In vitro mycorrhization of micropropagated plants: studies on Castanea sativa Mill.

In vitro mycorrhization can be made by several axenic and nonaxenic techniques but criticism exists about their artificiality and inability to reproduce under natural conditions. However, artificial mycorrhization under controlled conditions can provide important information about the physiology of symbiosis. Micropropagated Castanea sativa plants were inoculated with the mycorrhizal fungus Pisolithus tinctorius after in vitro rooting. The mycorrhizal process was monitored at regular intervals in order to evaluate the mantle and hartig net formation, and the growth rates of mycorrhizal and nonmycorrhizal plants. Plant roots show fungal hyphae adhesion at the surface after 24 hours of mycorrhizal induction. After 20 days a mantle can be observed and a hartig net is forming although the morphology of the epidermal cells remains unaltered. At 30 days of root–fungus contact the hartig net is well developed and the epidermal cells are already enlarged. After 50 days of mycorrhizal induction, growth was higher for mycorrhizal plants than for nonmycorrhizal ones. The length of the major roots was lower in mycorrhizal plants after 40 days. Fresh and dry weights were higher in mycorrhizal plants after 30 days. The growth rates of chestnut mycorrhizal plants are in agreement with the morphological development of the mycorrhizal structures observed at each mycorrhizal time. The assessment of symbiotic establishment takes into account the formation of a mantle and a hartig net that were already developed at 30 days, when differences between fresh and dry weights of mycorrhizal and nonmycorrhizal plants can be quantified. In vitro conditions, mycorrhization influences plant physiology after 20 days of root–fungus contact, namely in terms of growth rates. Fresh and dry weights, heights, stem diameter and growth rates increased while major root growth rate decreased in mycorrhizal plants.Springe

Monotropastrum humile var. humile is associated with diverse ectomycorrhizal Russulaceae fungi in Japanese forests

The original publication is available at www.springerlink.comArticleECOLOGICAL RESEARCH. 23(6):983-993(2008)journal articl

Identification of cytoskeletal components in pine ectomycorrhizas

Ectomycorrhizal associations were synthesized between pine seedlings and the fungi Suillus bovinus (L.) ex Fr. or Paxillus involutus (Batsch ex Fr.) Fr. Immunoblotting of polypeptides separated electrophoretically from crude tissue extracts revealed the abundant presence of tubulin and actin in ectomycorrhiza and lower amounts in the fungal strands surrounding the ectomycorrhizal roots. In ectomycorrhiza the alpha-tubulins from fungal hyphae and plant cells were clearly distinguishable but such discrimination was not possible for beta-tubulin or actin due to the similar mobility of proteins originating from the conifer and fungal tissues. Young ectomycorrhizal short roots were fixed while still attached to the seedlings and, using indirect immunofluorescence microscopy with tubulin antibodies, microtubules were detected in both the conifer cells and in fungal hyphae. In the host plant cytoplasmic and spindle microtubules were visualized in meristem cells and in differentiating vascular tissue but not in the cortical cells. In symbiotic hyphae the microtubule tracks and spindles of dividing nuclei were clearly distinguished in the mantle hyphae in the tip region of the short roots. In the Hartig net hyphae microtubule tracks changed to a less clear, reticulate structure. Actin was visualized as long filaments in vascular tissue cells and as small microfilament bundles in mantle hyphae. Short microtubules and actin dots were detected in cytoplasm-containing hyphae on the strand surface. The possible role of the cytoskeletal elements in the maintenance of the ectomycorrhizal association is discussed

Nitrogen translocation between Alnus glutinosa (L.) Gaertn. seedlings inoculated with Frankia sp and Pinus contorta Doug ex Loud seedlings connected by a common ectomycorrhizal mycelium

Uptake and translocation of nitrogen was studied in laboratory microcosms consisting of Alnus glutinosa (L.) Gaertn., Frankia sp., Paxillus involutus (Fr.) Fr. and Pinus contorta Dougl. ex Loud. P. involutus was shown to form a fully functional ectomycorrhizal association with alder as well as pine, and the seedlings thus became interconnected by a common mycelium. When microcosms were exposed to N-15(2) gas, interplant translocation of N-15 was observed in two out of three experiments. N-15(2) was fixed by Frankia and translocated to all other parts of the system. In the two experiments in which interplant translocation occurred, between 5 and 15 % of the N-15 recovered was found in the pine seedlings. Within seven days, fixed N2 was incorporated into amino acids in the Frankia nodules, translocated to both the A. glutinosa and P. contorta seedlings and incorporated into macromolecules. In alder seedlings, citrulline and ornithine were the free amino acids that had both the highest N-15 enrichment levels and concentrations. In pine, glutamine and citrulline had the highest N-15 concentrations, and glutamine had the highest level of N-15 enrichment. N-15 enrichment levels were greatest in the nodules, at between 5.5 and 29 % in the different amino acids and 12 % in the macromolecular fraction. Enrichment levels decreased with increasing distance from the nodules. The uptake and translocation of N-15 applied as (NH4Cl)-N-15 to the mycelium was also studied. N-15 was incorporated into amino acids in the mycelium and translocated further in this form. Generally, free amino acids had high N-15 enrichment levels in the mycelium, decreasing along the translocation pathway. Citrulline and glutamine were the amino acids with highest N-15 concentrations in all parts of the system. N-15 was also found in the macromolecular fraction