Bacterial associations with the mycorrhizosphere and hyphosphere of the arbuscular mycorrhizal fungus Glomus mosseae

Roots and mycorrhizal fungi may not associate with soil bacteria randomly, but rather in a hierarchical structure of mutual preferences. Elucidation of such structures would facilitate the management of the soil biota to enhance the stability of the plant-soil system. We conducted an experiment utilizing two isolates of soil bacteria to determine their persistence in distinct mycorrhizal regions of the root zone, and their effects on general rhizosphere populations of fluorescent pseudomonads (FP). Split-root sorghum (Sorghum bicolor L.) plants were grown in four-compartment containers, constructed so that the soils in individual compartments held either (1) roots colonized by the arbuscular-mycorrhizal (AM) fungus Glomus mosseae (M), (2) nonAM roots only (R), (3) hyphae of G. mosseae (H), or (4) no mycorrhizal structures (S). The soils were inoculated (10⁷ cells g^-1 dry soil) with antibiotic-resistant (rifampicin, rif; streptomycin, sm) strains of the soil bacteria, Alcaligenes eutrophus (rif^r50) or Arthrobacter globiformis (sm^r250), or were left uninoculated as control. A. eutrophus had been isolated from a specific source (hyphosphere soil of G. mosseae), and A. globiformis from mycorrhizosphere soils of two AM fungi. After 10 wk of growth, the presence of A. eutrophus was barely detectable (<10 cfu g^-1 dry soil) in nonAM (R and S) soils, but persisted well (10⁴ cfu g^-1 dry soil) in AM (H and M) soils. Numbers of A. globiformis were more evenly distributed between all soils, but were highest in the presence of AM roots (M soil). There were varied bacterial effects on root and AM-hyphal development: A. eutrophus decreased hyphal length in H soil, while A. globiformis stimulated root length in M soil. The two bacterial inoculants did not affect numbers of FP in H, R, and M soils, but the AM status of the soils did: the numbers of FP increased in the order M>R>H>S. There was a positive correlation of FP numbers with both bacterial inoculants in M and H soils. Numbers of FP changed with root or hyphal lengths, an effect that was related to changes in the numbers of the inoculated bacteria. The results indicate that the hyphosphere-specific A. eutrophus depended on the presence of G. mosseae, but that the nonspecific A. globiformis did not. The mycorrhizal status of soils may selectively influence persistence of bacterial inoculants as well as affecting the numbers of other native bacteria.     

Lack of congruence between morphometric evolution and genetic differentiation suggests a recent dispersal and local habitat adaptation of the Madeiran lizard Lacerta dugesii

Genetic differentiation among nine populations of the endemic lizard Lacerta dugesii Milne-Edwards 1829 (Lacertidae) from four groups of islands constituting the Archipelago of Madeira, was investigated by protein electrophoresis at 23 enzyme loci. Among twenty polymorphic loci, the total genetic diversity was due primarily to intra-population variation. The allele and genotypic frequencies among populations showed some heterogeneity, allowing the species to present a structuring pattern compatible with their geographical clustering. Some evidence suggests that selection acting on some loci in different ecological conditions may be responsible for the clustering of the populations studied. There was no apparent isolation effect expected under an "island" model of population divergence, and no correlation was found between genetic and geographic distances among populations. Morphological variation of the proposed three L. dugesii subspecies is not congruent with the allozyme analysis. This most probably suggests a rapid colonization of the islands followed by a strong effect of selection operating over the morphological characters used to define the subspecies.     

Endoproteolytic activities in pea roots inoculated with the arbuscular mycorrhizal fungus Glomus mosseae and/or Aphanomyces euteiches in relation to bioprotection

Arbuscular mycorrhizal (AM) symbioses are known to play a role in increased resistance of plants against soilborne pathogens. Mechanisms involved in this phenomenon are not yet well understood. This work investigates possible roles of endoproteolytic activities in bioprotection of Pisum sativum roots by Glomus mosseae against Aphanomyces euteiches . First, it is demonstrated that bioprotection occurs only in pre-mycorrhizal plants. Second, endoproteolytic activities were analysed qualitatively and quantitatively during AM symbiosis, in plants infected with either zoospores or mycelium of A. euteiches , and in mycorrhizal plants infected with the pathogen. In mycorrhizal symbiosis a progressive increase in endoproteolytic activities was observed following root colonization by G. mosseae . By contrast, in roots inoculated with A. euteiches , a drastic increase in endoproteolytic activities was observed which was correlated with the amount of pathogen occurring in roots. Qualitative differences were seen among the endoproteolytic activities detected in roots inoculated with zoospores or mycelium. The constitutive as well as mycorrhizal and pathogen-induced activities were further characterized as 'trypsin-like' serine endoproteases. Interestingly, in a situation of bioprotection, only low levels of the activities normally associated with the infection by A. euteiches were detected, suggesting that the synthesis of these proteins is directly linked to the growth or virulence of the pathogen.     

