Axenic mycorrhization of wild type and transgenic hybrid aspen expressing T-DNA indoleacetic acid-biosynthetic genes

 We describe and document the in vitro synthesis of ectomycorrhiza between roots of wild type and transgenic aspen (Populus tremula × P. tremuloides), expressing Agrobacterium tumefaciens T-DNA indoleacetic acid (IAA)-biosynthetic genes, and Amanita muscaria. Plantlets were raised from tissue culture. The root system of approximately 4-week-old plantlets was transferred to Petri dishes and incubated together with fungal mycelia under sterile conditions. Ectomycorrhiza showing both a well developed hyphal mantle and Hartig net were established within 3 to 4 weeks. Formation and morphology of ectomycorrhiza were not affected by the transformation of aspen, expressing the IAA biosynthetic genes in roots. As both hybrid aspen and fungal cells can be genetically engineered, this system offers a new approach to the study of mycorrhizal symbioses. Received: 19 January 1996 / Accepted: 23 January 1996     

Compatible and incompetent Paxillus involutus isolates for ectomycorrhiza formation in vitro with poplar (Populus x canescens) differ in H2O2 production

Abstract: Isolates of Paxillus involutus (Batsch) Fr. collected from different hosts and environmental conditions were screened for their ability to form ectomycorrhizal symbiosis with hybrid poplar P. × canescens ( = Populus tremula L. × P. alba) in vitro. The ability to form ectomycorrhiza varied between the fungal isolates and was not correlated with the growth rate of the fungi on agar-based medium. The isolate MAJ, which was capable of mycorrhiza synthesis under axenic conditions, and the incompetent isolate NAU were characterized morphologically and anatomically. MAJ formed a typical hyphal mantle and a Hartig net, whereas NAU was not able to penetrate the host cell walls and caused thickenings of the outer cell walls of the host. MAJ, but not NAU, displayed strong H₂O₂ accumulation in the outer hyphal mantle. Increases in H₂O₂ in the outer epidermal walls and adjacent hyphae of the incompetent isolate were moderate. No increases of H₂O₂ in response to the mycobionts were found inside roots. Suggested functions of H₂O₂ production in the outer hyphal mantle of the compatible interaction are: growth regulation of the host's roots, defence against other invading microbes, or increasing plant-innate immunity. The system established here for P. × canescens compatible and incompetent fungal associations will be useful to take advantage of genomic information now available for poplar to study tree-fungal interactions at the molecular and physiological level.     

Biosynthesis and Secretion of Indole-3-Acetic Acid and Its Morphological Effects on Tricholoma vaccinum-Spruce Ectomycorrhiza

Fungus-derived indole-3-acetic acid (IAA), which is involved in development of ectomycorrhiza, affects both partners, i.e., the tree and the fungus. The biosynthesis pathway, excretion from fungal hyphae, the induction of branching in fungal cultures, and enhanced Hartig net formation in mycorrhiza were shown. Gene expression studies, incorporation of labeled compounds into IAA, heterologous expression of a transporter, and bioinformatics were applied to study the effect of IAA on fungal morphogenesis and on ectomycorrhiza. Tricholoma vaccinum produces IAA from tryptophan via indole-3-pyruvate, with the last step of this biosynthetic pathway being catalyzed by an aldehyde dehydrogenase. The gene ald1 was found to be highly expressed in ectomycorrhiza and induced by indole-3-acetaldehyde. The export of IAA from fungal cells is supported by the multidrug and toxic extrusion (MATE) transporter Mte1 found in T. vaccinum. The addition of IAA and its precursors induced elongated cells and hyphal ramification of mycorrhizal fungi; in contrast, in saprobic fungi such as Schizophyllum commune, IAA did not induce morphogenetic changes. Mycorrhiza responded by increasing its Hartig net formation. The IAA of fungal origin acts as a diffusible signal, influencing root colonization and increasing Hartig net formation in ectomycorrhiza.     

