Eucalyptus root and shoot chitinases, induced following root colonization by pathogenic versus ectomycorrhizal fungi, compared on one- and two-dimensional activity gels

The present work deals with the effect of root fungal colonization on chitinases activities in Eucalyptus seedlings. Plant chitinases indiced during pathogenic infection are thought to be directed against the fungus, but chitinases induced by ectomycorrhizal fungi may contribute to ectomycorrhizal ontogenesis. Plant responses were compared to determine whether plants induce different chitinases activities in contact with symbionts and pathogens, and whether chitinases are induced systemically in both cases. Despite 2-D analysis of Eucalyptus root chitinolytic activities, induced following root colonization by pathogenic or ectomycorrhizal fungi, it was not possible to differentiate between both infections. Moreover, ectomycorrhizal colonization, as pathogenic infections, led to systemic induction of chitinase activities far from the site of inoculation. Contrasting with roots, the chitinase activities induced in shoots were not positively correlated with ectomycorrhizal strain aggressiveness. The differential stimulation of root chitinase activity by aggressive or non-aggressive ectomycorrhizal strains was related to induction of five additional isoforms in response to contact with the most aggressive strains.     

New acidic chitinase isoforms induced in tobacco roots by vesicular-arbuscular mycorrhizal fungi

Summary Chitinase activities have been compared in tobacco roots (Nicotiana tabacum cv. Xanthi nc) infected by the pathogenic fungus Chalara elegans or three species of vesicular arbuscular mycorrhizal (VAM) fungi: Glomus versiforme, G. intraradix and G. fasciculatum, using native polyacrylamide gel electrophoresis (PAGE). All previously known acidic chitinase isoforms were stimulated in roots by the pathogenic fungus and by the VAM fungi, while two new acidic chitinase isoforms were specifically induced in response to the endomycorrhizal association. After separation in sodium dodecyl sulphate polyacrylamide denaturing gels (SDS-PAGE) under non-reducing conditions, the estimated apparent molecular mass for these additional acidic chitinase isoforms from VAM-colonized samples was 33 kDa, compared to 30 kDa for the main activity stimulated in C. elegans-infected root extracts.     

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     

The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) inhibits the stimulation of in vitro lateral root formation and the colonization of the tap-root cortex of Norway spruce (Picea abies) seedlings by the ectomycorrhizal fungus Laccaria bicolor

Norway spruce ( Picea abies (L.) Karst.) seedlings were inoculated with the ectomycorrhizal fungus Laccaria bicolor ((Marie) Orton), strain S238 N, in axenic conditions. The presence of the fungus slowed tap–root elongation by 26% during the first 15 d after inoculation and then stimulated it by 136%. In addition, it multiplied in vitro lateral root formation by 4.3, the epicotyl growth of the seedlings by 8.4 and the number of needles by 2. These effects were maintained when the fungus was separated from the roots by a cellophane membrane preventing symbiosis establishment, thus suggesting that the fungus acted by non-nutritional effects. We tested the hypothesis that IAA produced by L. bicolor S238 N would be responsible for the stimulation of fungal induced rhizogenesis. We showed in previous work that L. bicolor S238 N can synthesize IAA in pure culture. Exogenous IAA supplies (100 and 500 μ m ) reproduced the stimulating effect of the fungus on root branching but inhibited root elongation. The presence of 2,3,5-triiodobenzoic acid (TIBA) in the culture medium significantly depressed lateral root formation of inoculated seedlings. As TIBA had no significant effect on IAA released in the medium by L. bicolor S238 N, but counteracted the stimulation of lateral rhizogenesis induced by an exogenous supply of IAA, we suggest that TIBA inhibited the transport of fungal IAA in the root. Furthermore TIBA blocked the colonization of the main root cortex by L. bicolor S238 N and the formation of the Hartig net. These results specified the role of fungal IAA in the stimulation of lateral rhizogenesis and in ectomycorrhizal symbiosis establishment.     

Frozen in time: a new method using cryo-scanning electron microscopy to visualize root-fungal interactions

A new method of sample preparation for cryo-scanning electron microscopy was used to visualize internal infection of wheat (Triticum aestivum) roots by the pathogenic fungus Rhizoctonia solani AG-8. The new method retained fungal hyphae and root cells in situ in disintegrating root tissues, thus avoiding the distortions that can be introduced by conventional preparation by chemical fixation, dehydration and embedding. Infected roots frozen in liquid nitrogen were cryo-planed and etched (sublimed) at -80 degrees C for a critical length of time (up to 9 min) in the microscope column to reveal plant and fungal structures in three dimensions. Root and fungal structures were well preserved irrespective of infection severity. Root and hyphal cell walls were clearly seen and hyphal architecture within and between root cells was preserved. This rapid method permits three-dimensional in situ visualization of fungal invasion within roots and has broad application for examination of diseases caused by other necrotrophic fungi.     

