Mucoid mutants of the biocontrol strain pseudomonas fluorescens CHA0 show increased ability in biofilm formation on mycorrhizal and nonmycorrhizal carrot roots

Extracellular polysaccharides play an important role in the formation of bacterial biofilms. We tested the biofilm-forming ability of two mutant strains with increased production of acidic extracellular polysaccharides compared with the wild-type biocontrol strain Pseudomonas fluorescens CHA0. The anchoring of bacteria to axenic nonmycorrhizal and mycorrhizal roots as well as on extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices was investigated. The nonmucoid wild-type strain P. fluorescens CHA0 adhered very little on all surfaces, whereas both mucoid strains formed a dense and patchy bacterial layer on the roots and fungal structures. Increased adhesive properties of plant-growth-promoting bacteria may lead to more stable interactions in mixed inocula and the rhizosphere.     

Specific interactions between arbuscular mycorrhizal fungi and plant growth-promoting bacteria: as revealed by different combinations

The interactions between two plant growth-promoting rhizobacteria (PGPR, Pseudomonas fluorescens SBW25 and Paenibacillus brasilensis PB177), two arbuscular mycorrhizal (AM) fungi (Glomus mosseae and Glomus intraradices) and one pathogenic fungus (Microdochium nivale) were investigated on winter wheat (Triticum aestivum cultivar Tarso) in a greenhouse trial. PB177, but not SBW25, had strong inhibitory effects on M. nivale in dual culture plate assays. The results from the greenhouse experiment show very specific interactions; for example, the two AM fungi react differently when interacting with the same bacteria on plants. Glomus intraradices (single inoculation or together with SBW25) increased plant dry weight on M. nivale-infested plants, suggesting that the pathogenic fungus is counteracted by G. intraradices, but PB177 inhibited this positive effect. This is an example of two completely different reactions between the same AM fungus and two species of bacteria, previously known to enhance plant growth and inhibit pathogens. When searching for plant growth-promoting microorganisms, it is therefore important to test for the most suitable combination of plant, bacteria and fungi in order to achieve satisfactory plant growth benefits.     

Influence of an arbuscular mycorrhizal fungus on Pseudomonas fluorescens DF57 in rhizosphere and hyphosphere soil

The influence of Glomus intraradices (BEG87) on Pseudomonas fluorescens DF57 in hyphosphere and rhizosphere soil was examined. Cucumis sativus (Aminex, F₁ hybrid) was grown in symbiosis with the arbuscular mycorrhizal fungus G. intraradices in PVC tubes, consisting of a central root compartment and two lateral root-free compartments. Two Tn 5 - lux AB-marked strains of P. fluorescens DF57 were used. Strain DF57-P2, which has an insertion of Tn 5::lux AB in a phosphate starvation-inducible locus, was used as a phosphate starvation reporter. Another lux -tagged strain DF57-40E7, which carries a constitutively expressed lux AB fusion, was used as control for strain DF57-P2 and for measuring the metabolic activity of P. fluorescens DF57. A strain of P. fluorescens DF57, which carries a constitutively expressed gfp gene, was used in studies of attachment between the bacteria and the hyphae. G. intraradices decreased the culturability of P. fluorescens DF57 significantly, both in rhizosphere and hyphosphere soil, whereas the total number of P. fluorescens DF57 measured by immunofluorescence microscopy was decreased in hyphosphere soil only. G. intraradices did not induce a phosphorus starvation response in P. fluorescens DF57, and the metabolic activity of the bacteria was not affected by the fungus after 48 h. P. fluorescens DF57 did not attach to G. intraradices hyphae and was not able to use the hyphae as carbon substrate. The negative effect of G. intraradices on culturability and on number of P. fluorescens DF57 in hyphosphere soil is discussed.     

