Rhodotorulic acid enhances root colonization of tomato plants by arbuscular mycorrhizal (AM) fungi due to its stimulatory effect on the pre-symbiotic stages of the AM fungi

Exudates of Rhodotorula mucilaginosa, a yeast commonly found in the rhizosphere, increased hyphal length of the arbuscular mycorrhizal (AM) fungi Gigaspora rosea and Gigaspora margarita. Rhodotorulic acid (RA), a siderophore compound obtained from R. mucilaginosa exudates, increased hyphal length and branching. Thus, the increase in the number of entry points and the higher AM root colonization of tomato plants in the presence of RA can at least partially be explained by the positive effect of RA on the pre-symbiotic stages of the AM fungi.     

Glycosidation of apigenin results in a loss of its activity on different growth parameters of arbuscular mycorrhizal fungi from the genus Glomus and Gigaspora

The effect of different concentrations (0.5, 2 and 8 μM) of apigenin and its glycosidated form 5,7,4′-hydroxy flavone glycoside on arbuscular mycorrhizal (AM) fungal spore germination, hyphal growth, hyphal branching, the formation of entry points and root colonization of Gigaspora. rosea, Gi. margarita, Glomus mosseae and G. intraradices was tested. The lowest apigenin concentration (0.5 μM) nearly doubled hyphal branching, the formation of entry points and root colonization of all four tested fungi, whereas higher concentrations (2 and 8 μM) nearly doubled the hyphal growth of Gi. margarita, G. mosseae and G. intraradices. In none of the treatments with the apigenin-glycoside any effect on AM fungi could be observed. Our data show that apigenin exhibits an AM fungal genus and even species activity and we provide strong evidence that glycosidation results in a loss of its activity towards AM fungi.     

Mycorrhizae, biocides, and biocontrol 3. Effects of three different fungicides on developmental stages of three AM fungi

The effects of biocide use on nontarget organisms, such as arbuscular mycorrhizal (AM) fungi, are of interest to agriculture, since inhibition of beneficial organisms may counteract benefits derived from pest and disease control. Benomyl, pentachloronitrobenzene (PCNB) and captan were tested for their effects on the germination and early hyphal growth of the AM fungiGlomus etunicatum (Becker & Gerd.),Glomus mosseae (Nicol. & Gerd.). Gerd. and Trappe andGigaspora rosea (Nicol & Schenck) in a silty-clay loam soil placed in petri plates. Application of fungicides at 20 mg active ingredient (a.i) kg^?1 soil inhibited spore germination by all three AM-fungal isolates incubated on unsterilized soil for 2 weeks. However, fungicides applied at 10 mg a.i. kg^?1 soil had variable effects on AM-fungal isolates. Fungicide effects on germination and hyphal growth of G.etunicatum were modified by soil pasteurization and CO₂ concentration in petri plates and also by placing spores below the soil surface followed by fungicide drenches. Effects of fungicides on mycorrhiza formation and sporulation of AM fungi, and the resulting host-plant response, were evaluated in the same soil in associated pea (Pisum sativum L.) plants. Fungicides applied at 20 mg a.i. kg^?1 soil did not affect the root length colonized byG. etunicatum, but both benomyl and PCNB reduced sporulation by this fungus. Benomyl and PCNB reduced the root length colonized byG. rosea at 48 and 82 days after transplanting. PCNB also reducedG. mosseae-colonized root length at 48 and 82 days, but benomyl only affected root length colonized byG. mosseae at the earlier time point. Only PCNB reduced sporulation byG. mosseae, consistent with its effect on root length colonized by this fungus. captan reduced the root length colonized by G. rosea at 48 days, but not at 82 days, and reduced colonization byG. mosseae at 82 days, but not at 48 days. Captan did not affect sporulation by any of the fungi.G. rosea spore production was highly variable, but benomyl appeared to reduce sporulation by this fungus. Overall,G. etunicatum was the most tolerant to fungicides in association with pea plants in this soil, andG. rosea the most sensitive. Benomyl and PCNB were overall more toxic to these fungi than captan. Interactions of AM fungi and fungicides were highly variable and biological responses depended on fungus-fungicide combinations and on environmental conditions.     