Cleavage of chitinous elicitors from the ectomycorrhizal fungus Hebeloma crustuliniforme by host chitinases prevents induction of K+ and Cl− release, extracellular alkalinization and H2O2 synthesis of Picea abies cells

Rapid reactions comprising efflux of K⁺ and Cl⁻, phosphorylation of a 63-kDa protein (pp63), extracellular alkalinization and synthesis of H₂O₂ are equally induced in cells of Picea abies (L.) Karst. by chitotetraose, colloidal chitin and cell wall elicitors from the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. an ectomycorrhizal partner of spruce. Cleavage of fungal cell wall elicitors and of artificial chitin elicitors to monomeric and dimeric fragments by apoplasmic spruce chitinases (36-kDa class I chitinase, pI 8.0, and 28-kDa chitinase, pI 8.7; EC 3.2.1.14) equally prevented induction of these rapid reactions. Also, N-acetylglucosamine oligomers and elicitors from the fungal cell walls showed a similar dependence of their activity on the degree of polymerisation. From these results it is suggested that, during ectomycorrhiza formation, only some of the chitin-derived elicitors reach their receptors at the plant plasma membrane, initiating reactions of the hypersensitive response in the host cells. The remaining fungal elicitors will be degraded to varying extents by wall-localized chitinases of the host root, reducing the defence reactions of the plant and allowing symbiotic interactions of both organisms. Received: 6 January 1997 / Accepted: 14 March 1997     

Rooting and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris are enhanced by the ectomycorrhizal fungus Hebeloma cylindrosporum

The effect of different strains of the ectomycorrhizal fungus Hebeloma cylindrosporum on rooting in vitro and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris was studied. Two clones of P. pinaster and one of P. sylvestris were unable to root in the absence of auxin, but were induced to root on a medium devoid of auxin by all the fungal strains. Wild-type and indoleacetic acid (IAA)-overproducing mutant strains of the fungus stimulated rooting of clones showing a good reactivity to auxin to the same extent. In contrast, with a clone of P. sylvestris that showed low reactivity to auxin, IAA-overproduction by the fungus was advantageous for the induction of rooting of cuttings. Adventitious roots formed in the presence of a fungal strain were completely surrounded by a loosely packed network of hyphae which formed mycorrhizas as soon as roots grew outside the agar medium. During acclimatization, fungal inoculation improved the survival of rooted cuttings. At the end of acclimatization, fungal mycelia could be easily detected in the culture substrate of cuttings inoculated with dikaryotic strains and most of the pines' short roots were mycorrhizal. Monokaryotic mycelia, which have a lower growth rate and a lower infectivity, displayed poor ability to colonize the substrate and to form mycorrhizas. Two months after the end of acclimatization, fungal inoculation frequently depressed the growth of acclimatized cuttings of the clone J of P. pinaster . No depressive effect was observed with clone 78 and growth stimulation could even be observed with the infective dikaryon D1 which formed numerous mycorrhizas. From these studies, it was concluded that ectomycorrhizal fungi could be a suitable tool for improving rooting in vitro and survival at acclimatization of micropropagated conifer cuttings.     

Morphological and molecular typing of the below-ground fungal community in a natural Tuber magnatum truffle-ground

The aims of the work were to elucidate the distribution of the ectomycorrhizal fungus Tuber magnatum Pico during its symbiotic stage, and to identify the root-associated fungi in a natural truffle-ground located in North Italy. Ectomycorrhizal root tips were harvested in the truffle ground, sorted in morphotypes and analyzed by ITS. Morphological and molecular analyses revealed that (i) T. magnatum mycorrhizae were rare and independent on the fruitbody productions and (ii) the dominant fungal species belonged to Thelephoraceae, followed by Tuberaceae and Sebacinaceae.     