Global patterns of gene regulation associated with the development of ectomycorrhiza between birch (Betula pendula Roth.) and Paxillus involutus (Batsch) Fr

The formation of ectomycorrhizal (ECM) root tissue is characterized by distinct morphological and developmental stages, such as preinfection and adhesion, mantle, and Hartig net formation. The global pattern of gene expression during these stages in the birch (Betula pendula)-Paxillus involutus ECM association was analyzed using cDNA microarrays. In comparison with nonsymbiotic conditions, 251 fungal (from a total of 1,075) and 138 plant (1,074 in total) genes were found to be differentially regulated during the ECM development. For instance, during mantle and Hartig net development, there were several plant genes upregulated that are normally involved in defense responses during pathogenic fungal challenges. These responses were, at later stages of ECM development, found to be repressed. Other birch genes that showed differential regulation involved several homologs that usually are implicated in water permeability (aquaporins) and water stress tolerance (dehydrins). Among fungal genes differentially upregulated during stages of mantle and Hartig net formation were homologs putatively involved in mitochondrial respiration. In fully developed ECM tissue, there was an upregulation of fungal genes related to protein synthesis and the cytoskeleton assembly machinery. This study highlights complex molecular interactions between two symbionts during the development of an ECM association.     

Ectomycorrhiza formation between Pseudotsuga menziesii seedling roots and monokaryotic and dikaryotic isolates of Laccaria bicolor

Seedling roots of Pseudotsuga menziesii were colonized with three monokaryotic isolates and one dikaryotic isolate of Laccaria bicolor to assess the effect of fungal genotype on ectomycorrhiza formation. Ectomycorrhizas resulting from colonization by the dikaryotic isolate had a multilayered mantle and a cortical Hartig net. One monokaryotic isolate (ss7) formed ectomycorrhizas comparable in anatomy to those induced by the dikaryotic isolate. Two other monokaryotic isolates (ss5, ss1) failed to form mantles or Hartig nets. Roots colonized by these isolates developed characteristics indicating an incompatible reaction.     

Induced defence responses limit Hartig net formation in ectomycorrhizal birch roots

Roots of clonal birches ( Betula pendula ) were inoculated with the ectomycorrhizal fungi Paxillus involutus (isolates P0 and Mi) and Hebeloma cylindrosporum (strains D1 and D105). These fungi showed different rates of mycorrhiza formation in vitro . Mature mycorrhizas were obtained after only 2–4 d with H. cylindrosporum , whereas 6–8 d were necessary with P. involutus isolate P0, and P. involutus isolate Mi was not able to form mature mycorrhiza during the 10 d of the experiment. Temporal changes in PAL activity and the expression of genes encoding intracellular pathogenesis-related proteins were followed after inoculating birch roots with these fungi. Transient increase of PAL activity, and transient induction of expression of the wound-inducible Bet v1-SC1 gene, were observed in roots challenged with both H. cylindrosporum strains and the P. involutus isolate P0. These changes were found to coincide with hyphal penetration between root cells during Hartig net formation, and were never observed in roots inoculated with the poorly aggressive P. involutus isolate Mi. Examination of mycorrhizal root sections under u.v. light indicated the presence of phenolic compounds in the host cell walls at the vicinity of the Hartig net. These results strongly suggest that hyphal penetration between the root cells triggers a transient defence response which, in turn, could limit Hartig net formation to the outer layer of the root cortex.     

赤松不定根和愈伤组织与松口蘑外生菌根菌的相互作用

本实验报道了以简单的离体培养方式来诱导赤松不定根和愈伤组织与松口蘑的菌根反应。不定根和愈伤组织均起源于无菌苗的下胚轴,接种2周后,菌丝体开始包围不定根。接种3周后,菌丝体出现在不定根皮层细胞间,哈蒂氏网型的形成也同时被确认。在愈伤组织培养物中,细胞间也能观察到菌丝体及拟-哈蒂氏网结构。这是第一个离体条件下成功地诱导赤松培养组织与松口蘑形成外生菌根的报道。     

Cytolocalization of glycogen, starch, and other insoluble polysaccharides during ontogeny of Paxillus involutus–Betula pendula ectomycorrhizas

The Paxillus involutus (Fries) Karsten– Betula pendula Roth association was studied during the early stages of formation. Cytological studies revealed fungal colonization behind the root cap and gradually around the entire root apex. Ultrastructural investigations were carried out and insoluble polysaccharide distribution was followed. The density of starch grains increased in plant cells especially after 4 d of contact between the two partners, but later on decreased strongly in the root cap. Large amounts of glycogen were revealed in the hyphae in certain mycorrhizal regions after 6 d of contact: in the Hartig net, in the inner sheath but only near the net, and all along the outer sheath surrounding the mycorrhiza. Thickenings of the epidermal cell walls were detected as early as 2 d after contact and then varied according to the distance from the root tip. Such polysaccharide distributions are assumed to show a transfer of carbohydrates from the root to the fungus and are discussed in terms of carbon requirements for both partners.     