Evidence for specificity of cultivable bacteria associated with arbuscular mycorrhizal fungal spores

Bacteria associated with arbuscular mycorrhizal (AM) fungal spores may play functional roles in interactions between AM fungi, plant hosts and defence against plant pathogens. To study AM fungal spore-associated bacteria (AMB) with regard to diversity, source effects (AM fungal species, plant host) and antagonistic properties, we isolated AMB from surface-decontaminated spores of Glomus intraradices and Glomus mosseae extracted from field rhizospheres of Festuca ovina and Leucanthemum vulgare. Analysis of 385 AMB was carried out by fatty acid methyl ester (FAME) profile analysis, and some also identified using 16S rRNA gene sequence analysis. The AMB were tested for capacity to inhibit growth in vitro of Rhizoctonia solani and production of fluorescent siderophores. Half of the AMB isolates could be identified to species (similarity index 0.6) within 16 genera and 36 species. AMB were most abundant in the genera Arthrobacter and Pseudomonas and in a cluster of unidentified isolates related to Stenotrophomonas. The AMB composition was affected by AM fungal species and to some extent by plant species. The occurrence of antagonistic isolates depended on AM fungal species, but not plant host, and originated from G. intraradices spores. AM fungal spores appear to host certain sets of AMB, of which some can contribute to resistance by AM fungi against plant pathogens.     

Functional diversity of arbuscular mycorrhizal fungal isolates in relation to extraradical mycelial networks

We investigated the functional significance of extraradical mycorrhizal networks produced by geographically different isolates of the arbuscular mycorrhizal fungal (AMF) species Glomus mosseae and Glomus intraradices. A two-dimensional experimental system was used to visualize and quantify intact extraradical mycelium (ERM) spreading from Medicago sativa roots. Growth, phosphorus (P) and nitrogen (N) nutrition were assessed in M. sativa plants grown in microcosms. The AMF isolates were characterized by differences in extent and interconnectedness of ERM. Phenotypic fungal variables, such as total hyphal length, hyphal density, hyphal length per mm of total or colonized root length, were positively correlated with M. sativa growth response variables, such as total shoot biomass and plant P content. The utilization of an experimental system in which size, growth rate, viability and interconnectedness of ERM extending from mycorrhizal roots are easily quantified under realistic conditions allows the simultaneous evaluation of different isolates and provides data with a predictive value for selection of efficient AMF.     

植物磷营养状况对丛枝菌根真菌生长及代谢活性的调控*

采用四室隔网培养装置,以玉米为宿主植物,通过在植物生长室设0、50、250和500 mgPkg-1 4个施磷水平,研究了植株体内的磷营养状况对AM真菌Glomus sinuosum和Glomus intraradices生长及活性的影响。研究发现在不施磷条件下,接种AM真菌G. intraradices显著促进了植物生长和磷的吸收;低磷条件(50 mgPkg-1)下,接种菌根真菌显著促进了植物对磷的吸收,但对植物生长没有明显的影响;而在高磷条件(250 mgPkg-1 和500 mgPkg-1)下,接种菌根真菌不但没有促进植物的生长和磷的吸收,反而对其有抑制作用。随着施磷水平的提高, AM真菌根内菌丝的碱性磷酸酶活性显著下降;与不施磷相比,低量(50 mgPkg-1)供磷增加了AM真菌土壤中根外菌丝的密度,高磷(250 mgPkg-1 和500 mgPkg-1)降低了土壤中根外菌丝的密度。上述结果说明:⑴ 给宿主植物施用磷肥引起的植物磷营养状况的改变,对AM真菌生长和代谢活性具有一定的调控作用;⑵ G. sinuosum和G. intraradices两种AM真菌的生长和代谢活性对施磷水平的响应程度无显著性差异;⑶ 高磷抑制AM真菌生长和代谢活性,使真菌吸磷量减少,可能是造成菌根效应降低的原因之一     

Different nitrogen sources modulate activity but not expression of glutamine synthetase in arbuscular mycorrhizal fungi

