differential effects on root tissue were observed with fungal development within the cortex: ingress of strain Foeu1 was accompanied by severe host-cell alterations whilst no tissue damage occurred with development of strain Fo47.

Isolation from the Sorghum bicolor mycorrhizosphere of a bacterium compatible with arbuscular mycorrhiza development and antagonistic towards soilborne fungal pathogens.

A gram-positive bacterium with antagonistic activity towards soilborne fungal pathogens has been isolated from the mycorrhizosphere of Sorghum bicolor inoculated with Glomus mosseae. It has been identified as Paenibacillus sp. strain B2 based on its analytical profile index and on 16S ribosomal DNA analysis. Besides having antagonistic activity, this bacterium stimulates mycorrhization.

Cellular localization of a plant protein PSAM 1 in arbuscular mycorrhizas of Pisum sativum.

Psam 1 is a single-copy gene which is activated during early plant-fungal interaction in wild-type pea inoculated with Glomus mosseae and which codes for PSAM 1, a putative protein of 108 amino acids. A synthetic peptide was designed of 108 amino acids. A synthetic peptide was designed in an antigenic region of this protein to produce a polyclonal antibody against PSAM 1 and to investigate its cellular localization. Western blot analysis revealed that a polypeptide of about 14.5 kDa accumulated more in mycorrhizal than non-mycorrhizal pea roots. The PSAM 1 antigen was immunolocated in planta in arbuscule-containing cells of mycorrhizal roots and especially in the cytoplasm surrounding young arbuscules in cortical cells, which suggests that its accumulation is somehow related to the symbiotic state of these cells.

Differential display analysis of RNA accumulation in arbuscular mycorrhiza of pea and isolation of a novel symbiosis-regulated plant gene.

Differential RNA display was used to analyze gene expression during the early steps of mycorrhiza development on Pisum sativum following inoculation with Glomus mosseae. Seven out of 118 differentially displayed cDNA fragments were subcloned and sequenced. One fragment corresponded to part of the fungal 25S ribosomal RNA gene and a second one showed similarity to a human Alu element. The others were derived from plant genes of unknown function. One of the fragments was used for the isolation of a full-length cDNA clone. It corresponded to a single-copy gene (psam1) which is induced during early symbiotic interactions, and codes for a putative transmembrane protein. Northern and RNA dot blot analyses revealed enhanced accumulation of psam1 RNA after inoculation with G. mosseae of wild-type pea and an isogenic mutant deficient for nodule development (Nod-, Myc+).

First report of the inhibition of arbuscular mycorrhizal infection of Pisum sativum by specific and irreversible inhibition of polyamine biosynthesis or by gibberellic acid treatment.

DFMO (alpha-DL-difluoromethylornithine), a specific irreversible inhibitor of ornithine decarboxylase (ODC), a polyamine biosynthetic pathway enzyme, strongly inhibits root growth and arbuscular mycorrhizal infection of Pisum sativum (P56 myc+, isogenic mutant of cv. Frisson). This inhibition is reversed when exogenous polyamine (putrescine) is included in the DFMO treatment, showing that the effect of DFMO on arbuscular mycorrhizal infection is indeed due to putrescine limitation and suggesting that ODC may have a role in root growth and mycorrhizal infection. However, treatment with gibberellic acid (GA3) which increased root titers of polyamines strongly inhibited arbuscular mycorrhizal development. The possible role of polyamines in the regulation of the development of arbuscular mycorrhizal infection is discussed.

Arbuscular mycorrhizal induced changes to plant growth and root system morphology in Prunus cerasifera.

