Influence of soil impoverishment on the interaction between Glomus mosseae and saprobe fungi

 The effect of the saprobe fungi Wardomyces inflatus (Marchal) Hennebert, Paecilomyces farinosus (Holm & Gray) A. H. S. Brown & G. Sm., Gliocladium roseum Bain., Trichoderma pseudokoningii Rifai and T. harzianum Rifai, isolated from sporocarps of Glomus mosseae, on arbuscular mycorrhizal (AM) colonisation and plant dry matter of soybean was studied in 2/3 and 1/5 diluted soils in a greenhouse trial. Soil dilution to 1/5 had no effect on shoot dry matter of soybean but decreased AM colonisation and root dry weight of plants. CFU of saprobe fungi, except T. harzianum, were higher in 1/5 than in 2/3 diluted soils. W. inflatus and Gliocladium roseum decreased the shoot dry weight of soybean plant when inoculated together with Glomus mosseae. The saprobe fungi P. farinosus and T. pseudokoningii increased the shoot dry weights of plants grown in 1/5 diluted soil. The shoot dry weight and AM colonisation in 1/5 diluted soil were also increased when T. harzianum was inoculated together with Glomus mosseae. Thus, saprobe fungi increased AM colonisation of soybean plants by indigenous endophytes. The AM colonisation of plants at both soil dilutions was increased by Glomus mosseae. The highest level of AM colonisation was observed when P. farinosus and T. pseudokoningii were inoculated together Glomus mosseae. The dilution of soils influenced the interaction between inoculated microorganisms and their effect on plant growth. Accepted: 7 June 1999     

Interactions between Glomus mosseae and arbuscular mycorrhizal sporocarp-associated saprophytic fungi

The saprophytic fungi Wardomyces inflatus (Marchal) Hennebert, Paecilomyces farinosus (Holm & Gray) A. H. S. Brown & G. Sm., Gliocladium roseum Bain., sterile dark mycelium (SDM-54), Trichoderma pseudokoningii Rifai and Trichoderma harzianum Rifai were isolated from sporocarps of Glomus mosseae. The effect of saprophytic fungi on G. mosseae spore germination was tested on water agar. Wardomyces inflatus decreased the percent germination of G. mosseae spores; G. roseum, T. pseudokoningii and T. harzianum had no effect on germination; and P. farinosus and SDM-54 increased the percentage of spore germination of G. mosseae after 4 d. Wardomyces inflatus significantly decreased hyphal length of spores which germinated, but no other saprophytic fungi affected hyphal growth. Trichoderma pseudokoningii, T. harzianum, P. farinosus and SDM-54 increased the number of auxiliary cells formed by G. mosseae. The effect of saprophytic fungi on arbuscular mycorrhizal colonization of soybean was studied in a greenhouse trial. The percentage of soybean root length colonized was decreased by W. inflatus, unaffected by SDM-54 and T. harzianum, and increased by P. farinosus. Gliocladium roseum decreased root length colonized when plants were 12 wk old, and T. pseudokoningii increased colonization of roots when plants were 4 wk old. Antagonistic, synergistic and neutral actions of G. mosseae upon the saprophytic fungi were observed. The population of T. harzianum decreased and the populations of T. pseudokoningii and SDM-54 increased in the presence of G. mosseae. Our results indicate a complex interaction between G. mosseae and associated saprophytic fungi.     

Interaction between arbuscular mycorrhizal fungus Glomus mosseae and plant growth promoting fungus Phoma sp. on their root colonization and growth promotion of cucumber (Cucumis sativus L.)