Clonal diversity and population genetic structure of arbuscular mycorrhizal fungi (Glomus spp.) studied by multilocus genotyping of single spores

A nested multiplex PCR (polymerase chain reaction) approach was used for multilocus genotyping of arbuscular mycorrhizal fungal populations. This method allowed us to amplify multiple loci from Glomus single spores in a single PCR amplification. Variable introns in the two protein coding genes GmFOX2 and GmTOR2 were applied as codominant genetic markers together with the LSU rDNA. Genetic structure of Glomus spp. populations from an organically and a conventionally cultured field were compared by hierarchical sampling of spores from four plots in each field. Multilocus genotypes were characterized by SSCP (single stranded conformation polymorphism) and sequencing. All spore genotypes were unique suggesting that no recombination was taking place in the populations. There were no overall differences in the distribution of genotypes in the two fields and identical genotypes could be sampled from both fields. Analysis of gene diversity indicated that Glomus populations are subdivided between plots within each field. There were however, no subdivision between the fields.     

Low genetic diversity and strong but shallow population differentiation suggests genetic homogenization by metapopulation dynamics in a social spider

Mating systems and population dynamics influence genetic diversity and structure. Species that experience inbreeding and limited gene flow are expected to evolve isolated, divergent genetic lineages. Metapopulation dynamics with frequent extinctions and colonizations may, on the other hand, deplete and homogenize genetic variation, if extinction rate is sufficiently high compared to the effect of drift in local demes. We investigated these theoretical predictions empirically in social spiders that are highly inbred. Social spiders show intranest mating, female‐biased sex ratio, and frequent extinction and colonization events, factors that deplete genetic diversity within nests and populations and limit gene flow. We characterized population genetic structure in Stegodyphus sarasinorum, a social spider distributed across the Indian subcontinent. Species‐wide genetic diversity was estimated over approximately 2800 km from Sri Lanka to Himalayas, by sequencing 16 protein‐coding nuclear loci. We found 13 SNPs in 6592 bp (π = 0.00045) indicating low species‐wide nucleotide diversity. Three genetic lineages were strongly differentiated; however, only one fixed difference among them suggests recent divergence. This is consistent with a scenario of metapopulation dynamics that homogenizes genetic diversity across the species' range. Ultimately, low standing genetic variation may hamper a species' ability to track environmental change and render social inbreeding spiders ‘evolutionary dead‐ends’.     

Expression studies of plant genes differentially expressed in leaf and root tissues of tomato colonised by the arbuscular mycorrhizal fungus Glomus mosseae

Arbuscular mycorrhizal (AM) fungi are a multifaceted group of mutualistic symbionts that are common to terrestrial ecosystems. The interaction between AM fungi and plant roots is of environmental and agronomic importance. Understanding the molecular changes within the host plant upon AM fungal colonisation is a pre-requisite to a greater understanding of the mechanisms underlying the interaction. Differential mRNA display was conducted on leaf tissue of tomato plants colonised and non-colonised by the AM fungus Glomus mosseae and five putative differentially regulated cDNAs were identified. All cDNAs isolated shared high sequence similarity to known plant genes. Differential screening was initially used to establish whether the cDNAs were differentially expressed. Semi-quantitative RT-PCR was used to establish gene expression patterns for all five clones within leaf and root tissue of mycorrhizal and non-mycorrhizal colonised tomato plants. Differential regulation was observed for all five cDNAs. Down-regulation within the leaf tissue of mycorrhizal plants was observed for 4 out of the 5 cDNAs with an up-regulation observed only for one. Tissue specific regulation was observed for several cDNAs, with down-regulation observed in mycorrhizal leaf tissue and up-regulation observed within mycorrhizal root tissue as compared to non-mycorrhizal tissue.     