Changes in the activity of enzymes involved with primary nitrogen metabolism due to ectomycorrhizal symbiosis on jack pine seedlings

Jack pine (Pinus banksiana Lamb.) seedlings were inoculated with either one of the ectomycorrhizal (ECM) fungi, Laccaria bicolor (Maire) Orton or Pisolithus tinctorius (Pers.) Coker and Couch, and grown for 16 weeks in a growth chamber along with non-ECM controls. Five enzymes involved with the assimilation of nitrogen or the synthesis of amino acids were measured in the 3 jack pine root systems as well as in the pure fungal cultures. Pisolithus tinctorius in pure culture had no detectable activity of nitrate reductase (NR. EC 1.6.6.1), glutamate dehydrogenase (GDH. EC 1.4.1.2), glutamate decarboxylase (GDCO. EC 4.1.1.15) or glutamate oxoglutarate aminotransferase (GOGAT, EC 1.4.1.13) but did have some glutamine synthetase (GS, EC 6.3.1.2) activity. Laccaria bicolor in pure culture had no NR activity, small levels of GDCO activity, and high GS, GDH and GOGAT activity. The high levels of enzymatic activity present in L. bicolor indicate that it may play a greater role in the nitrogen metabolism of its host plant than P. tinctorius. ECM infection clearly altered the enzymatic activity in jack pine roots but the nature of these changes depended on the fungal associate. Non-ECM root systems had higher specific activities than ECM root systems for NR, GS, GDH and GDCO but GOGAT activites were the same for both the ECM and non-ECM roots. Root systems infected with L. bicolor had significantly greater NR and GDCO activity than those infected with P. tinctorius. Differences in the GS activity of the two fungi in pure culture corresponded to the GS activity of jack pine roots in symbiotic association with these fungi. While the free amino acid profiles in roots were significantly affected by ECM infection, the profile of free amino acids exported to the stem was the same for all treatments. High asparagine and low glutamine in roots infected with P. tinctorius indicates that asparagine synthetase (EC x.x.x.x) activity should be higher within this symbiotic association than in the L. bicolor association or in the non-mycorrhizal roots.     

Salinity effects on germination,growth, and seed production of the halophyte Cakile maritima

The growth ofEucalyptus regnans seedlings in forest soil is enhanced when it has been air-dried. In undried forest soil seedlings grow poorly and develop purple coloration in the foliage, indicating P deficiency. This paper reports the results of pot experiments designed to investigate the relationship between growth and P acquisition, ectomycorrhizal infection and age of seedlings grown in air-dried and undried soil. The effect on seedling growth of their inoculation with air-dried or undried soil or with ectomycorrhizal roots from plants growing in air-dried or undried soil was also investigated. Ectomycorrhizal root tips were detected in 3-week-oldE. regnans seedlings in both air-dried and undried soil, and from then on the frequency of ectomycorrhizal root tips increased rapidly. In air-dried soil, seedlings were fully ectomycorrhizal at 9 weeks, and the occurrence of maximum ectomycorrhizal infection coincided with enhanced P acquisition and the initiation of rapid seedling growth. In undried forest soil, seedling growth remained poor, even though the seedlings had well-developed ectomycorrhizae and the incidence of ectomycorrhizal root tips was the same as in air-dried soil. The dominant ectomycorrhizae in airdried soil were associated with an ascomycete fungus, whereas in undried, undisturbed soil they were commonly associated with basidiomycete fungi. Inoculation of sterile soil/sand mix with washed ectomycorrhizal roots from air-dried soil increased the P acquisition and growth of the seedlings significantly compared with controls, whereas ectomycorrhizal inocula from undried soil had no effect on seedling growth, although both inocula resulted in a similar incidence of ectomycorrhizal root tips. Similarly, addition of a small amount of air-dried soil into sterile soil/sand mix resulted in a significantly greater increase in the P content and dry weight of the seedlings, compared with the control, than addition of undried soil. In both treatments, the incidence of ectomycorrhial root tips was similar. As (i) the differentiation in seedling growth between air-dried and undried soil occurred after seedlings became ectomycorrhizal, (ii) the dominant ectomycorrhizae in air-dried soil were different from those in undried soil, and (iii) inocula from air-dried soil, but not from undried soil, stimulated seedling growth in sterile soil/sand mix, it is concluded that development of particular ectomycorrhizae may be involved in seedling growth stimulation and enhanced P acquisition associated with air drying of forest soil.     

INTERACTION BETWEEN THE ECTOMYCORRHIZAL FUNGUS PISOLITHUS TINCTORIUS AND ROOT HAIRS OF PICEA MARIANA (PINACEAE)

The ectomycorrhizal fungus Pisolithus tinctorius interacts with roots of Picea mariana to form a typical mantle and Hartig net. Hyphae alter their growth pattern when in contact with susceptible root hairs in the mycorrhizal infection zone and grow acropetally, gradually covering the length of the hair to form a mantlelike structure. Initial contact with the hair may be influenced by a fibrillar material on the root hair surface. Although many root hairs become surrounded by fungal hyphae, they are not penetrated, and therefore are not entry points for this symbiotic fungus.     