Relative importance of the endomycorrhizal and (or) ectomycorrhizal associations in Allocasuarina and Casuarina genera

This work was carried out to determine the relative importance of the endomycorrhizal and (or) ectomycorrhizal association in species of Casuarina and Allocasuarina. Under axenic conditions, Pisolithus and Scleroderma isolates formed ectomycorrhizas with a mantle and a Hartig net on Allocasuarina verticillata but failed to form a Hartig net on Casuarina glauca. In a controlled soil system, C. glauca was inoculated with the endomycorrhizal fungus Glomus intraradices Schenck & Smith, and A. verticillata was inoculated with Pisolithus albus IR100 Bougher & Smith and (or) G. intraradices. Both symbionts significantly stimulated growth in both plant species. For A. verticillata, its growth response to ectomycorrhizal inoculation was higher than to endomycorrhizal inoculation. When both symbionts were inoculated, antagonism among the fungal isolates was observed with a higher ectomycorrhizal colonization. These results showed that A. verticillata was ectomycorrhizal dependent, whereas C. glauca was endomycorrhizal dependent. From a practical point of view, this study shows the importance of selecting compatible mycorrhizal fungi for developing successful inoculation programmes. In addition, it would help to further research and determine the effect of ecto- and endo-mycorrhizal symbiosis on the formation and function of N2-fixing actinorhizal nodules.     

Paxillus involutus/Pinus sylvestris Mycorrhizae from Heavily Polluted Forest II. Ultrastructural and Cytochemical Observations

Paxillus involutus/Pinus sylvestris mycorrhizae, collected from experimental plots (situated in a Pino-Quercetum forest) treated with cadmium dust, were previously reported to accumulate metals like Cd, P, S, N, Al and Si in vacuolar bodies. The Gomori-Swift test was applied to the same material to reveal if cysteine-rich proteins might be involved in element detoxification. Vacuolar deposits of high and low phosphate level were both stained. Higher intensity of the Swift reaction was found in the fungal mycelium from mycorrhizae sampled from polluted plots than in mycorrhizae from unpolluted sites. In both cases a higher intensity of the test was revealed in the host cytoplasm close to the Hartig net than in internal parts of roots. Different intensities of the Gomori-Swift reaction were also observed when the Hartig net was compared to the fungal mantle. A PATAg test was additionally processed and revealed an accumulation of glycogen in rosettes and net-like structures when samples from cadmium dust treated plots were observed.     

A sheathing mycorrhiza between the tropical bolete Phlebopus spongiosus and Citrus maxima

Phlebopus (Ph.) spongiosus was recently described from several pomelo orchards (Citrus maxima) in southern Vietnam. This fungus was suspected to associate with pomelo plants as an ectomycorrhiza, although members of the genus Phlebopus have previously been presumed saprotrophic. To clarify this association, pomelo roots collected from the orchard (in situ roots), and those cultured with Ph. spongiosus (in vitro roots) in test tubes for 12 wk, were examined for ectomycorrhizal colonization. Both in vitro and in situ roots were analyzed for colonization using fungal LSU nuclear ribosomal DNA sequencing. The in situ roots exhibited the anatomical features of ectomycorrhizae: a thick fungal mantle, Hartig net, and extramatrical hyphae. The Hartig net, however, was very rare and showed discontinuous development. The in vitro association between Ph. spongiosus and C. maxima showed ectomycorrhiza-like structures, i.e., mantles and rhizomorphs in the plant roots, but no Hartig net development in the roots. Continuous hyphal penetration was restricted to the exodermis in both in situ and in vitro roots. Although the association between Ph. spongiosus and C. maxima could be considered ectomycorrhizal, its anatomy matches the unique feature known as sheathing mycorrhiza.     