Use of specific phospholipid fatty acids for identifying and quantifying the external hyphae of the arbuscular mycorrhizal fungus Gigaspora rosea

The external hypha of arbuscular mycorrhizal (AM) fungi, extending from roots out into soil, is an important structure in the uptake of phosphate from the depletion zone around each root. In this paper, we analysed some phospholipid fatty acids (PLFAs) derived from external hyphae of four AM fungi (Glomus etunicatum, Glomus clarum, Gigaspora margarita and Gigaspora rosea) to find fatty acids which may be useful as specific markers for identifying and quantify the external hyphae of Gigaspora species. Leek (Allium porrum L.) seedlings inoculated with each AM fungus were grown in river sand. Sand samples were collected and four PLFAs (16:1ω5, 18:1ω9, 20:1ω9 and 20:4) in the sand were analysed. In addition, the hyphal biomass in the sand was determined by the direct microscopic method. PLFAs 18:1ω9 and 20:4 were found in all the AM-inoculated and non-inoculated sand samples. PLFA 16:1ω5 was detected in the sand inoculated with G. etunicatum, G. clarum and Gi. rosea. PLFA 20:1ω9 was detected only in the sand inoculated with Gi. rosea. PLFAs 16:1ω5 and 20:1ω9 were not found in the sand inoculated with Gi. margarita. The amount of PLFA 20:1ω9 was closely correlated with the amount of biomass of external hyphae of Gi. rosea (r=0.937, P<0.001), whereas no correlation was observed for PLFA 16:1ω5. The 20:1ω9 content of Gi. rosea was approximately 6.56 nmol mg⁻¹ hyphal biomass. We suggest that PLFA 20:1ω9 can be used as a specific marker for identifying and quantifying the external hyphae of Gi. rosea, at least in controlled experimental systems.     

Mycorrhizae, biocides, and biocontrol 3. Effects of three different fungicides on developmental stages of three AM fungi

The effects of biocide use on nontarget organisms, such as arbuscular mycorrhizal (AM) fungi, are of interest to agriculture, since inhibition of beneficial organisms may counteract benefits derived from pest and disease control. Benomyl, pentachloronitrobenzene (PCNB) and captan were tested for their effects on the germination and early hyphal growth of the AM fungiGlomus etunicatum (Becker & Gerd.),Glomus mosseae (Nicol. & Gerd.). Gerd. and Trappe andGigaspora rosea (Nicol & Schenck) in a silty-clay loam soil placed in petri plates. Application of fungicides at 20 mg active ingredient (a.i) kg^–1 soil inhibited spore germination by all three AM-fungal isolates incubated on unsterilized soil for 2 weeks. However, fungicides applied at 10 mg a.i. kg^–1 soil had variable effects on AM-fungal isolates. Fungicide effects on germination and hyphal growth of G.etunicatum were modified by soil pasteurization and CO₂ concentration in petri plates and also by placing spores below the soil surface followed by fungicide drenches. Effects of fungicides on mycorrhiza formation and sporulation of AM fungi, and the resulting host-plant response, were evaluated in the same soil in associated pea (Pisum sativum L.) plants. Fungicides applied at 20 mg a.i. kg^–1 soil did not affect the root length colonized byG. etunicatum, but both benomyl and PCNB reduced sporulation by this fungus. Benomyl and PCNB reduced the root length colonized byG. rosea at 48 and 82 days after transplanting. PCNB also reducedG. mosseae-colonized root length at 48 and 82 days, but benomyl only affected root length colonized byG. mosseae at the earlier time point. Only PCNB reduced sporulation byG. mosseae, consistent with its effect on root length colonized by this fungus. captan reduced the root length colonized by G. rosea at 48 days, but not at 82 days, and reduced colonization byG. mosseae at 82 days, but not at 48 days. Captan did not affect sporulation by any of the fungi.G. rosea spore production was highly variable, but benomyl appeared to reduce sporulation by this fungus. Overall,G. etunicatum was the most tolerant to fungicides in association with pea plants in this soil, andG. rosea the most sensitive. Benomyl and PCNB were overall more toxic to these fungi than captan. Interactions of AM fungi and fungicides were highly variable and biological responses depended on fungus-fungicide combinations and on environmental conditions.     

