Effect of phosphate and the arbuscular mycorrhizal fungus Glomus intraradices on disease severity of root rot of peas (Pisum sativum) caused by Aphanomyces euteiches

 The effects of inorganic phosphate levels and the presence of arbuscular mycorrhiza on disease severity of Aphanomyces euteiches in pea roots were studied. Disease severity on roots and epicotyl as well as the oospore number within infected root tissue were correlated with the phosphorus (P) level in the growth medium. The arbuscular mycorrhizal fungus Glomus intraradices increased P uptake and the P concentration in the plant but reduced disease development in peas. Polyacrylamide gel electrophoresis followed by densitometry of glucose-6-phosphate dehydrogenase specific to A.euteiches was used to measure the activity of the pathogen in roots. The enzyme activity increased with disease severity and disease incidence, except in plants supplemented with P at the highest level, where a peak in activity was seen 12 days after inoculation with the pathogen, followed by a decrease in activity. The epicotyl of mycorrhizal plants showed a reduction in disease severity although this part of the plants was not mycorrhizal. Thus, an induced systemic factor may be responsible for increased resistance in mycorrhizal plants. Accepted: 21 August 1998     

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.     

The arbuscular mycorrhizal fungus, Glomus intraradices, induces the accumulation of cyclohexenone derivatives in tobacco roots

Tobacco (Nicotiana tabacum L.) plants were grown with and without the arbuscular mycorrhizal fungus, Glomus intraradices Schenk & Smith. High-performance liquid chromatographic analyses of methanolic extracts from mycorrhizal and non-mycorrhizal tobacco roots revealed marked fungus-induced changes in the patterns of UV-detectable products. The UV spectra of these products, obtained from an HPLC photodiode array detector, indicated the presence of several blumenol derivatives. The most predominant compound among these derivatives was spectroscopically identified as 13-hydroxyblumenol C 9-O-gentiobioside (“nicoblumin”), i.e. the 9-O-(6′-O-β-glucopyranosyl)-β-glucopyranoside of 13-hydroxy-6-(3-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one, a new natural product. This is the first report on the identification of blumenol derivatives in mycorrhizal roots of a non-gramineous plant. Received: 28 August 1998 / Accepted: 26 October 1998     

Production and specificity of polyclonal antibodies against soluble proteins from the arbuscular mycorrhizal fungus Glomus intraradices

Rabbit polyclonal antibodies were produced against a soluble protein fraction from a vesicle and spore mixture of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices. The protocol for isolation of vesicles and spores from plant roots was optimized to minimize debris contamination. Protein extract purification and preparation for immunization was adapted to increase protein content and immunogenicity. Active antisera were produced starting from the second boost immunization. Antibodies obtained were specific for surface antigens of AMF and revealed different patterns of soluble protein antigens in G. intraradices, G. constrictum and an unidentified Glomus species. Accepted: 6 December 2000     

Hydrogen peroxide accumulation in Medicago truncatula roots colonized by the arbuscular mycorrhiza-forming fungus Glomus intraradices

The diaminobenzidine (DAB) staining technique was used to examine the accumulation of H₂O₂ in parts of roots of Medicago truncatula Gaertn. colonized by the arbuscular mycorrhiza (AM)-forming fungus Glomus intraradices Schenk and Smith. At the cellular level, the combination of bright-field and fluorescence microscopy revealed that a brownish stain, indicative of H₂O₂ accumulation was present within cortical root cells in the space occupied by arbuscules. Accumulation of H₂O₂ was especially pronounced in cells containing arbuscules that were clumped and less branched. Moreover, H₂O₂ accumulated around hyphal tips attempting to penetrate a host cell. In contrast, no H₂O₂ accumulation was observed in hyphal tips growing along the middle lamella, or in appressoria or vesicles. On the basis of these findings we suggest that a locally restricted oxidative burst is involved in the temporal and spatial control of the intracellular colonization of M. truncatula cells by the AM-forming fungus G. intraradices. Received: 1 October 1998 / Accepted: 22 December 1998     

Effect of root exudate fractions from P-deficient and P-sufficient onion plants on root colonisation by the arbuscular mycorrhizal fungus Gigaspora margarita