Early developmentally regulated genes in the arbuscular mycorrhizal fungus Glomus mosseae: identification of GmGIN1, a novel gene with homology to the C-terminus of metazoan hedgehog proteins

The life cycle of the obligate biotrophic arbuscular mycorrhizal fungi comprises several well-defined developmental stages whose genetic determinants are still unknown. With the aim of understanding the molecular processes governing the early developmental phase of the AM fungal life cycle, a subtractive cDNA library was constructed using a suppressive subtractive hybridization technique. The library contains more than 600 clones with an average size of 500 bp. The isolated cDNAs correspond to genes up-regulated during the early development of the AM fungus Glomus mosseaeversus genes expressed in extraradical hyphae. The expression of several of the isolated genes was further confirmed by RT-PCR analysis. Among the isolated clones, a novel gene named GmGIN1 only expressed during early development in G. mosseae was found. The full-length GmGIN1 cDNA codes for a protein of 429 amino acids. The most interesting feature of the deduced protein is its two-domain structure with a putative self-splicing activity. The N-terminal domain shares sequence similarity with a novel family of GTP binding proteins while the C-terminus has a striking homology to the C-terminal part of the hedgehog protein family from metazoa. The C-terminal part of hedgehog proteins is known to participate in the covalent modification of the N-terminus by cholesterol, and in the self-splicing activity which renders the active form of the protein with signalling function. We speculate that the N-terminal part of GmGIN1, activated through a similar mechanism to the hedgehog proteins, has GTP-binding activity and participates in the signalling events prior to symbiosis formation.     

Growth and sporulation of the arbuscular mycorrhizal fungus Glomus caledonium in dual culture with transformed carrot roots

Glomus caledonium was established in a dual culture with Ri T-DNA-transformed carrot roots. A modification of the minimal M medium buffered at pH 6.50 with 10 mM MES and solidified with 0.4% unpurified gellan gum allowed spore germination and formation of the symbiosis, together with the development of an extensive extramatrical mycelium and sporulation. Spore production increased with culture generation and most spores were viable. These spores colonized carrot roots and completed the fungal life cycle. In many cultures, sporulation was accompanied by the formation of arbuscule-like structures on short and thickened lateral branches of main hyphae. Root colonization was of the Paris-type with hyphae spreading intracellularly. Most colonized root cells contained coils of thickened hyphae, sometimes surrounded by fine hyphae, but no typical arbuscules were observed. Accepted: 26 January 2000     

Root colonization by the arbuscular mycorrhizal fungus Glomus mosseae and enhanced phosphorous levels in cucumber do not affect host acceptance and development of Frankliniella occidentalis

Abstract

We tested the effect of root colonization of cucumber (Cucumis sativus L.) by the arbuscular mycorrhizal fungus (AMF) Glomus mosseae on different parameters of a plant-thrips (Frankliniella occidentalis Pergande) interaction. In leaf disc bioassays, the feeding activity, the oviposition rate, the settling preference of adult females and the developmental time (first instar larva to adult) on leaves of mycorrhizal and non-mycorrhizal plants were studied. To distinguish between a nutritional effect through an improved phosphorous (P) status of the mycorrhizal plant and other effects caused by mycorrhization, non-mycorrhizal plants watered with a nutrient solution with (+P) or without P (?P) were included in the study. Mycorrhization did not affect any of the parameters on host acceptance tested, whereas on plants with a higher P-level the duration of the non-feeding stages (pronymphae, nymphae) of F. occidentalis was shortened, but all other developmental parameters were similar as in the control and the mycorrhizal plants. Our data indicate that the polyphagous thrips F. occidentalis is neither affected by mycorrhization of cucumber plants nor by enhanced P-levels.     

Effect of elevated atmospheric CO2 on mycorrhizal colonization, external mycorrhizal hyphal production and phosphorus inflow in Plantago lanceolata and Trifolium repens in association with the arbuscular mycorrhizal fungus Glomus mosseae

Plantago lanceolata and Trifolium repens were grown under ambient (400 μmol mol^–1) and elevated (650 μmol mol^–1) atmospheric CO₂ conditions. Plants were inoculated with the arbuscular mycorrhizal fungus Glomus mosseae and given a phosphorus supply in the form of bonemeal. Six sequential harvests were taken in order to determine whether the effect of elevated CO₂ on internal mycorrhizal colonization and external hyphal production was independent of the stimulatory effect of elevated CO₂ on plant growth. At a given time, elevated CO₂ increased the percentage of root length colonized (RLC), the total length of colonized root and the external mycorrhizal hyphal (EMH) density and decreased the ratio of EMH to total length of colonized root. When plant size was taken into account, the CO₂ effect on RLC and total length of colonized root was greatly reduced (and only apparent for early harvests in T. repens) and the effects on the EMH parameters disappeared. Root tissue P concentration was unchanged at elevated CO₂, but there was a decrease in shoot P at the later harvests. There was no direct effect of elevated CO₂ on P inflow for the earlier period (< 50 d) of the experiment. However, over the last period, there was a significant negative effect of elevated CO₂ on P inflow for both species, independent of plant size. It is concluded that elevated CO₂ had no direct effect on mycorrhizal colonization or external hyphal production, and that any observed effects on a time basis were due to faster growing plants at elevated CO₂. However, for older plants, elevated CO₂ had a direct negative effect on P inflow. This decrease in P inflow coincides with the observed decrease in shoot P concentration. This is discussed in terms of downregulation of photosynthesis often seen in elevated CO₂ grown plants, and the potential for mycorrhizas (via external hyphal turnover) to alleviate the phenomenon. The direction for future research is highlighted, especially in relation to carbon flow to and storage in the soil.     