Interaction between arbuscular mycorrhizal fungus Glomus mosseae and plant growth promoting fungus Phoma sp. was studied for its effect on their root colonization and plant growth of cucumber. Two isolates of Phoma sp. (GS8-2 and GS8-3) were tested with G. mosseae. The percent root length colonized by G. mosseae was not adversely affected by the presence of Phoma isolates. In contrast, the root colonization of both isolates GS8-2 and GS8-3 in 4-week-old plants was significantly reduced (80.7% and 84.3%, respectively) by added G. mosseae. Inoculating plants with each Phoma isolate significantly increased the shoot dry weight. However, dual inoculation of each Phoma isolate with G. mosseae had no significant effect on growth enhancement.     

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.     

Interactions between Pseudomonas fluorescens biocontrol agents and Glomus mosseae, an arbuscular mycorrhizal fungus, within the rhizosphere

Interactions between Pseudomonas fluorescens biocontrol agents and Glomus mosseae , an arbuscular mycorrhizal fungus, were studied. The biocontrol agents included the genetically modified strains CHA96 and CHA0 pME3424 which produced enhanced levels of antifungal compounds. Tomato ( Lycopersicum esculentum ) and leek ( Allium porrum ) host plants were grown in sterile Terra-Green (calcined attapulgite clay) with limited nutrients. Mycorrhizal activity was indicated by shoot dry weight and phosphorus content. In all experiments, plants grown in the presence of G. mosseae had a significantly higher shoot dry weight than those grown in the absence of G. mosseae . Colonisation and activity of G. mosseae was unaltered in the presence of P. fluorescens isolates and presence of G. mosseae increased the population of P. fluorescens in the rhizosphere.     

Growth and arsenic uptake by Chinese brake fern inoculated with an arbuscular mycorrhizal fungus

A split-root experiment investigated the effects of inoculation with the arbuscular mycorrhizal fungus Glomus mosseae and arsenic (As) addition on As uptake by Pteris vittata L. Either part or all of the root system was inoculated with G. mosseae or exposed to As addition (50 ml 1000 μmol L⁻¹ As 1 week before harvest). Mycorrhizal colonization substantially increased frond and root dry weight and P and As contents irrespective of As addition. Frond As contents in mycorrhizal plants were highest when the whole root system was exposed to As. Frond As concentrations and contents were higher when inoculation and As addition were in the same parts of the root system than when spatially separate. There were positive effects of arbuscular mycorrhiza inoculation on plant growth and As uptake, and inoculation of part of the roots seemed to be as effective as inoculation of the whole root system.     

Flavonoids in roots of white clover: interaction of arbuscular mycorrhizal fungi and a pathogenic fungus

The effects of two arbuscular mycorrhizal fungi (AMF) (Glomus mosseae and G. claroideum) and a pathogenic fungus (Pythium ultimum) on the production of eight flavonoids in roots of two white clover (Trifolium repens L.) cultivars were evaluated. Quantification of AM and pathogenic fungi in the roots showed that the AM symbiosis significantly reduced P. ultimum biomass and in some cases prevented infection. The flavonoid productions in clover roots varied depending on the presence of beneficial and/or pathogenic fungi, fungal isolate or plant cultivar. Only plants colonized with G. claroideum showed detectable concentrations of either coumestrol or kaempferol (cultivar-dependant). In addition, inoculation with G. claroideum resulted in significantly higher concentrations of coumestrol in cv. Sonja and medicarpin in cv. Milo. A low production of coumestrol and kaempferol in mycorrhizal plants may be G. mosseae-specific. Only the concentrations of formononetin and daidzein increased in clover roots in response to infection with P. ultimum. These flavonoids are supposedly stress metabolites, synthesized or produced from glycosides in response to pathogen infection. However, the presence of one or both AMF significantly lowered the formononetin and daidzein concentrations, and overruled the inductive effect of P. ultimum. Therefore the antagonistic action of AM against the pathogen must take place through another mechanism.     