Organic acid exudation by mycorrhizal Andropogon virginicus L. (broomsedge) roots in response to aluminum

Elevated aluminum (Al) availability limits plant growth on acidic soils. Although this element is found naturally in soils, acidic conditions create an environment where Al solubility increases and toxic forms of Al impact plant function. Plant resistance to Al is often attributed to organic acid exudation from plant roots and the chelation of cationic Al in the rhizosphere. The association of arbuscular mycorrhizal (AM) fungi with the roots of plants may alleviate Al toxicity by altering soil Al availability or plant exposure through the binding of Al to fungal structures or through the influence of fungi on exudation from roots. Diverse communities of AM fungi are found in soil ecosystems and research suggests that AM fungi exhibit functional diversity that may influence plant performance under varying edaphic environments. In the present study, we evaluated acidic isolates of six AM species in their responses to Al. Andropogon virginicus (broomsedge), a warm-season grass that commonly grows in a range of stressful environments including acidic soils, was used as a plant host for Acaulospora morrowiae, Glomus claroideum, Glomus clarum, Glomus etunicatum, Paraglomus brasilianum, and Scutellospora heterogama. Fungal spores were germinated and exposed to 0 or 100 μM Al on filter paper in sand culture or were grown and exposed to Al in sand culture in association with A. virginicus. Short- and long-term responses to Al were evaluated using direct measurements of fungal spore germination, hyphal elongation, and measurements of A. virginicus colonization and plant growth as a phytometer of AM function in symbio. Spore germination and hyphal elongation varied among AM species in response to Al, but patterns were not consistent with the influences of these AM species on A. virginicus under Al exposure. Exposure to Al did not influence colonization of roots, although large differences existed in colonization among fungal species. Plants colonized by G. clarum and S. heterogama exhibited the least reduction in growth when exposed to Al, produced the highest concentrations of Al-chelating organic acids, and had the lowest concentrations of free Al in their root zones. This pattern provides evidence that variation among AM fungi in Al resistance conferred to their plant hosts is associated with the exudation of Al-binding organic acids from roots and highlights the role that AM fungal diversity may play in plant performance in acidic soil environments.     

Diversity of arbuscular mycorrhizal fungi in red tangerine (Citrus reticulata Blanco) rootstock rhizospheric soils from hillside citrus orchards

Root colonization, abundance of spores and hyphae, as well as species diversity of arbuscular mycorrhizal (AM) fungi were analyzed in citrus orchards along an altitudinal gradient. The citrus trees were heavily colonized (50.87–77.45%) by native AM fungi. In citrus orchards located at <600 m above sea level (asl), we recorded more extensive hyphal and arbuscular colonization, and higher spore and hyphal length density. AM fungal colonization, spore density, and hyphal length density were closely correlated with edaphic factors such as available phosphorus, pH, and organic matter. A total of 18 AM fungal species belonging to 3 different orders, Archaeosporales (1 species), Diversisporales (7 species) and Glomerales (10 species), were identified on the basis of spore morphological characteristics. In orchards located at higher altitudes (≥700 m asl), we observed a significant decrease in species richness and Shannon–Wiener index values. However, in all of the surveyed orchards, Glomus aggregatum, Funneliformis mosseae and Rhizophagus intraradices were the dominant species. Isolate frequency and relative abundance of AM fungi exhibited clearly distinct distribution patterns among taxonomic families. Canonical correspondence analysis revealed that the AM fungal community structure was significantly influenced by environmental factors, especially altitude, pH, soil moisture, and available nitrogen. Our data indicated that environmental factors are important in determining AM fungal root colonization, propagule numbers, and species diversity in citrus orchards.     

