Salt stress affects in vitro growth and in situ symbioses of ectomycorrhizal fungi

Ten isolates of six species of ectomycorrhizal fungi were grown in vitro at nine concentrations of three sodium salts (NaCl, Na₂SO₄, Na₃C₆H₅O₇) for 4 weeks. Colony diamater, biomass and protein content of fungi were evaluated. Isolates of Pisolithus tinctorius and Suillus luteus were more tolerant of NaCl and Na₂SO₄ than of Na₃C₆H₅O₇. Fungi in the genera Cenococcum, Laccaria, and Thelephora were highly intolerant of Na₃C₆H₅O₇ and Na₂SO₄ in vitro. Biomass and protein content of fungi generally declined with increasing substrate salinity in solution culture. In situ ectomycorrhizal colonization by Laccara laccata and P. tinctorius and the dry weight of Pinus taeda seedlings were significantly reduced by 80 mM NaCl after 14 weeks. Only select ectomycorrhizal fungi appear capable of growth and symbiosis in saline soils.     

Effect of a synthetic pyrethroid on the growth of ectomycorrhizal fungi and mycorrhiza formation in Pinus patula

Th effect of fenvalerate 20% EC (a synthetic pyrethroid) on the growth of two ectomycorrhizal fungi, Thelephora terrestris and Laccaria laccata, was studied in vitro. In the case of T. terrestris, mycelial growth was promoted at lower concentrations of the insecticide but inhibited at higher concentrations; mycelial growth of L. laccata decreased with increase in concentration. The effect of the insecticide at three concentrations (0.25, 0.5, 1.5 ppm) was also studied on ectomycorrhizal formation in 4-month-old seedlings of Pinus patula in nursery trials. At lower concentrations the insecticide had no significant effect on mycorrhizal formation.     

Inoculation and field testing of Sitka spruce and Douglas fir with ectomycorrhizal fungi in the United Kingdom

Picea sitchensis and Pseudotsuga menziesii seedlings were grown in containers, inoculated with ectomycorrhizal fungi, and planted in British forestry sites. Root samples taken during the year after planting were assessed for mycorrhiza formation. Survival and shoot height were assessed at the end of each year. Observations were made each autumn on the occurrence of sporophores of ectomycorrhizal fungi. Pot experiments were used to assess the colonization potential of soils from the experimental locations. Assessment of mycorrhiza formation by the inoculant fungi both before planting and the following year showed much variation among the fungi used. Similar variation was found among field sites. Inoculation with Laccaria isolates was most successful. Height measurements are reported for the first 2 years after planting, at which time there were few significant effects on growth of Picea sitchensis or Pseudotsuga menziesii seedlings. Experimental assessment of colonization potential was of little value in this work for predicting events in the forest.     

Effects of red pine (Pinus resinosa Ait.) mycorrhizoplane-associated actinomycetes onin vitro growth of ectomycorrhizal fungi

Mycorrhizoplane-associated actinomycetes were isolated using an enrichment technique from red pine (Pinus resinosa Ait.) roots of seedlings recently outplanted onto cleared northern hardwood sites in the Upper Peninsula of Michigan, USA. Interactions were assessedin vitro between actinomycete isolates and three commonly occurring ectomycorrhizal fungi (Laccaria bicolor (Maire) Orton,L. laccata (Scop.: Fr.) Berk. and Br., andThelephora terrestris Fr.). Most actinomycete isolates exerted a range of effects on the growth of the three fungus isolates during the four week test period, inhibiting some while stimulating others; several inhibited growth of all three fungus isolates. Mycorrhizoplane-associated actinomycetes show potential for use as coinoculants with selected ectomycorrhizal fungi to optimize the soil microflora for developing seedlings.     

Inoculation of pines in a nursery withPisolithus tinctorius andThelephora terrestris in Korea

Summary Mycelial inocula of the two ectomycorrhizal fungi,Pisolithus tinctorius (Pt) andThelephora terrestris (Tt) were artificially introduced into fumigated nursery soil before seed sowing. At the end of the first growing season, Pt stimulated the height growth ofPinus densiflora, P. thunbergii, P. rigida, andP. rigida×taeda by 55, 36, 69, and 37%, respectively, compared with control seedlings (with no fumigation and no inoculation). When growth stimulation was expressed by dry weight, Pt increased dry weight ofP. densiflora andP. rigida×taeda by 143% and 128%, respectively, over control seedlings. Total absorption (per plant top) of nitrogen and phosphorus of inoculatedP. densiflora seedlings was also increased by 188 and 144%, respectively, even though the concentration of these two elements in the tissue was not significantly different from that of control seedlings.Tt failed to stimulate growth of the inoculated plants possibly due to its lower level of infection (40–60% mycorrhizal tips) compared with the relatively high infection (over 80%) of Pt.Pinus koraiensis did not respond to inoculation during the first growing season, probably due to its inherent characteristics of producing a small number of short roots and its slow shoot growth during the first few years after germination.This study was supported by the International Foundation for Science in Stockholm, Sweden.     