Genetic diversity and host plant preferences revealed by simple sequence repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi (AMF) are important symbionts of plants that improve plant nutrient acquisition and promote plant diversity. Although within-species genetic differences among AMF have been shown to differentially affect plant growth, very little is actually known about the degree of genetic diversity in AMF populations. This is largely because of difficulties in isolation and cultivation of the fungi in a clean system allowing reliable genotyping to be performed. A population of the arbuscular mycorrhizal fungus Glomus intraradices growing in an in vitro cultivation system was studied using newly developed simple sequence repeat (SSR), nuclear gene intron and mitochondrial ribosomal gene intron markers. The markers revealed a strong differentiation at the nuclear and mitochondrial level among isolates. Genotypes were nonrandomly distributed among four plots showing genetic subdivisions in the field. Meanwhile, identical genotypes were found in geographically distant locations. AMF genotypes showed significant preferences to different host plant species (Glycine max, Helianthus annuus and Allium porrum) used before the fungal in vitro culture establishment. Host plants in a field could provide a heterogeneous environment favouring certain genotypes. Such preferences may partly explain within-population patterns of genetic diversity.     

A comparison of wild-type, old and modern tomato cultivars in the interaction with the arbuscular mycorrhizal fungus Glomus mosseae and the tomato pathogen Fusarium oxysporum f. sp. lycopersici

The effect of the arbuscular mycorrhizal symbiosis (AM) varies in plant cultivars. In the present study, we tested whether wild-type, old and modern tomato cultivars differ in the parameters of the AM interaction. Moreover, the bioprotective effect of AM against the soilborne tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol) was tested in the different cultivars. Ten tomato cultivars were inoculated with the arbuscular mycorrhizal fungus (AMF) Glomus mosseae alone or in combination with Fol. At the end of the experiment, AM root colonization, Fusarium infection, and the plant fresh weight was determined. The tomato cultivars differed in their susceptibility to AMF and Fol, but these differences were not cultivar age dependent. In all the cultivars affected by Fol, mycorrhization showed a bioprotective effect. Independent of the cultivar age, tomato cultivars differ in their susceptibility to AMF and Fol and the bioprotective effect of mycorrhization, indicating that the cultivar age does not affect the AM parameters tested in this study.     

The distribution of the ancestral haplotype in finite stepping-stone models with population expansion

Recent extensive analyses of human DNA polymorphism reveal that the ancestral haplotype at various genetic loci occurs almost exclusively in African samples. We develop a coalescence-based simulation method in stepping-stone models with population expansion and examine the probability (P(A)) that the ancestral haplotype is found in African samples and the probability (Q(A)) that the most recent common ancestor of sampled genes occurs in Africa. These probabilities and other summary statistics are used to infer the human demographic history. It is shown that the high observed P(A) value cannot be explained simply by sampling bias. Rather, it suggests that the African population has been more strongly subdivided and isolated from each other than the non-African population and that there must have been some African populations which were not directly involved in the Out-of-Africa expansion in the late Pleistocene.     

Chitin synthase genes in the arbuscular mycorrhizal fungus Glomus versiforme: full sequence of a gene encoding a class IV chitin synthase

Chitin synthase genes of the arbuscular mycorrhizal fungus Glomus versiforme were sought in an investigation of the molecular basis of fungal growth. Three DNA fragments (Gvchs1, Gvchs2 and Gvchs3) corresponding to the conserved regions of distinct chitin synthase (chs) genes were amplified by means of the polymerase chain reaction (PCR) with two sets of degenerate primers. Gvchs1 and Gvchs2 encode two class I chitin synthases, whereas Gvchs3 encodes a class IV chitin synthase. A genomic library was used to obtain the Gvchs3 complete gene (1194 amino acids), which shows a very close similarity to the class IV chitin synthase from Neurospora crassa.     