Changes in soil aggregation and glomalin-related soil protein content as affected by the arbuscular mycorrhizal fungal species Glomus mosseae and Glomus intraradices

Arbuscular mycorrhizal (AM) fungi are key organisms of the soil/plant system, influencing soil fertility and plant nutrition, and contributing to soil aggregation and soil structure stability by the combined action of extraradical hyphae and of an insoluble, hydrophobic proteinaceous substance named glomalin-related soil protein (GRSP). Since the GRSP extraction procedures have recently revealed problems related to co-extracting substances, the relationship between GRSP and AM fungi still remains to be verified. In this work the hypothesis that GRSP concentration is positively correlated with the occurrence of AM fungi was tested by using Medicago sativa plants inoculated with different isolates of Glomus mosseae and Glomus intraradices in a microcosm experiment. Our results show that (i) mycorrhizal establishment produced an increase in GRSP concentration - compared to initial values - in contrast with non-mycorrhizal plants, which did not produce any change; (ii) aggregate stability, evaluated as mean weight diameter (MWD) of macroaggregates of 1-2 mm diameter, was significantly higher in mycorrhizal soils compared to non-mycorrhizal soil; (iii) GRSP concentration and soil aggregate stability were positively correlated with mycorrhizal root volume and weakly correlated with total root volume; (iv) MWD values of soil aggregates were positively correlated with values of total hyphal length and hyphal density of the AM fungi utilized.The different ability of AM fungal isolates to affect GRSP concentration and to form extensive and dense mycelial networks, which may directly affect soil aggregates stability by hyphal enmeshment of soil particles, suggests the possibility of selecting the most efficient isolates to be utilized for soil quality improvement and land restoration programs.     

Nickel-tolerant Brevibacillus brevis and arbuscular mycorrhizal fungus can reduce metal acquisition and nickel toxicity effects in plant growing in nickel supplemented soil

The growth of clover (Trifolium repens ) and its uptake of N, P and Ni were studied following inoculation of soil with Rhizobium trifolii, and combinations of two Ni-adapted indigenous bacterial isolates (one of them was Brevibacillus brevis) and an arbuscular mycorrhizal (AM) fungus (Glomus mosseae). Plant growth was measured in a pot experiment containing soil spiked with 30 (Ni I), 90 (Ni II) or 270 (Ni III) mg kg⁻¹ Ni-sulphate (corresponding to 11.7, 27.6 and 65.8 mg kg⁻¹ available Ni on a dry soil basis). Single inoculation with the most Ni-tolerant bacterial isolate (Brevibacillus brevis) was particularly effective in increasing shoot and root biomass at the three levels of Ni contamination in comparison with the other indigenous bacterial inoculated or control plants. Single colonisation of G. mosseae enhanced by 3 fold (Ni I), by 2.4 fold (Ni II) and by 2.2 fold (Ni III) T. repens dry weight and P-content of the shoots increased by 9.8 fold (Ni I), by 9.9 fold (Ni II) and by 5.1 fold (Ni III) concomitantly with a reduction in Ni concentration in the shoot compared with non-treated plants. Coinoculation of G. mosseae and the Ni-tolerant bacterial strain (B. brevis) achieved the highest plant dry biomass (shoot and root) and N and P content and the lowest Ni shoot concentration. Dual inoculation with the most Ni-tolerant autochthonous microorganisms (B. brevis and G. mosseae) increased shoot and root plant biomass and subtantially reduced the specific absorption rate (defined as the amount of metal absorbed per unit of root biomass) for nickel in comparison with plants grown in soil inoculated only with G. mosseae. B. brevis increased nodule number that was highly depressed in Ni I added soil or supressed in Ni II and Ni III supplemented soil. These results suggest that selected bacterial inoculation improved the mycorrhizal benefit in nutrients uptake and in decreasing Ni toxicity. Inoculation of adapted beneficial microorganisms (as autochthonous B. brevis and G. mosseae) may be used as a tool to enhance plant performance in soil contaminated with Ni.     