Spermidine and the ectomycorrhizal fungus Pisolithus tinctorius synergistically induce maturation of Scots pine embryogenic cultures

Exogenous spermidine (Spd) and the ectomycorrhizal (ECM) fungus Pisolithus tinctorius (Pers.) Coker and Couch had a synergistic effect on the maturation of Scots pine (Pinus sylvestris L.) somatic embryos. Induced maturation was expressed as a higher number of cell masses able to form embryos and a greater number of embryos formed per cell mass. In contrast, treatment with P. tinctorius alone on the hormone-free medium resulted in the lowest embryo-forming capacity. Retarded proliferation growth appeared to be required for maturation, but did not explain the synergistic effect of the fungus and exogenous Spd. Simultaneous treatment did not result in lower concentrations of putrescine (Put), Spd or spermine (Spm) in the embryogenic cell masses relative to the separate treatments. Our study is the first report on the use of a specific ECM fungus to induce maturation of somatic embryos, and it indicates that P. tinctorius was able to modify the maturation media in a way that, together with exogenous Spd, positively affected embryogenic cultures of Scots pine. Our study also shows that it is possible to enhance plant development other than root formation by using specific ECM fungi.     

Coinoculation of aseptically grown Douglas fir with pairs of ectomycorrhizal fungi

Douglas fir seedlings grown under aseptic conditions in a peat-vermiculite substrate were inoculated with four pairs of ectomycorrhizal fungi to assess the relative inoculum dosages needed to establish two mycorrhizal fungi simultaneously in the same root system. The dual fungal combinations tested were: Pisolithus arhizus + Rhizopogon subareolatus, P. arhizus + R. roseolus, Laccaria bicolor + P. arhizus and L. bicolor + R. subareolatus. A total of 12 ml of inocula per plant was applied at the rates: 0+12, 3+9, 6+6, 9+3, 12+0, and 0+0 (v+v) for each combination. After 3 months growth, the number of mycorrhizas and uninfected short roots as well as the total plant biomass produced were recorded. Inoculations were successful with the fungal combinations P. arhizus + R. subareolatus and L. bicolor + P. arhizus. Plants developed P. arhizus and R. subareolatus mycorrhizas only at the rate 9Pa + 3Rs; at other rates tested, only monospecific mycorrhizas were formed. Plants developed L. bicolor and P. arhizus mycorrhizas at the three rates containing both fungi. L. bicolor behaved as an aggressive root colonizer and its level of root colonization remained constant at increasing rates of P. arhizus inoculum. L. bicolor displaced R. subareolatus at all inocula rates. P. arhizus displaced R. roseolus except at the rate 3Pa + 9Rr, with only a low number of mycorrhizas formed by either fungus. Total plant biomass was significantly increased by the presence of any fungal combination up to four times the values for uninoculated controls. P. arhizus and R. subareolatus were more effective in promoting plant growth and stimulating short root formation than either L. bicolor or R. roseolus.     

Lignosulfonate promotes the interaction between Scots pine and an ectomycorrhizal fungus Pisolithus tinctorius in vitro

Lignosulfonate (LS) is a lignin-based polymer obtained as a by-product from paper industry, which may have potential as an amendment with macronutrients. We studied effects of LS on the interaction between Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings and the ectomycorrhizal (ECM) fungusPisolithus tinctorius (Pers.) Coker and Couch. The experiments were performed in vitroon the MMN agar medium containing Fe–LS chelate at the concentrations of 0, 5, 10 and 25 mg/L. Inoculation with P. tinctoriusincreased root growth of the seedlings. Fe–LS enhanced P. tinctorius induced formation of lateral roots and had a dose-dependent positive effect on the establishment of mycorrhizas on the seedlings. The growth of the fungal mycelium was improved by Fe–LS, which might cause faster and more intensive contact with the roots and, thus, better root growth and mycorrhiza formation. P.tinctorius enhanced also adventitious root formation and subsequent root growth of the hypocotyl cuttings but without any synergistic effect with Fe–LS. Our study with P. tinctorius and Scots pine in vitro indicates that a low-cost by-product Fe–LS, obtained from paper industry, may be a potential tool to improve the efficiency of fungal inoculations, thus, facilitating the early interaction between an ECM fungus and host seedling.     