外生菌根菌不同接种方法对樟子松苗木生长的影响

 以提高外生菌根真菌对樟子松(Pinus sylvestris var. mongolica)苗木促生长效果为目的, 在前期研究的基础上, 采用菌株配对培养的方法对获得的樟子松外生菌根真菌进行混合接种菌株组合筛选; 采用苗木截根-菌液浸根和沟施接种方法分别对2年生和3年生樟子松苗木进行野外单接种及混合接种。研究外生菌根真菌不同接种方法、菌株单接种及混合接种对樟子松苗木生长的影响。试验结果表明: 供试菌株及菌株组合对樟子松苗木生长均有一定的促进作用。菌株GT005和菌株035为供试菌株中对樟子松苗木促生长效果最佳的菌株。采用苗木截根-菌液浸根方法接种2年生樟子松苗木130 d, GT005接种的苗木高生长提高54%, 地径生长提高15%, 过氧化氢酶活性提高48%, 而根系活力降低3%; 菌株035接种的苗木高生长提高42%, 地径生长提高56%, 过氧化氢酶活性提高47%, 根系活力提高11%。沟施接种方法接种3年生樟子松苗木100 d, GT005接种苗木高生长提高10%, 地径生长提高15%, 过氧化氢酶活性提高90%, 而根系活力降低34%; 菌株035接种苗木高生长提高7%, 地径生长提高9%, 过氧化氢酶活性提高6%, 而根系活力降低46%; 菌株组合044/GT001和GT001/GT005 接种的苗木高生长仅比对照提高3.47%和2.07%, 而菌株组合 044/025 和044/009 接种的苗木高生长低于对照; 混合接种的苗木其地径生长高于对照0.16%～7.98%。综上所述, 苗木截根-菌液浸根接种方法对苗木的促生长效果显著高于沟施接种方法; 外生菌根菌高效菌株与一般菌株混合接种会弱化高效菌株自身接种效果; 苗木过氧化氢酶活性、苗木根系活力与苗木的生物量间无相关性。     

Lateral root stimulation in the early interaction between Arabidopsis thaliana and the ectomycorrhizal fungus Laccaria bicolor: Is fungal auxin the trigger?

Lateral root (LR) stimulation during early signal exchange between plant roots and ectomycorrhizal (ECM) fungi has recently been shown to be achieved by modulation of auxin gradients. We suggested that this modulation could occur through altered polar auxin transport (PAT) and through activation of auxin signalling pathways in the root. However, it remains unclear, which fungal molecules alter auxin pathways inside the plant partner. It has been suggested in previous studies that auxin released by the fungus could trigger observed plant responses during early signal exchange and later on during root colonization. Here we focus on the early interaction and we provide evidence for an alternative mechanism. Indeed, LR stimulation by the fungus in Arabidopsis thaliana followed a totally different timing than with exogenously applied auxin. Furthermore, experimental conditions that excluded the exchange of soluble molecules while allowing exchange of volatile(s) between the plant and the fungus were sufficient for LR induction, therefore questioning the role of secreted fungal auxin. These data suggest that volatiles released by the fungus and sensed by the plant may act upstream of altered auxin signaling in the plant.Key words: mycorrhiza, ectomycorrhiza, lateral root, auxin, volatiles, ethylene, jasmonic acidInteractions of plant roots with symbiotic, ectomycorrhizal soil fungi lead to lateral root (LR) stimulation during the very early interaction phase.¹ This LR stimulation has recently been shown to be independent of root colonization and to occur as well in non-mycorrhizal plants, such as Arabidopsis suggesting that fungal signals have a broad perception spectrum.^1,2 However, little is known about the type of signals exchanged between fungi and their plant partners during this early interaction phase. Several studies have proposed a role for the phytohormone auxin produced and secreted by ECM fungi as the signalling molecule during ECM fungus/plant signaling.^2–7 Recently we studied changes in auxin response and auxin transport in poplar and Arabidopsis thaliana roots during contact with the ECM fungus Laccaria bicolor.¹ We demonstrated that the presence of the fungus enhances the auxin response and distribution at the root apex and that this, as well as LR stimulation, is reliant on polar auxin transport through AtPIN2 and probably through PtPIN9 in poplar. Here, using Arabidopsis thaliana, whose LR stimulation by Laccaria bicolor has been demonstrated, we propose that not yet identified fungal volatiles may regulate auxin homeostasis in the plant, questioning the contribution of the auxin released by the fungus on the induction of LR.     