Soil inoculation with the biocontrol agent Clonostachys rosea and the mycorrhizal fungus Glomus intraradices results in mutual inhibition, plant growth promotion and alteration of soil microbial communities

Interactions between the biocontrol fungus Clonostachys rosea IK 726 and a tomato/Glomus intraradices BEG87 symbiosis were examined with and without wheat bran, which served as a food base for C. rosea. In soil without wheat bran amendment, inoculation with C. rosea increased plant growth and altered shoot nutrient content resulting in an increase and decrease in P and N content, respectively. Inoculation with G. intraradices had no effect on plant growth, but increased the shoot P content. Dual inoculation with G. intraradices and C. rosea followed the pattern of C. rosea in terms of plant growth and nutrient content. Wheat bran amendment resulted in marked plant growth depressions, which were counteracted by both inoculants and dual inoculation increased plant growth synergistically. Amendment with wheat bran increased the population density of C. rosea and reduced mycorrhizal fungus colonisation of roots. The inoculants were mutually inhibitory, which was shown by a reduction in root colonisation with G. intraradices in treatments with C. rosea and a reduction in colony-forming units (cfu) of C. rosea in treatments with G. intraradices, irrespective of wheat bran amendment. Moreover, both inoculants markedly influenced soil microbial communities examined with biomarker fatty acids. Inoculation with G. intraradices increased most groups of microorganisms irrespective of wheat bran amendment, whereas the influence of C. rosea on other soil microorganisms was affected by wheat bran amendment. Overall, inoculation with C. rosea increased and decreased most groups of microorganisms without and with wheat bran amendment, respectively. In conclusion, despite mutual inhibition between the two inoculants this interaction did not impair their observed plant growth promotion. Both inoculants also markedly influenced other soil microorganisms, which should be further studied in relation to their plant growth-promoting features.     

The bacterial community of tomato rhizosphere is modified by inoculation with arbuscular mycorrhizal fungi but unaffected by soil enrichment with mycorrhizal root exudates or inoculation with Phytophthora nicotianae

Arbuscular mycorrhizal (AM) fungi have been shown to induce the biocontrol of soilborne diseases, to change the composition of root exudates and to modify the bacterial community structure of the rhizosphere, leading to the formation of the mycorrhizosphere. Tomato plants were grown in a compartmentalized soil system and were either submitted to direct mycorrhizal colonization or to enrichment of the soil with exudates collected from mycorrhizal tomato plants, with the corresponding negative controls. Three weeks after planting, the plants were inoculated or not with the soilborne pathogen Phytophthora nicotianae growing through a membrane from an adjacent infected compartment. At harvest, a PCR-Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments amplified from the total DNA extracted from each plant rhizosphere was performed. Root colonization with the AM fungi Glomus intraradices or Glomus mosseae induced significant changes in the bacterial community structure of tomato rhizosphere, compared to non-mycorrhizal plants, while enrichment with root exudates collected from mycorrhizal or non-mycorrhizal plants had no effect. Our results support that the effect of AM fungi on rhizosphere bacteria would not be mediated by compounds present in root exudates of mycorrhizal plants but rather by physical or chemical factors associated with the mycelium, volatiles and/or root surface bound substrates. Moreover, infection of mycorrhizal or non-mycorrhizal plants with P. nicotianae did not significantly affect the bacterial community structure suggesting that rhizosphere bacteria would be less sensitive to the pathogen invasion than to mycorrhizal colonization. Of 96 unique sequences detected in the tomato rhizosphere, eight were specific to mycorrhizal fungi, including two Pseudomonas, a Bacillus simplex, an Herbaspirilium and an Acidobacterium. One Verrucomicrobium was common to rhizospheres of mycorrhizal plants and of plants watered with mycorrhizal root exudates.     

Exudates of dark septate endophyte (DSE) modulate the development of the arbuscular mycorrhizal fungus (AMF) Gigaspora rosea

Exudates of a dark septate endophyte (DSE) identified as Dreschlera sp., a common endophyte isolated by the inner cortical cells of the grass Lolium multiflorum, were put in contact with the arbuscular mycorrhizal fungus (AMF) Gigaspora rosea. These exudates stimulated the hyphal length and the hyphal branching of the AMF. A negative effect on the extramatrical phase of the AMF was detected. This is the first report to show how exudates of DSE can affect the development of AMF. These results show that DSE could be modifying the mycorrhizal status of the plants, modulating a different symbiosis in the rhizosphere.     