Glutamine synthetase (GS) is a central enzyme of nitrogen metabolism that allows assimilation of nitrogen and biosynthesis of glutamine. We isolated the cDNA encoding GS from two arbuscular mycorrhizal fungi, Glomus mosseae (GmGln1) and Glomus intraradices (GiGln1). The deduced protein orthologues have a high degree of similarity (92%) with each other as well as with GSs from other fungi. GmGln1 was constitutively expressed during all stages of the fungal life cycle, i.e., spore germination, intraradical and extraradical mycelium. Feeding experiments with different nitrogen sources did not induce any change in the mRNA level of both genes independent of the symbiotic status of the fungus. However, GS activity of extraradical hypahe in G. intraradices was considerably modulated in response to different nitrogen sources. Thus, in a N re-supplementation time-course experiment, GS activity responded quickly to addition of nitrate, ammonium or glutamine. Re-feeding with ammonium produced a general increase in GS activity when compared with hyphae grown in nitrate as a sole N source.     

Branched absorbing structures (BAS): a feature of the extraradical mycelium of symbiotic arbuscular mycorrhizal fungi

The present work describes the morphogenesis and cytological characteristics of 'branched absorbing structures' (BAS, formely named arbuscule-like structures, ALS), small groups of dichotomous hyphae formed by the extraradical mycelium of arbuscular mycorrhizal (AM) fungi. Monoxenic cultures of the AM fungus Glomus intraradices Smith & Schenck and tomato ( Lycopersicum esculentum Mill.) roots allowed the continuous, non-destructive study of BAS development. These structures were not observed in axenic cultures of the fungus under different nutritional conditions or in unsuccessful (asymbiotic) monoxenic cultures. However, extraradical mycelium of G. intraradices formed BAS immediately after fungal penetration of the host root and establishment of the symbiosis. The average BAS development time was 7 d under our culture conditions, after which they degenerated, becoming empty septate structures. Certain BAS were closely associated with spore formation, appearing at the spore's substending hypha. Branches of these spore-associated BAS (spore-BAS) usually formed spores. Electron microscopy studies revealed that BAS and arbuscules show several ultrastructural similarities. The possible role of BAS in nutrient uptake by the mycorrhizal plant is discussed.     

Extraradical hyphae of the mycorrhizal fungus Glomus intraradices can hydrolyse organic phosphate

Organic phosphorus sources make up a large fraction of the total P in some soils. Vesicular–arbuscular mycorrhizal fungi provide a large surface area for the absorption of inorganic P. The question of whether or not they have direct access to organic P by producing extracellular phosphatases has hitherto been controversial because experiments had not been performed in the absence of other soil microorganisms. We used a split-dish in vitro carrot mycorrhiza system free from contaminating microorganisms. The extraradical hyphae of Glomus intraradices hydrolysed both 5-bromo-4-chloro-3-indolyl phosphate and phenolphthalein diphosphate. Moreover, they transferred significantly more P to roots when they had access to inositol hexaphosphoric acid (phytate) than when they did not. Thus we show unequivocally that extraradical hyphae of G. intraradices can hydrolyse organic P, and, further, that the resultant inorganic P can be taken up and transported to host roots.     

Expressed genes in the extraradical hyphae of an arbuscular mycorrhizal fungus, Glomus intraradices, in the symbiotic phase

To collect extraradical hyphae of arbuscular mycorrhizal (AM) fungi for RNA isolation, a PVDF membrane was laid on the hyphal compartment of a two-compartment culture system of transformed carrot hairy roots and Glomus intraradices. Extraradical hyphae free from host tissue were easily collected, and their RNA was rapidly extracted with a modified acid guanidinium thiocyanate-phenol-chloroform method. A 3'-RACE (rapid amplification of cDNA ends) of a known gene indicated that this protocol enabled the isolation of mRNA molecules as small as 2.3 kb. The cDNA libraries of an AM fungus from the aseptic extraradical hyphae in a symbiotic state were constructed for the first time. Three-fourth of 150 ESTs (expressed sequence tags) indicated low or no similarities to known sequences from other organisms.     