 The effect of root exudates from P-deficient onion on root colonisation by an arbuscular mycorrhizal fungus was examined. Onions (Allium cepa L.) were grown in solution culture at phosphorus concentrations of 0 (P0) and 2 (P2) mg P l^–1. Root exudates were collected and fractionated with Amberlite XAD-4 resin to give EtOH and water soluble fractions. Onions inoculated with the arbuscular mycorrhizal fungus Gigaspora margarita Becker & Hall were grown with or without (control) root exudates and exudate fractions in a growth chamber. After 24 days, arbuscular mycorrhiza levels and appressoria formation had increased in plants treated with P0-root exudate or the P0-EtOH fraction when compared to corresponding P2 treatments or control plants. P0 and P2 water-soluble fractions did not significantly affect either aspect of fungal development. These results suggest that hydrophobic compounds found in root exudates from P-deficient onion increase appressorium formation and, therefore, enhance mycorrhiza development. Accepted: 2 June 1998     

Fenpropimorph slows down the sterol pathway and the development of the arbuscular mycorrhizal fungus Glomus intraradices

The direct impact of fenpropimorph on the sterol biosynthesis pathway of Glomus intraradices when extraradical mycelia alone are in contact with the fungicide was investigated using monoxenic cultures. Bi-compartmental Petri plates allowed culture of mycorrhizal chicory roots in a compartment without fenpropimorph and exposure of extraradical hyphae to the presence of increasing concentrations of fenpropimorph (0, 0.02, 0.2, 2, 20 mg l⁻¹). In the fungal compartment, sporulation, hyphal growth, and fungal biomass were already reduced at the lowest fungicide concentration. A decrease in total sterols, in addition to an increase in the amount of squalene and no accumulation of abnormal sterols, suggests that the sterol pathway is severely slowed down or that squalene epoxidase was inhibited by fenpropimorph in G. intraradices. In the root compartment, neither extraradical and intraradical development of the arbuscular mycorrhizal (AM) fungus nor root growth was affected when they were not in direct contact with the fungicide; only hyphal length was significantly affected at 2 mg l⁻¹ of fenpropimorph. Our results clearly demonstrate a direct impact of fenpropimorph on the AM fungus by a perturbation of its sterol metabolism.     

Interactions between a mycophagous Collembola,dry yeast and the external mycelium of an arbuscular mycorrhizal fungus

 A plant growth system with root-free hyphal compartments was used to examine the interactions between a mycophagous Collembola (Folsomia candida Willem), dry yeast and an arbuscular mycorrhizal (AM) fungus [Glomus caledonium (Nicol. & Gerd.) Trappe and Gerdemann] in terms of Collembola reproduction, AM-hyphal length and AM-hyphal P transport. Collembola reproduction was unaffected by AM mycelium, but a supplement of dry yeast increased the Collembola population size. The addition of dry yeast increased AM-hyphal P transport by increasing hyphal length. Collembola without yeast affected neither AM-hyphal growth nor AM-hyphal P transport, whereas Collembola with yeast decreased AM-hyphal P transport by 75% after 8 weeks. The hyphal density of G. caledonium remained unaffected by Collembola except after 4 weeks in combination with yeast, when a 33% reduction was observed. The results of this experiment show that the interaction between F. candida and the external mycelium of G. caledonium is limited under the conditions imposed. Accepted: 27 February 1996     

Molecular characterization of chromosome termini of the arbuscular mycorrhizal fungus Glomus intraradices (Glomeromycota)

The minimum chromosome number of Glomus intraradices was assessed through cloning and sequencing of the highly divergent telomere-associated sequences (TAS) and by pulsed field gel electrophoresis (PFGE). The telomere of G. intraradices, as in other filamentous fungi, consists of TTAGGG repeats, this was confirmed using Bal31 nuclease time course reactions. Telomere length was estimated to be roughly 0.9 kb by Southern blots on genomic DNA and a telomere probe. We have identified six classes of cloned chromosomal termini based on the TAS. An unusually high genetic variation was observed within two of the six TAS classes. To further assess the total number of chromosome termini, we used telomere fingerprinting. Surprisingly, all hybridization patterns showed smears, which demonstrate that TAS are remarkably variable in the G. intraradices genome. These analyses predict the presence of at least three chromosomes in G. intraradices while PFGE showed a pattern of four bands ranging from 1.2 to 1.5 Mb. Taken together, our results indicate that there are at least four chromosomes in G. intraradices but there are probably more. The information on TAS and telomeres in the G. intradicies will be essential for making a physical map of the G. intraradices genome and could provide molecular markers for future studies of genetic variation among nuclei in these multigenomic fungi.     