Pre-symbiotic and symbiotic interactions between Glomus intraradices and two Paenibacillus species isolated from AM propagules. In vitro and in vivo assays with soybean (AG043RG) as plant host

Two indole-producing Paenibacillus species, known to be associated with propagules of arbuscular mycorrhizal (AM) fungi, were examined for their mycorrhization helper bacteria activity at pre-symbiotic and symbiotic stages of the AM association. The effects were tested under in vitro and in vivo conditions using an axenically propagated strain of the AM fungus Glomus intraradices and Glycine max (soybean) as the plant host. The rates of spore germination and re-growth of intraradical mycelium were not affected by inoculation with Paenibacillus strains in spite of the variation of indole production measured in the bacterial supernatants. However, a significant promotion in pre-symbiotic mycelium development occurred after inoculation of both bacteria under in vitro conditions. The Paenibacillus rhizosphaerae strain TGX5E significantly increased the extraradical mycelium network, the rates of sporulation, and root colonization in the in vitro symbiotic association. These results were also observed in the rhizosphere of soybean plants grown under greenhouse conditions, when P. rhizosphaerae was co-inoculated with G. intraradices. However, soybean dry biomass production was not associated with the increased development and infectivity values of G. intraradices. Paenibacillus favisporus strain TG1R2 caused suppression of the parameters evaluated for G. intraradices during in vitro symbiotic stages, but not under in vivo conditions. The extraradical mycelium network produced and the colonization of soybean roots by G. intraradices were promoted compared to the control treatments. In addition, dual inoculation had a promoting effect on soybean biomass production. In summary, species of Paenibacillus associated with AM fungus structures in the soil, may have a promoting effect on short term pre-symbiotic mycelium development, and little impact on AM propagule germination. These findings could explain the associations found between some bacterial strains and AM fungus propagules.     

Mycorrhizae,biocides, and biocontrol. 4. Response of a mixed culture of arbuscular mycorrhizal fungi and host plant to three fungicides

The effects of three commonly used fungicides on the colonization and sporulation by a mixture of three arbuscular mycorrhizal (AM) fungi consisting of Glomus etunicatum (Becker & Gerd.), Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe, and Gigaspora rosea (Nicol. & Schenck) in symbiosis with pea plants and the resulting response of the host-plant were examined. Benomyl, PCNB, and captan were applied as soil drenches at a rate of 20 mg active ingredient kg^-1 soil 2 weeks after transplanting pea seedlings in a silty clay-loam soil containing the mixed inocula of AM fungi (AM plants). Effects of fungicides were compared to untreated plants that were inoculated with fungi (AM control). The effect of mycorrhizal inoculation on plant growth was also examined by including nonmycorrhizal, non-fungicide-treated plants (non-AM control). Fungicides or inoculation with AM fungi had only a small effect on the final shoot weights of pea plants, but had greater effects on root length and seed yield. AM control plants had higher seed yields and lower root lengths than the corresponding non-AM plants, and the fungicide-treated AM plants had intermediate yields and root lengths. Seed N and P contents were likewise highest in AM control plants, lowest in non-AM plants, and intermediate in fungicide-treated AM plants. All three fungicides depressed the proportion (%) of root length colonized by AM fungi, but these differences did not translate to reductions in the total root length that was colonized, since roots were longer in the fungicide-treated AM plants. Pea plants apparently compensated for the reduction in AM-fungal metabolism due to fungicides by increasing root growth. Fungicides affected the population of the three fungi as determined by sporulation at the final harvest. Captan significantly reduced the number, relative abundance, and relative volume of G. rosea spores in the final population relative to the controls. The relative volume of G. etunicatum spores was greater in all the fungicide-treated soils, while G. mosseae relative volumes were only greater in the captan-treated soil. These findings show that fungicides can alter the species composition of an AM-fungal community. The results also show that AM fungi can increase seed yield without enhancing the vegetative shoot growth of host plants.     