Pochonia chlamydosporia fungal activity in a solid medium and its crude extract against eggs of Ascaridia galli

The present study aimed to evaluate the ovicidal activity (type 3 effect) of VC1 and VC4 isolates of Pochonia chlamydosporia in a solid medium and the action of a crude extract of P. chlamydosporia against eggs of Ascaridia galli. To evaluate ovicidal activity in culture medium, 1000 A. galli eggs were plated on Petri dishes containing 2% water-agar with grown fungal isolates (VC1 or VC4) and without fungus (control group) and were examined at 1, 3 and 5 days post-inoculation (assay A). Then, to test the action of crude extracts of P. chlamydosporia (VC1 or VC4), 500 eggs of A. galli were plated on Petri dishes of 4.5 cm diameter with 5 ml of fungal filtrate from each tested isolate. The control group consisted of 500 eggs of A. galli with 10 ml of distilled water on each Petri dish (assay B). Fungal isolates were effective (P < 0.01) at destroying these eggs, showing a type 3 effect at the studied intervals. On the other hand, the crude extract of isolates (VC1 or VC4) reduced the number of A. galli eggs in the treated group compared with the control group by 64.1% and 56.5%, respectively. The results of the present study show that P. chlamydosporia is effective at destroying eggs of A. galli and could therefore be used in the biological control of nematodes.     

Structure and function of the ectomycorrhizal association between Paxillus involutus and Betula pendula. II. Metabolic changes during mycorrhiza formation

Seedlings of Betula pendula Roth. were grown in the presence of Paxillus involutus (Batsch) Fr., and metabolic changes during mycorrhiza formation were examined by measuring organic acid and amino acid pools and related enzyme activities, following sequential harvests. Glutamine, aspartate and asparagine pools were always lower in infected roots than in non-infected roots, especially during Hartig net initiation and formation. Glutamate concentration was similar in both tissues. Citrate and malate were the two major organic acids detected and their concentrations were equal in infected and non-infected roots. Aspartate aminotransferase, glutamine synthetase, NAD-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activities were higher in infected roots than non-infected roots. For all enzymes revealed on polyacrylamide gels, both root and fungal isoforms were present in infected roots. Quantitative changes in enzyme capacities and metabolite pools indicated that mycorrhiza formation caused a re-arrangement of the main metabolic pathways during the very early stages following contact, which might be related to the structural changes.     

Distribution of lead in mycorrhizal and non-mycorrhizal Norway spruce seedlings

Non-mycorrhizal spruce seedlings (Picea abies Karst.) and spruce seedlings colonized with Lactarius rufus (Scop.) Fr. or two strains of Paxillits involutus (Batsch) Fr. were grown in an axenic silica sand culture system with frequently renewed nutrient solution. After successful mycorrhizal colonization, the seedlings were exposed to 1 μM PbCI₂ for 19 weeks. The degree of infection in all of the mycorrhizal treatments approached 100% during the experiment and was not affected by exposure to Pb. However, the number of root tips per root dry weight and the shoot: root ratio, both in the non-mycorrhizal and the mycorrhizal seedlings, had decreased after the 19 week treatment with PbCl₂ Using X-ray microanalysis, the distribution and concentration of Pb in the tissues of mycorrhizal and non-mycorrhizal root tips were compared. In the mycorrhizae of seedlings exposed to Pb no significant accumulation of Pb in the hyphal mantle or in fungal cell walls of the Hartig net were detected. Lead accumulated primarily in the cortex cell walls both of non-mycorrhizal and mycorrhizal root tips. No significant difference of Pb concentrations in root cortex cell walls of non-mycorrhizal and mycorrhizal seedlings was found; except for seedlings colonized with Paxillus involutus strain 537. However, at the endodermis no effect of mycorrhizal fungal colonization on the Pb tissue concentration was detected. The presence of the fungal sheath did not prevent Pb from reaching the root cortex. The endodermis acted as a barrier to Pb radial transport in both mycorrhizal and non-mycorrhizal seedling roots.     