The effects of insect pathogenic soil fungi and ectomycorrhizal inoculation of birch seedlings on the survival of Otiorhynchus larvae

• 1 Weevil larvae of the genus Otiorhynchus are a serious problem in agriculture and forestry, causing damage to a wide range of plant species, primarily by larval feeding on roots. Otiorhynchus larvae are a serious pest in forest plantations in Iceland, causing 10–20% mortality of newly‐planted seedlings.
• 2 We studied the effects of soil fungi on the survival of Otiorhynchus sulcatus larvae. The larvae were introduced into pots with birch seedlings grown in: (i) nursery peat; (ii) nursery peat inoculated with three different species of ectomycorrhizal fungi; (iii) nursery peat inoculated with insect pathogenic fungi; (iv) nursery peat inoculated with ectomycorrhizal fungi and insect pathogenic fungi; and (v) nursery peat inoculated with natural forest soil from Icelandic birch woodland.
• 3 Larval survival was negatively affected by inoculation of: (i) the ectomycorrhizal fungus Laccaria laccata; (ii) the ectomycorrhizal fungus Cenococcum geophylum; (iii) the insect pathogenic fungus Metarhizium anisopliae; and (iv) forest soil. Inoculation with the ectomycorrhizal fungus Phialophora finlandia did not have any significant effect on larval survival. No significant synergistic effect was found between insect pathogenic and ectomycorrhizal fungi.
• 4 It is concluded that ectomycorrhizal and insect pathogenic fungi have a significant potential in biological control of Otiorhynchus larvae in afforestation areas in Iceland. Further studies are needed to establish the effect of these fungi in the field and to analyse how mycorrhizal fungi affect root‐feeding larvae.

     

The auxin-inducible GH3 homologue Pp-GH3.16 is downregulated in Pinus pinaster root systems on ectomycorrhizal symbiosis establishment

In an attempt to determine whether auxin-regulated plant genes play a role in ectomycorrhizal symbiosis establishment, we screened a Pinus pinaster root cDNA library for auxin-upregulated genes. This allowed the identification of a cDNA, Pp-GH3.16, which encodes a polypeptide sharing extensive homologies with GH3 proteins of different plants. Pp-GH3.16 was specifically upregulated by auxins and was not affected by cytokinin, gibberellin, abscisic acid or ethylene, or by heat shock, water stress or anoxia. Pp-GH3.16 mRNAs were quantified in pine roots inoculated with two ectomycorrhizal fungi, Hebeloma cylindrosporum and Rhizopogon roseolus. Surprisingly, Pp-GH3.16 was downregulated following inoculation with both fungal species. The downregulation was most rapid on establishment of symbiosis with an indole-3-acetic acid (IAA)-overproducing mutant of H. cylindrosporum, which overproduced mycorrhizas characterized by a hypertrophic Hartig net. This indicates that, despite being auxin-inducible, Pp-GH3.16 can be downregulated on establishment of symbiosis with a fungus that releases auxin. By contrast, Pp-GH3.16 was not downregulated in pine root systems inoculated with a nonmycorrhizal mutant of H. cylindrosporum, suggesting that the downregulation we observed in mycorrhizal root systems was a component of the molecular cross-talk between symbiotic partners at the origin of differentiation of symbiotic structures.     

Partitioning of Intermediary Carbon Metabolism in Vesicular-Arbuscular Mycorrhizal Leek

Vesicular-arbuscular mycorrhizal fungi are symbionts for a large variety of crop plants; however, the form in which they take up carbon from the host is not established. To trace the course of carbon metabolism, we have used nuclear magnetic resonance spectroscopy with [13C]glucose labeling in vivo and in extracts to examine leek (Allium porrum) roots colonized by Glomus etunicatum (and uncolonized controls) as well as germinating spores. These studies implicate glucose as a likely substrate for vesicular-arbuscular mycorrhizal fungi in the symbiotic state. Root feeding of 0.6 mM 1-[13C]glucose labeled only the fungal metabolites trehalose and glycogen. The time course of this labeling was dependent on the status of the host. Incubation with 50 mM 1-[13C]glucose caused labeling of sucrose (in addition to fungal metabolites) with twice as much labeling in uncolonized plants. There was no detectable scrambling of the label from C1 glucose to the C6 position of glucose moieties in trehalose or glycogen. Labeling of mannitol C1,6 in the colonized root tissue was much less than in axenically germinating spores. Thus, carbohydrate metabolism of host and fungus are significantly altered in the symbiotic state.     

Effect of root exudates and extracts on rhizosphere fungi

Summary The effects of root exudates and extracts of Cassia tora L. and Crotalaria medicaginea Lamk., on some dominant rhizosphere fungi isolated from the plants were studied. Root extracts induced a high degree of stimulation in rate of growth of a majority of the fungi tested. Root exudates caused only a marginal effect which was positive on all the fungi except Trichoderma lignorum which was inhibited by the root exudate of C. medicaginea.