Accumulation of secondary compounds in barley and wheat roots in response to inoculation with an arbuscular mycorrhizal fungus and co-inoculation with rhizosphere bacteria

 Colonization of Hordeum vulgare L. cv. Salome (barley)and Triticum aestivum L. cv. Caprimus (wheat) roots by the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith leads to de novo synthesis of isoprenoid cyclohexenone derivatives with blumenin [9-O-(2′-O-β-glucuronosyl)-β-glucopyranoside of 6-(3-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one] as the major constituent and to transient accumulation of hydroxycinnamate amides (4-coumaroylagmatine and -putrescine). Accumulation of these compounds in mycorrhizal wheat roots started 2 weeks after sowing together with the onset of arbuscule formation and proceeded with mycorrhizal progression. Highest levels were reached in 3- to 4-week-old secondary roots (root branches of first and higher order) characterized by the formation of vesicles. In the final developmental stages, the fungus produced massive amounts of spores, enclosing the stele of older root parts (older than 5 weeks) characterized by cortical death. In these root parts, the secondary compounds were detected in trace amounts only, indicating that they were located in the cortical tissues. Some rhizosphere bacteria tested, i.e. Agrobacterium rhizogenes, Pseudomonas fluorescens, and Rhizobium leguminosarum, markedly stimulated both fungal root colonization and blumenin accumulation, thus, acting as mycorrhiza-helper bacteria (MHB). Application of blumenin itself strongly inhibited fungal colonization and arbuscule formation at early stages of mycorrhiza development. This was associated with a markedly reduced accumulation of the hydroxycinnamate amides 4-coumaroylputrescine and -agmatine. The results suggest that both the isoprenoid and the phenylpropanoid metabolism are closely linked to the developmental stage and the extent of fungal colonization. Their possible involvement in the regulation of mycorrhiza development is discussed. Accepted: 18 September 1998     

Response of 11 eucalyptus species to inoculation with three arbuscular mycorrhizal fungi

 Numerous publications have reported growth stimulation of Eucalyptus following ectomycorrhizal inoculation in nursery or field conditions. Although Eucalyptus species can also form arbuscular mycorrhiza, their dependency on this type of mycorrhiza is still debatable. This paper presents information on the effect of inoculation of arbuscular mycorrhizal fungi on eucalypt growth. Twenty weeks after mycorrhizal inoculation, Eucalyptus seedlings' stem dry weight could be increased up to 49% compared to non-inoculated control plants. Intensity of root colonization by a given fungus depended on the host species, but it was not related to a plant growth response. Leaf phosphorus concentration of non-inoculated Eucalyptus seedlings varied greatly between species. Increases in leaf phosphorus concentration following mycorrhizal infection were not necessarily associated with plant growth stimulation. The most mycorrhiza-dependent Eucalyptus species tended to be those having the highest leaf phosphorus concentration in the absence of a fungal symbiont. These mycorrhiza-dependent Eucalyptus species seem to have greater phosphorus requirements and consequently to rely more on the symbiotic association. Accepted: 1 September 1995     

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.     

The BEG Expert System – a multimedia identification system for arbuscular mycorrhizal fungi

 Knowledge of the biodiversity of arbuscular mycorrhizal fungi is limited and keys for their identification are not in a user-friendly format for the non-expert. An Expert System, linked to the BEG (La Banque Européenne des Glomales), has been developed on a CD-ROM to produce a multimedia identification system for these fungi. Accepted: 12 March 1996     

Localized alteration in lateral root development in roots colonized by an arbuscular mycorrhizal fungus

 The morphological responses of root systems to localized colonization by endophytes is not well understood. We examined the responses of lateral roots to the arbuscular mycorrhizal (AM) fungus Gigaspora margarita Becker & Hall inoculated locally into the soil. Peanut (Arachis hypogaea L.) and pigeon pea (Cajanus cajan (L.) Millsp.) were examined. Root boxes filled with nutrient-poor soil in were inoculated in one half with the fungus and in the other half with a sterilized inoculum. Responses were apparent after 30 days but not after 20 days. Overall, lateral root development was more advanced in inoculated soil. This was clearly observed for 2nd- and 3rd-order lateral roots, but less clear for 1st-order lateral roots in both species, although percentage of colonized root length was higher in 1st-order lateral roots. Whilst in peanut the responses were clearly evident at the level of lateral roots initiated on more proximal parts of the tap root axis, they occurred on more distal parts in pigeon pea. We conclude that plants under nutrient-poor conditions give priority to mycorrhizal roots when partitioning assimilation products within the root system. Thus, AM formation may induce local morphological alteration of root systems. Accepted: 29 August 1996