真菌营养亲和群鉴定方法的改进

真菌营养不亲和鉴定的通常方法是将菌株两两对接,观察它们之间是否产生拮抗反应,此方法简单且成本低,但是因每个菌株要与其余菌株配对,若测试菌株多,则工作量大。为了减少工作量,提高效率,将配对方式加以优化,仅利用3个培养皿即可得到7个菌株之间的营养亲和关系,结果直观清晰,可以大大减少工作量,节省了材料和时间,并且降低了接种过程中污染风险。     

An Improved Growth Medium to Assess Mycelial Compatibility Groups in Sclerotium rolfsii

Mycelial compatibility is assayed mainly by pairing mycelial plugs of field isolates on Petri dishes with agar media. Although methodologically simple, mycelial compatibility testing requires an artificial growth medium that permits the identification of compatible and incompatible interactions. In this work, several growth media were studied to assess consistently mycelial interactions between Sclerotium rolfsii isolates. A modification of Patterson’s medium with an increment of 25% glucose from the original concentration at a rate of 23.4 g/l and amended with 180 μl/l of red food colouring was the most effective combination for enhancing the size, density and distinctiveness of the aversion zone between incompatible isolates. This medium allowed the unequivocal identification of compatible and incompatible reactions of a set of five S. rolfsii isolates, which could be determined quickly after 5 days of incubation in the dark at 25°C. This new formulation improved significantly and consistently the assessment of the aversion zone reaction that was visible as a red line on the colony reverse as compared to that assessed using previous media formulations, for which the visualization of aversion zones was scarcely discernible. The utility of the improved growth medium was validated by microscopic observations of the contact area of hyphal pairings between isolates of S. rolfsii in microscope slide cultures.     

Root soluble carbohydrates of Afzelia africana Sm. seedlings and modifications of mycorrhiza establishment in response to the excision of cotyledons

Stem length, number of secondary lateral roots, shoot dry weight and reducing sugar concentrations of root were significantly reduced when translocation of reserves from cotyledons to the roots of Afzelia africana seedlings was interrupted by complete or partial cotyledon excision. The sucrose but not the glucose concentration of lateral roots also decreased significantly after complete cotyledon excision. Hartig net development rather than fungal sheath formation was affected after inoculation with the early fungal isolate E1 and by both late-stage fungal isolates L1 and L2 after partial or complete cotyledon excision. However, mycorrhizal colonization by the early fungal isolate E2 was not affected by cotyledonary reserves, suggesting that this fungal isolate has a lower carbohydrate requirement than fungal isolates E1, L1 and L2. The late-stage fungal isolates L1 and L2 induced a hypersensitivity reaction by epidermal cell walls of the host plant after complete cotyledon excision, suggesting they are more dependent than the early fungal isolate E1 on available root carbohydrate substrates for ectomycorrhizal colonization. These results are discussed in the light of the hypothesis that early and late-stage fungi were different carbohydrate requirements, and that the time sequence of colonization was related to the root carbohydrate status, which increased with time.     

In vitro synthesis of Pisolithus-Eucalyptus ectomycorrhizae: synchronization of lateral tip emergence and ectomycorrhizal development

 A simple and reproducible in vitro system is described for the synthesis of Pisolithus-Eucalyptus grandis ectomycorrhizae. Hyphal discs from actively growing colonies were placed in large petri dishes containing minimum nutrient agar overlaid with cellophane and allowed to grow for 7 days. Seeds were then surface sterilized and placed above the expanding fungal colonies and the plates slanted. Seedlings that germinated and grew in the presence of fungal hyphae had twice as many lateral root tips as seedlings that germinated before they were transferred onto hyphal mats. In addition, the lateral root tips of inoculated seedlings had a faster maturation rate and emerged closer to the primary root apex than non-inoculated seedlings. All lateral tips emerged in contact with fungal hyphae and the differentiation of ectomycorrhizae was followed by examining lateral tips basipetally along a single primary root. Typical ectomycorrhizae had formed on 4-day-old lateral tips, i.e. a mantle, radially elongated epidermal cells and a Hartig net commencing about 0.3 mm behind the lateral root apex. Thereafter, the mantle continued to thicken and the apical meristem diminished. The Hartig net often surrounded the apex of 11- to 12-day-old lateral root tips. This model system will facilitate detailed studies on synchronized ectomycorrhizal development and associated molecular and biochemical changes. Accepted: 12 January 1996