Ultrastructural localization of heavy metals in the extraradical mycelium and spores of the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi, obligate symbionts of most plant species, are able to accumulate heavy metals, thereby, protecting plants from metal toxicity. In this study, the ultrastructural localization of Zn, Cu, and Cd in the extraradical mycelium and spores of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic cultures was investigated. Zinc, Cu, or Cd was applied to the extraradical mycelium to final concentrations of 7.5, 5.0, or 0.45 mmol/L, respectively. Samples were collected at time 0, 8 h, and 7 days after metal application and were prepared for rapid freezing and freeze substitution. Metal content in different subcellular locations (wall, cytoplasm, and vacuoles), both in hyphae and spores, was determined by energy-dispersive X-ray spectroscopy. In all treatments and fungal structures analysed, heavy metals accumulated mainly in the fungal cell wall and in the vacuoles, while minor changes in metal concentrations were detected in the cytoplasm. Incorporation of Zn into the fungus occurred during the first 8 h after metal addition with no subsequent accumulation. On the other hand, Cu steadily accumulated in the spore vacuoles over time, whereas Cd steadily accumulated in the hyphal vacuoles. These results suggest that binding of metals to the cell walls and compartmentalization in vacuoles may be essential mechanisms for metal detoxification.     

Identification of a novel genetically controlled step in mycorrhizal colonization: plant resistance to infection by fungal spores but not extra-radical hyphae

Vesicular arbuscular mycorrhizal fungi infect plants by means of both spores and vegetative hyphae at early stages of symbiosis. Using 2500 M2 fast-neutron-mutagenized seeds of the miniature tomato (Lycopersicon esculentum) cultivar, Micro-Tom, we isolated a mutant, M161, that is able to resist colonization in the presence of Glomus intraradices spores. The myc(-) phenotype of the mutant was stable for nine generations, and found to segregate as a single Mendelian recessive locus. The mutant exhibited morphological and growth-pattern characteristics similar to those of wild-type plants. Alterations of light intensity and day/night temperatures did not eliminate the myc(-) characteristic. Resistance to mycorrhizal fungal infection and colonization was also evident following inoculation with the fungi Glomus mosseae and Gigaspora margarita. Normal colonization of M161 was evident when mutant plants were grown together with arbuscular mycorrhizal-inoculated wild-type plants in the same growth medium. During evaluation of the pre-infection stages in the mutant rhizosphere, spore germination and appressoria formation of G. intraradices were lower by 45 and 70%, respectively, than the rates obtained with wild-type plants. These results reveal a novel, genetically controlled step in the arbuscular mycorrhizal colonization process, governed by at least one gene, which significantly reduces key steps in pre-mycorrhizal infection stages.     

Absence of carbon transfer between Medicago truncatula plants linked by a mycorrhizal network, demonstrated in an experimental microcosm

Carbon transfer between plants via a common extraradical network of arbuscular mycorrhizal (AM) fungal hyphae has been investigated abundantly, but the results remain equivocal. We studied the transfer of carbon through this fungal network, from a Medicago truncatula donor plant to a receiver (1) M. truncatula plant growing under decreased light conditions and (2) M. truncatula seedling. Autotrophic plants were grown in bicompartmented Petri plates, with their root systems physically separated, but linked by the extraradical network of Glomus intraradices. A control Myc-/Nod- M. truncatula plant was inserted in the same compartment as the receiver plant. Following labeling of the donor plant with 13CO2, 13C was recovered in the donor plant shoots and roots, in the extraradical mycelium and in the receiver plant roots. Fatty acid analysis of the receiver's roots further demonstrated 13C enrichment in the fungal-specific lipids, while almost no label was detected in the plant-specific compounds. We conclude that carbon was transferred from the donor to the receiver plant via the AM fungal network, but that the transferred carbon remained within the intraradical AM fungal structures of the receiver's root and was not transferred to the receiver's plant tissues.     