Lack of genetic and plumage differentiation in the red-billed quelea Quelea quelea across a migratory divide in southern Africa

A migratory divide usually signals the presence of a geographical region over which other traits, such as morphology and genotypes, also undergo rapid change. A migratory divide has been hypothesized in central southern Africa for the abundant migratory weaver, the red-billed quelea Quelea quelea. The positioning of the divide in the region is based on the patterns of rainfall in the region that stimulate the annual migrations of queleas. Evidence indicates that premigratory queleas near the divide show two distinct preferred directions for migration. We used eight polymorphic microsatellite loci and a range of plumage characters to determine whether there was population structure among red-billed queleas in southern Africa, and specifically whether this structure coincided with the location of the migratory divide. There was no evidence of population genetic structure. An amova revealed no significant differences between samples taken either side of the migratory divide. Similarly, there was no geographical variation in plumage patterns across southern Africa. For both microsatellites and plumage characteristics, the variation that does exist occurs within each sampled site, with little differentiation between sites. We were therefore unable to find any evidence that either plumage or microsatellite genotypes varied in a similar way to migratory direction preference in red-billed queleas in southern Africa. This is perhaps because the migratory divide does not act to separate individuals into populations within which genetic and plumage differentiation can be maintained.     

Understanding the recent colonization history of a plant pathogenic fungus using population genetic tools and Approximate Bayesian Computation

Understanding the processes by which new diseases are introduced in previously healthy areas is of major interest in elaborating prevention and management policies, as well as in understanding the dynamics of pathogen diversity at large spatial scale. In this study, we aimed to decipher the dispersal processes that have led to the emergence of the plant pathogenic fungus Microcyclus ulei, which is responsible for the South American Leaf Blight (SALB). This fungus has devastated rubber tree plantations across Latin America since the beginning of the twentieth century. As only imprecise historical information is available, the study of population evolutionary history based on population genetics appeared most appropriate. The distribution of genetic diversity in a continental sampling of four countries (Brazil, Ecuador, Guatemala and French Guiana) was studied using a set of 16 microsatellite markers developed specifically for this purpose. A very strong genetic structure was found (F_st=0.70), demonstrating that there has been no regular gene flow between Latin American M. ulei populations. Strong bottlenecks probably occurred at the foundation of each population. The most likely scenario of colonization identified by the Approximate Bayesian Computation (ABC) method implemented in 𝒟ℐ𝒴𝒜ℬ𝒞 suggested two independent sources from the Amazonian endemic area. The Brazilian, Ecuadorian and Guatemalan populations might stem from serial introductions through human-mediated movement of infected plant material from an unsampled source population, whereas the French Guiana population seems to have arisen from an independent colonization event through spore dispersal.     

The plant growth‐promoting fungus Fusarium equiseti and the arbuscular mycorrhizal fungus Glomus mosseae stimulate plant growth and reduce severity of anthracnose and damping‐off diseases in cucumber (Cucumis sativus) seedlings

To alleviate the environmental contamination due to persistent chemical usage, approaches to integrated pest management were conceived. In this perspective, microbe–microbe interactions such as mycorrhizal relationships with other soil microbiota in the rhizosphere like the plant growth‐promoting fungi (PGPF) are particularly important. Better understanding of the interactions between beneficial microbial groups is imperative in the identification of possible synergistic or antagonistic effects to improve their practical usage as biocontrol agents or biofertilizers. In this study, the consequence of co‐inoculation of the arbuscular mycorrhizal fungus (AMF) Glomus mosseae (Gm) and the PGPF Fusarium equiseti (isolates GF18‐3 and GF19‐1) in terms of plant growth enhancement, root and rhizosphere colonisation, and development of anthracnose (Colletotrichum orbiculare) and damping‐off (Rhizoctonia solani AG‐4) diseases in cucumber plants was investigated under controlled conditions. The amendment of either GF18‐3 or GF19‐1 singly or in combination with Gm indicated a general tendency to significantly enhance the shoot dry weight (SDW) of cucumber plants at 4 weeks after planting (WAP). Similarly, Gm alone significantly enhanced SDW at 4 WAP. Gm showed a tendency to depress root colonisation by F. equiseti but such antagonistic effect was not observed in the rhizosphere soil. Both GF18‐3 and GF19‐1 significantly reduced percent root colonisation of Gm. However, these general tendencies may vary with the inoculum densities of AMF and PGPF. Both F. equiseti and Gm inoculated singly significantly increased percent of protection against anthracnose, but the combined inoculation was more effective in controlling the disease compared to single inoculation. The inoculation of the cucumber seedlings with GF18‐3, GF19‐1 or Gm, 6 or 12 days prior to damping‐off pathogen inoculation, increased percent of protection against damping‐off disease. This study shows that the co‐inoculation of F. equiseti and Gm resulted in additive effect on the suppression of anthracnose disease in cucumber.