Differential effects of soil disturbance and plant residue retention on function of arbuscular mycorrhizal (AM) symbiosis are not reflected in colonization of roots or hyphal development in soil

The effects of soil disturbance and residue retention on the functionality of the symbiosis between medic (Medicago truncatula L.) and arbuscular mycorrhizal fungi (AMF) were assessed in a two-stage experiment simulating a crop rotation of wheat (Triticum aestivum L.) followed by medic. Plants were inoculated or not with the AMF, Glomus intraradices and Gigaspora margarita, separately or together. The contribution of the arbuscular mycorrhizal (AM) pathway for P uptake was determined using ³²P-labeled soil in a small hyphal compartment accessible only to hyphae of AMF. In general AM colonization was not affected by soil disturbance or residue application and disturbance did not affect hyphal length densities (HLDs) in soil. At 4 weeks disturbance had a negative effect on growth and phosphorus (P) uptake of plants inoculated with G. margarita, but not G. intraradices. By 7 weeks disturbance reduced growth of plants inoculated with G. margarita or AMF mix and total P uptake in all inoculated plants. With the exception of plants inoculated with G. margarita in disturbed soil at 4 weeks, the AM pathway made a significant contribution to P uptake in all AM plants at both harvests. Inoculation with both AMF together eliminated the negative effects of disturbance on AM P uptake and growth, showing that a fungus insensitive to disturbance can compensate for loss of contribution of a sensitive one. Application of residue increased growth and total P uptake of plants but decreased ³²P in plants inoculated with the AMF mix in disturbed soil, compared with plants receiving no residue. The AMF responded differently to disturbance and G. intraradices, which was insensitive to disturbance, compensated for lack of contribution by the sensitive G. margarita when they were inoculated together. Colonization of roots and HLDs in soil were not good predictors of the outcomes of AM symbioses on plant growth, P uptake or P delivery via the AM pathway.     

Effects of soil application of olive mill wastewaters on the structure and function of the community of arbuscular mycorrhizal fungi

Recycling of olive mill wastewaters (OMW) into agricultural soils is a controversial issue since benefits to soil fertility should counterbalance potential short-term toxicity effects. We investigated the short-term effects of OMW on the soil-plant system, regarding the diversity, structure and root colonization capacity of arbuscular mycorrhizal (AM) fungi and the respective growth response of Vicia faba L, commonly used as green manure in olive-tree plantations. A compartmentalized pot system was used that allowed the establishment of an AM fungal community in one compartment (feeder) and the application of three OMW dose levels in an adjacent second compartment (receiver). At 0, 10, and 30 days after OMW treatment (DAT), V. faba pre-germinated seeds were seeded in the receiver compartment. At harvest, shoot and root dry weights, AM fungal root colonization, soil hyphal length and P availability were recorded in the receiver compartment. In addition, OMW effects on AM fungal diversity in plant roots were studied by DGGE. A transient effect of OMW application was observed; plant growth and AM fungal colonization were initially inhibited, whereas soil hyphal length was stimulated, but in most cases differences were absent when seeding was performed 30 DAT. Similarly, changes induced in the structure of the root AM fungal community were of transient nature. Cloning and sequencing of all the major DGGE bands showed that roots were colonized by Glomus spp. The transient effects of OMW on the structure and function of AM fungi could be attributed to OMW-derived phytoxicity to V. faba plants or to an indirect effect via alteration of soil nutritional status. The high OMW dose significantly increased soil P availability in the presence of AM fungi, suggesting efficient involvement of AM fungi in organic-P minerilization. Overall our results indicate that soil application of OMW would cause transient changes in the AM fungal colonization of V. faba plants, which, would not impair their long-term plant growth promoting ability.     

The bioprotective effect of AM root colonization against the soil-borne fungal pathogen Gaeumannomyces graminis var. tritici in barley depends on the barley variety

The systemic effect of root colonization by the arbuscular mycorrhizal fungus (AMF) Glomus mosseae on the susceptibility of old and modern barley varieties to the soil-borne fungal pathogen Gaeumannomyces graminis var. tritici (Ggt) was studied in a split-root system. Plants were precolonized on one side of the split-root system with the AMF and thereafter the other side of the split-root system was inoculated with the pathogen. At the end of the experiment the level of bioprotection was estimated by quantifying lesioned roots and the determination of the root fresh weight. AM root colonization provided protection in some of the barley genotypes tested, but not in others. This protective effect seemed to vary in the oldest and the most modern barley variety tested.     