Glomus intraradices dominates arbuscular mycorrhizal communities in a heavy textured agricultural soil

Arbuscular mycorrhizal fungal (AMF) spore communities were surveyed in a long-term field fertilization experiment in Switzerland, where different amounts of phosphorus (P) were applied to soil. Plots receiving no P as well as plots systematically fertilized in excess to plant needs for 31 years were used to test the hypothesis that application of P fertilizer changes the composition and diversity of AMF communities. AMF spores were isolated from the field soil, identified, and counted so as to quantify the effect of P fertilization on AMF spore density, composition, and diversity. Trap cultures were established from field soil with four host plants (sunflower, leek, maize, and Crotalaria grahamiana), and the spore communities were then analyzed in substrate samples from the pots. Altogether, nine AMF species were detected in the soil. No evidence has been acquired for effect of P fertilization on spore density, composition, and diversity of AMF in both the field soil and in trap cultures. On the other hand, we observed strong effect of crop plant species on spore densities in the soil, the values being lowest under rapeseed and highest under Phacelia tanacetifolia covercrop. The identity of plant species in trap pots also significantly affected composition and diversity of associated AMF communities, probably due to preferential establishment of symbiosis between certain plant and AMF species. AMF spore communities under mycorrhizal host plants (wheat and Phacelia in the fields and four host plant species in trap pots) were dominated by a single AMF species, Glomus intraradices. This resulted in exceptionally low AMF spore diversity that seems to be linked to high clay content of the soil.Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

Effects of the root-lesion nematode Pratylenchus vulnus and the mycorrhizal fungus Glomus mosseae on the growth of EMLA-26 apple rootstock

The interaction between Pratylenchus vulnus and the endomycorrhizal fungus Glomus mosseae on the growth of EMLA 26 apple rootstock was studied under shadehouse conditions in the field during the first 6 months of growth. Fresh top weights, fresh root weights, and shoot lengths of mycorrhizal plants with and without P. vulnus were significantly higher than those of nonmycorrhizal plants. Addition of P to non-mycorrhizal controls had little overall effect. Mycorrhizal treatments with the nematode showed a significantly lower amount of nematodes per gram of root than nonmycorrhizal treatments with P. vulnus. Root colonization by G. mosseae was not affected by the presence of the nematode. No nutrient deficiencies were detected in foliar analyses, although low levels of K, A1, and Fe were detected in nematode treatments. The highest levels of S, Mg, Mn and Zn were detected in P. vulnus inoculated plants. Mycorrhizal plants had the highest levels of N, Na, P, K, and Fe. The importance of early mycorrhizal infection of EMLA 26 apple root-stock in the presence of the nematode is discussed.     

Associations between arbuscular mycorrhizal fungi and Rhynchrelyrum repens in abandoned quarries in southern China

The association between arbuscular mycorrhizal fungi (AMF) and Rhynchrelyrum repens was investigated. In six abandoned quarries in the Pearl River Delta area, R. repens was found to be associated with nine AMF species, including Glomus versiforme, G. brohultii, G. microaggregatum, G. clarum and G. claroideum, Acaulospora delicata, A. mellea, A. mollowae and Entrophospora infrequens. The genus Glomus and the species G. brohultii were recorded at the highest frequencies. Three typical arbuscular mycorrhizal (AM) structures, i.e. hyphae, vesicles and arbuscules, were found in the roots of the R. repens specimens collected from all the quarries investigated. Vesicles were the most frequently recorded structure. Results of a container-based experiment showed that R. repens had very high mortality (83.3%) in the absence of AMF in soil containing sufficient P (phosphorus); this indicates that R. repens is an obligate mycotroph. The presence of AMF significantly increased the biomass accumulation of R. repens seedlings (p < 0.01). It was also observed that AMF colonization was related to soil P and K (potassium) utilization by R. repens seedlings. It is, therefore, important to inoculate with AMF when using R. repens for the restoration of damaged ecosystems.     