Phosphorus effects on metabolic processes in monoxenic arbuscular mycorrhiza cultures

The influence of external phosphorus (P) on carbon (C) allocation and metabolism as well as processes related to P metabolism was studied in monoxenic arbuscular mycorrhiza cultures of carrot (Daucus carota). Fungal hyphae of Glomus intraradices proliferated from the solid minimal medium containing the colonized roots into C-free liquid minimal medium with different P treatments. The fungus formed around three times higher biomass in P-free liquid medium than in medium with 2.5 mM inorganic P (high-P). Mycelium in the second experiment was harvested at an earlier growth stage to study metabolic processes when the mycelium was actively growing. P treatment influenced the root P content and [(13)C]glucose administered to the roots 7 d before harvest gave a negative correlation between root P content and (13)C enrichment in arbuscular mycorrhiza fungal storage lipids in the extraradical hyphae. Eighteen percent of the enriched (13)C in extraradical hyphae was recovered in the fatty acid 16:1omega5 from neutral lipids. Polyphosphate accumulated in hyphae even in P-free medium. No influence of P treatment on fungal acid phosphatase activity was observed, whereas the proportion of alkaline-phosphatase-active hyphae was highest in high-P medium. We demonstrated the presence of a motile tubular vacuolar system in G. intraradices. This system was rarely seen in hyphae subjected to the highest P treatment. We concluded that the direct responses of the extraradical hyphae to the P concentration in the medium are limited. The effects found in hyphae seemed instead to be related to increased availability of P to the host root.     

Influence of a Bacillus sp. on physiological activities of two arbuscular mycorrhizal fungi and on plant responses to PEG-induced drought stress

The effects of bacterial inoculation (Bacillus sp.) on the development and physiology of the symbiosis between lettuce and the arbuscular mycorrhizal (AM) fungi Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe and Glomus intraradices (Schenck and Smith) were investigated. Plant growth, mineral nutrition and gas-exchange values in response to bacterial inoculation after PEG-induced drought stress were also evaluated. In AM plants, inoculation with Bacillus sp. enhanced fungal development and metabolism, measured as succinate dehydrogenase (SDH) and alkaline phosphatase (ALP) activities, more than plant growth. Under non-stressed conditions, G. intraradices colonization increased all plant physiological values to a higher extent when in dual inoculation with the bacterium. Under stress conditions, the bacterium had an important stimulatory effect on G. intraradices development. Under such conditions, the effects of the bacterium on photosynthetic rate, water use efficiency (WUE) and stomatal conductance of lettuce plants differed with the fungus species. Plant-gas exchange was enhanced in G. intraradices- and reduced in G. mosseae-colonized plants when co-inoculated with Bacillus sp. Thus, the effects of each fungus on plant physiology were modulated by the bacterium. Stress was detrimental, particularly in G. intraradices-colonized plants without the bacterium, reducing intra and extraradical mycelium growth and vitality (SDH), as well as plant-gas exchange. Nevertheless, Bacillus sp. inoculation improved all these plant and fungal parameters to the same level as in non-stressed plants. The highest amount of alive and active AM mycelium for both fungi was obtained after co-inoculation with Bacillus sp. These results suggest that selected free-living bacteria and AM fungi should be co-inoculated to optimize the formation and functioning of the AM symbiosis in both normal and adverse environments.     

13C incorporation into signature fatty acids as an assay for carbon allocation in arbuscular mycorrhiza

The ubiquitous arbuscular mycorrhizal fungi consume significant amounts of plant assimilated C, but this C flow has been difficult to quantify. The neutral lipid fatty acid 16:1omega5 is a quantitative signature for most arbuscular mycorrhizal fungi in roots and soil. We measured carbon transfer from four plant species to the arbuscular mycorrhizal fungus Glomus intraradices by estimating (13)C enrichment of 16:1omega5 and compared it with (13)C enrichment of total root and mycelial C. Carbon allocation to mycelia was detected within 1 day in monoxenic arbuscular mycorrhizal root cultures labeled with [(13)C]glucose. The (13)C enrichment of neutral lipid fatty acid 16:1omega5 extracted from roots increased from 0.14% 1 day after labeling to 2.2% 7 days after labeling. The colonized roots usually were more enriched for (13)C in the arbuscular mycorrhizal fungal neutral lipid fatty acid 16:1omega5 than for the root specific neutral lipid fatty acid 18:2omega6,9. We labeled plant assimilates by using (13)CO(2) in whole-plant experiments. The extraradical mycelium often was more enriched for (13)C than was the intraradical mycelium, suggesting rapid translocation of carbon to and more active growth by the extraradical mycelium. Since there was a good correlation between (13)C enrichment in neutral lipid fatty acid 16:1omega5 and total (13)C in extraradical mycelia in different systems (r(2) = 0.94), we propose that the total amount of labeled C in intraradical and extraradical mycelium can be calculated from the (13)C enrichment of 16:1omega5. The method described enables evaluation of C flow from plants to arbuscular mycorrhizal fungi to be made without extraction, purification and identification of fungal mycelia.     