Hydrolytic enzyme activities in maize (Zea mays) and sorghum (Sorghum bicolor) roots inoculated with Gluconacetobacter diazotrophicus and Glomus intraradices

Two strains of Gluconacetobacter diazotrophicus (Pal 5, UAP5541) and the arbuscular mycorrhizal fungus Glomus intraradices increased both the shoot and root dry weight of sorghum 45 days after inoculation, whereas they had no effect on the shoot and root dry weight of maize. Co-inoculation (Gluconacetobacter diazotrophicus plus Glomus mosseae) did not increase the shoot and root dry weight of either plant. There was a synergistic effect of Gluconacetobacter diazotrophicus on root colonization of maize by Glomus intraradices, whereas an antagonistic interaction was observed in the sorghum root where the number of Gluconacetobacter diazotrophicus and the colonization by Glomus intraradices were reduced. Plant roots inoculated with Gluconacetobacter diazotrophicus and Glomus intraradices, either separately or together, significantly increased root endoglucanase, endopolymethylgalacturonase and endoxyloglucanase activities. The increase varied according to the plant. For example, in comparison with non-inoculated plants, there were higher endoglucanase (+328%), endopolymethylgalacturonase (+180%) and endoxyloglucanase (+125%) activities in 45-day old co-inoculated maize, but not in 45-day old sorghum. The possibility is discussed that hydrolytic enzyme activities were increased as a result of inoculation with Gluconacetobacter diazotrophicus, considering this to be one of the mechanisms by which these bacteria may increase root colonization by AM fungi.     

Lyophyllum sp. strain Karsten alleviates pH pressure in acid soil and enhances the survival of Variovorax paradoxus HB44 and other bacteria in the mycosphere

In previous work, Variovorax paradoxus strain HB44, next to Burkholderia terrae BS001 and Dyella japonica BS003, were found to be selected in the mycosphere of the tricholomataceous fungi Laccaria proxima and Lyophyllum sp. strain Karsten in an acid soil denoted G. V. paradoxus HB44 showed poor survival in G bulk soil, irrespective of prior soil sterilization, and this poor survival also occurred for B. terrae BS001 and D. japonica BS003. In contrast, the survival rate of strain HB44 in two other soils, with pH values > 5.5, was significantly raised. Also, significantly enhanced strain HB44 survival in G soil was found if the pH was raised to 5.5 or 6.5, and it was even shown to grow (in the presence of the exogenous carbon source glycerol) at such pH values in the sterile G soil. This behaviour was similar to that of the V. paradoxus type strain. Strikingly, Lyophyllum sp. strain Karsten, when colonizing the sterilized G soil, significantly raised the soil pH from about 4.6 to ≥5.0. The pH raise was dependent on time, hyphal development, as well as on initial soil pH, but was consistent throughout. The modulated soil pH conditions were shown to be permissive for the survival and growth of strain HB44, and this was extended to strains BS001 and BS003. These findings corroborate the hypothesis that L. sp. strain Karsten provides a suitable habitat for acid-sensitive strains like HB44, BS001 and BS003 in its mycosphere in acid soil, which is strongly defined by the establishment of a growth-permissive pH.     

Fraxinus-Glomus-Pisolithus symbiosis: Plant growth and soil aggregation effects

It has been established that arbuscular mycorrhizal (AM) fungi are involved in the conservation of soil structure. However, the effect of ectomycorrhizal (EM) fungi alone or in interaction with AM fungi in soil structure has been much less studied. This experiment evaluated EM and AM fungi effects on soil aggregation and plant growth. Ash plants (Fraxinus uhdei) were grown in pots, and were inoculated with Glomus intraradices and Pisolithus tinctorius separately but also in combination. Our results showed that F. uhdei established a symbiotic association with EM and AM fungi, and that these organisms, when interacting, showed synergistic and additive effects on plant growth compared to singly inoculated treatments. EM and AM fungi prompted changes in root morphology and increased water-stable aggregates. AM fungi affect mainly small-sized macroaggregates, while EM and EM-AM fungi interaction mainly affected aggregates bigger than 0.5 mm diameter. These results suggest that ectomyccorrhizal as well as arbuscular mycorrhizal fungi should be considered in restoration programs with Fraxinus plants.