31P NMR for the study of P metabolism and translocation in arbuscular mycorrhizal fungi

³¹P nuclear magnetic resonance (NMR) spectroscopy was used to study phosphate (P) metabolism in mycorrhizal and nonmycorrhizal roots of cucumber (Cucumis sativus L) and in external mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith. The in vivo NMR method allows biological systems to be studied non-invasively and non-destructively. ³¹P NMR experiments provide information about cytoplasmic and vacuolar pH, based on the pH-dependent chemical shifts of the signals arising from the inorganic P (P_i) located in the two compartments. Similarly, the resonances arising from α, β and γ phosphates of nucleoside triphosphates (NTP) and nucleoside diphosphates (NDP) supply knowledge about the metabolic activity and the energetic status of the tissue. In addition, the kinetic behaviour of P uptake and storage can be determined with this method. The ³¹P NMR spectra of excised AM fungi and mycorrhizal roots contained signals from polyphosphate (PolyP), which were absent in the spectra of nonmycorrhizal roots. This demonstrated that the P_i taken up by the fungus was transformed into PolyP with a short chain length. The spectra of excised AM fungi revealed only a small signal from the cytoplasmic P_i, suggesting a low cytoplasmic volume in this AM fungus. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Mass production of Glomus mosseae spores

Five crops inoculated with Glomus mosseae were grown for 10 weeks and the development of mycorrhizal infection and sporulation were assessed. Infected roots from pot cultures of different ages were used to examine the host effect on the development of mycorrhizae. The effectiveness of each host was assessed by measuring spore numbers. For all hosts, the percentage of root length infected increased rapidly up to 10 weeks after sowing. Infectivity of root inocula increased with increasing percentage of root length infected with the inoculum for all crops, except where large numbers of mature spores (1755) had been produced on barley. The highest spore numbers were achieved in the rhizosphere of barley plants, followed by chickpea and beans. The lowest spore numbers were found in the rhizosphere of corn and okra plants. The type of the crop as well as the harvest date greatly influenced the size of the spore population and the extent of root colonization of G. mosseae.     

Effects of the root-lesion nematode Pratylenchus vulnus and the vesicular-arbuscular mycorrhizal fungus Glomus mosseae on the growth of three plum rootstocks

The effects of Pratylenchus vulnus and the endomycorrhizal fungus Glomus mosseae on growth of Myrobalan 605, Marianna 2624 and San Julian 655-2 plum rootstocks were measured under shadehouse conditions in the field for two growing seasons (1990–91). Shoot dry weights were higher in the majority of the vesicular-arbuscular mycorrhizal (VAM) alone inoculated plants after both growing seasons. Root weights of mycorrhizal Myrobalan and Marianna were higher than root weights of the same rootstocks lacking mycorrhizae, inoculated with P. vulnus, and VAM in combination with the nematode. Mycorrhizal Marianna inoculated with the nematode showed a considerably higher final nematode population in relation to non-inoculated VAM treatments. No correlation was found in the number of nematodes per gram of root between mycorrhizal and non-mycorrhizal treatments. P. vulnus adversely affected the mycorrhizal colonization in Marianna, but not in Myrobalan and San Julian. Marianna appears to be more mycorrhizal dependent than the two other rootstocks.     

Biolistic transformation of arbuscular mycorrhizal fungi

Gene transfer systems have proved effective for the transformation of a range of organisms for both fundamental and applied studies. Biolistic transformation is a powerful method for the gene transfer into various organisms and tissues that have proved recalcitrant to more conventional means. For fungi, the biolistic approach is particularly effective where protoplasts are difficult to obtain and/or the organisms are difficult to culture. This is particularly applicable to arbuscular mycorrhizal (AM) fungi, being as they are obligate symbionts that can only be propagated in association with intact plants or root explants. Furthermore, these fungi are aseptate and protoplasts cannot be released. Recent advancements in gene transformation systems have enabled the use of biolistic technology to introduce foreign DNA linked to molecular markers into these fungi. In this review we discuss the development of transformation strategies for AM fungi by biolistics and highlight the areas of this technology which require further development for the stable transformation of these elusive organisms.