We compared root system morphogenesis of micropropogated transplants of Prunus cerasifera L. inoculated with either of the arbuscular mycorrhizal (AM) fungi Glomus mosseae or Glomus intraradices or with the ericoid mycorrhizal species Hymenoscyphus ericae. All plants were grown in sand culture, irrigated with a nutrient solution that included a soluble source of phosphorus, for 75 days after transplanting. Arbuscular mycorrhizal colonization increased both the survival and growth (by over 100%) of transplants compared with either uninoculated controls or transplants inoculated with H. ericae. Arbuscular mycorrhizal colonization increased root, stem and leaf weights, leaf area, root length and specific leaf area, and it decreased root length/leaf area ratio, root/shoot weight ratio and specific root length. Both uptake of phosphorus and its concentration in leaves were increased by AM infection, although the time course of the relationships between intensity of AM infection and P nutrition were complex and suggested a role for factors other than nutrition. The time course for the development of infection varied. It was most rapid with G. mosseae, but it was ultimately higher with G. intraradices. None of the treatments significantly affected the lengths of adventitious roots or the first-, second- or third-order laterals that developed from them. Arbuscular mycorrhizal colonization increased the intensity of branching in all root orders with the effect being most obvious on first-order lateral roots where the number of branches increased from under 100 to over 300 brances m(-1). As a result, although first-order laterals made up 55% of the root systems of control plants, the comparable value was 36% in AM-infected plants. In contrast, second-order laterals represented 25% of control root systems, but 50% of AM-colonized root systems. Glomus intraradices but not G. mosseae increased root diameter. Anatomical studies revealed no changes in the overall form of the root tip, although there were changes in the diameter of the root cap, cell numbers and cell size. Hymenoscyphus ericae increased the duration of the metaphase index. Both AM fungal treatments increased the concentrations of soluble proteins in root extracts and modified the protein profiles by the elimination and addition of protein bands detected by PAGE analysis. We conclude that AM fungal inoculation influenced processes in the root system at different levels, but not all effects were due to improved P nutrition or increased physiological age.

Vesicular-arbuscular mycorrhizae in relation to plant disease.

Vesicular-arbuscular mycorrhizae (VAM) enhance plant growth through increased nutrient uptake, stress tolerance and disease resistance. As an integral part of the root system, they interact with other microorganisms in soil and result in increased root exudation approaching about 25% of the plant dry matter production. Roots support a multitude of microorganisms that, in concert, can have profound influence on growth and survival of the plant. VAM fungi can alter the root exudation pattern, enhance chitinolytic activity and alter photosynthetic/respiratory deficiencies. VAM-positive plants are known to exhibit varied resistance towards soil-borne and foliar pathogens. The known interactions include a number of mechanisms, such as exclusion of the pathogen, lignification of plant cell walls, changed phosphate nutrition resulting in altered exudation by roots, and formation of inhibitory low molecular weight compounds. The purpose of this review is to discuss VAM-plant-pathogen interactions and the possible mechanisms involved in altered resistance. Based on these observations, a working model is proposed to explain the VAM-disease interaction under varied environmental conditions.

Detection of PR-b proteins in tobacco roots infected with Chalara elegans.

All previous studies on pathogenesis-related (b) protein (PR-b) induction in tobacco have been carried out on leaves or callus tissue. This paper reports the production of PR-b proteins also in roots of tobacco plants (Nicotiana tabacum cv. Xanthi NC) infected with Chalara elegans. Antiserum against PR-b1 reacted with PR-b1, PR-b2 and PR-b3 and gave the same pattern of reaction as for leaves. Antiserum against PR-b5 revealed the presence of PR-b4, PR-b5 and, very weakly, PR-b6 which have been shown to be beta-1,3 glucanases. Antiserum against PR-b7 reacted with both PR-b7 and PR-b8 which are chitinases.

Electrophoretic and immunological studies on acid phosphatase from a mycorrhizal fungus of Erica hispidula L.

Wall-bound acid phosphatases of an endomycorrhizal fungus of Erica hispidula L. were separated by PAGE, electrotransferred to nitrocellulose membranes and glycoprotein characteristics studied using Concanavalin A. An antiserum was raised against the major high-molecular-weight acid phosphatase and its specificity demonstrated by immunoblotting and ELISA. The antiserum reacted very little with protein extracts of other cellular fractions and was unable to bind to wall-bound protein antigens of a mycorrhizal isolate from K. mauritanica. Ultrastructural localization of acid phosphatase by cytoenzymology and indirect immunogold labelling showed it to be almost exclusively associated with the fungal wall and septa of living hyphae, whilst in senescent hyphae it was also present within the disorganized cytoplasm.

DEPRESSED METABOLIC ACTIVITY OF VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI AFTER FUNGICIDE APPLICATIONS.

Application to soil of either benomyl or captan significantly decreased the growth of vesicular-arbuscular mycorrhizal (VAM) onion (Allium cepa L. cv. Hyper) plants 4 weeks after treatment but non-VAM plants were not affected. Fungal colonization of the onion roots, as indicated by non-vital staining with chlorozole black E, was depressed 2 weeks after fungicide application. However, decreases in metabolically active VAM fungal tissue, revealed by a succinate dehydrogenase assay, could be detected as soon as 3 d after fungicide treatment. There was little difference between the fungicides in their effect on the VAM fungi used. The usefulness of the succinate dehydrogenase assay in predicting effects of fungicide is discussed.