Competition and substrate colonization strategies of three polyxenically grown arbuscular mycorrhizal fungi

Intra- and extraradical colonization competition and hyphal interactions among arbuscular mycorrhizal fungi (AMF) Glomus intraradices, Glomus proliferum and Gigaspora margarita were investigated in two in vitro experimental systems. AMF were polyxenically cultured with a Ri T-DNA transformed carrot root organ culture (ROC) in either big Petri plates containing three culture compartments and a common hyphal compartment (i.e. an independent host root for each AMF) or two by two in the culture compartment of regular bicompartmented Petri dishes (i.e. a common host root and a common hyphal compartment). Maps of the extraradical mycelial development of the three AMF were obtained. Two distinct substrate colonization strategies (Glomus-type and Gigaspora-type) were identified, reflecting intrinsic differences among AMF genera/families. Our data reveal a general lack of antagonism between the isolates when extraradical hyphae explore and exploit the substrate outside the root influence zone; however certain growth restrictions were imposed by Gi. margarita extraradical mycelium when developing near the host root and by G. proliferum intraradical hyphae. This work highlights once more the appropriateness of AM in vitro culture systems to perform in vivo studies on the biology of this symbiosis and opens new avenues to the formulation of in vitro AMF inoculants.     

Identification of heavy metal-induced genes encoding glutathione S-transferases in the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi are able to alleviate the stress for plants caused by heavy metal contamination of soil. To analyze the molecular response of arbuscular mycorrhizal fungi to these pollutants, a subtractive cDNA library was constructed using RNA from Glomus intraradices extraradical hyphae of a root organ culture treated with a mixture of Cd, Zn, and Cu. Screening by reverse Northern blot analysis indicated that, among 308 clones, 17% correspond to genes up-regulated by heavy metals. Sequence analysis of part of the clones resulted, amongst others, in the identification of six genes putatively coding for glutathione S-transferases belonging to two different classes of these enzymes. Expression analyses indicated that the genes are differentially expressed during fungal development and that their RNA accumulation dramatically increases in extraradical hyphae grown in a heavy metal-containing solution.     

Trichoderma harzianum might impact phosphorus transport by arbuscular mycorrhizal fungi

Trichoderma sp. is a biocontrol agent active against plant pathogens via mechanisms such as mycoparasitism. Recently, it was demonstrated that Trichoderma harzianum was able to parasitize the mycelium of an arbuscular mycorrhizal (AM) fungus, thus affecting its viability. Here, we question whether this mycoparasitism may reduce the capacity of Glomus sp. to transport phosphorus ((33)P) to its host plant in an in vitro culture system. (33)P was measured in the plant and in the fungal mycelium in the presence/absence of T. harzianum. The viability and metabolic activity of the extraradical mycelium was measured via succinate dehydrogenase and alkaline phosphatase staining. Our study demonstrated an increased uptake of (33)P by the AM fungus in the presence of T. harzianum, possibly related to a stress reaction caused by mycoparasitism. In addition, the disruption of AM extraradical hyphae in the presence of T. harzianum affected the (33)P translocation within the AM fungal mycelium and consequently the transfer of (33)P to the host plant. The effects of T. harzianum on Glomus sp. may thus impact the growth and function of AM fungi and also indirectly plant performance by influencing the source-sink relationship between the two partners of the symbiosis.