Polyphosphate granules are an artefact of specimen preparation in the ectomycorrhizal fungusPisolithus tinctorius

Summary Polyphosphate granules are precipitated in the vacuoles of the ectomycorrhizal fungusPisolithus tinctorius (Pers.) Coker & Couch by various treatments, including conventional specimen preparation. Granules are not produced by glutaraldehyde fixation but appear at early stages of ethanol dehydration and are visible with Nomarski DIC microscopy. They show -metachromasy with toluidine blue O at low pH, are extracted by cold trichloroacetic acid and contain phosphorus and calcium as demonstrated by X-ray microanalysis. The granules are surrounded by electron-lucent areas that do not contain these elements at detectable levels. In contrast, vacuoles of freeze-substituted hyphae contain evenly dispersed flocculent material. Phosphorus and potassium are distributed more or less uniformly throughout, but calcium is not detected. This indicates that polyphosphate is present in the vacuole of living hyphae in soluble form and is precipitated to form granules by various treatments. It is thought that granules form when membranes, including the tonoplast, become leaky and there is an influx of precipitating ions such as calcium.Abbreviations DIC differential interference contrast - GMA glycol methacrylate - MMN modified Melin Norkrans - NMR nuclear magnetic resonance - P_i inorganic phosphate - STEM scanning transmission electron microscope     

Effect of dual inoculation of Douglas fir with the ectomycorrhizal fungus Laccaria laccata and mycorrhization helper bacteria (MHB) in two bare-root forest nurseries

Douglas fir (Pseudotsuga menziesii) seedlings in two bare-root forest nurseries were inoculated with the ectomycorrhizal fungus Laccaria laccata, together or not with one of five mycorrhization helper bacteria isolated from L. laccata sporocarps or mycorrhizas and previously selected by in vitro and glasshouse screenings. With the most efficient MHB isolates, when compared to the control with no bacteria, the percent of mycorrhizal short roots was increased from 60 to 90 or from 80 to 100, depending on the nursery, with inoculation doses as low as 10⁶ living cells per m². A dual inoculum made of calcium alginate beads containing the two microorganisms appears to be a valuable technique for increasing the efficiency of ectomycorrhizal inoculation of planting stocks in forest nurseries.     

Motile tubular vacuoles in extramatrical mycelium and sheath hyphae of ectomycorrhizal systems

Summary Extramatrical mycelium and outer hyphae of the sheath ofEucalyptus pilularis-Pisolithus tinctorius mycorrhizas contain abundant motile tubular vacuoles which accumulate the carboxyfluorescein analogue Oregon Green 488 carboxylic acid. The fluorochrome accumulates in a system of small vacuoles, tubules, and larger vacuoles, which are interlinked, motile, and pleiomorphic, in external hyphae, cords, and hyphae of the outer sheath. There is often a difference in fluorescence between two neighbouring cells, indicating that the dolipore septum exercises control on the movement of material between cells. Generally the motile tubular vacuole system in mycorrhizas resembles that previously found in isolated mycelium. The majority of fungal cells in the sheath contain no fluorochrome even after long exposure of the mycorrhiza to the solution, but with differential interference optics the cells are clearly seen to be alive and to contain vacuoles resembling those in the outer hyphae. In translocation experiments, long-distance transport of the fluorochrome is slow and slight, or even nonexistent in some cases.Abbreviations carboxy-DFF Oregon Green 488 carboxylic acid - carboxy-DFFDA Oregon Green 488 carboxylic acid diacetate - DIC differential interference contrast Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

In vitro weathering of phlogopite by ectomycorrhizal fungi

The ways in which ectomycorrhizal fungi benefit tree growth and nutrition have not been fully elucidated. Whilst it is most probably due to improved soil colonization, it is also likely that ectomycorrhizal fungi could be directly involved in nutrient cycling of soil reserves. This study assessed access by two species of ectomycorrhizal fungi to soil nonexchangeable K⁺ reserves. The incubation of ectomycorrhizal fungi in bi-compartment Petri dishes with phlogopite led to cation exchange reactions and to crystal lattice weathering. Paxillus involutus COU led to irreversible phlogopite transformations, while Pisolithus tinctorius 441 led to reversible ones. Simultaneous depletion in K⁺ and Mg²⁺ led to an enhanced weathering of phlogopite by P. tinctorius 441. The observation of phlogopite evolution shows that some specific Al³⁺ immobilization occurred under P. tinctorius 441. The data suggest that these bio-weathering mechanisms could be related to the release of fungal organic acids or other complex forming molecules.     

Nitrogen nutrition,photosynthesis and carbon allocation in ectomycorrhizal pine

Summary Studies examined net photosynthesis (Pn) and dry matter production of mycorrhizal and nonmycorrhizalPinus taeda at 6 intervals over a 10-month period. Pn rates of mycorrhizal plants were consistently greater than nonmycorrhizal plants, and at 10 months were 2.1-fold greater. Partitioning of current photosynthate was examined by pulse-labelling with¹⁴CO₂ at each of the six time intervals. Mycorrhizal plants assimilated more¹⁴CO₂, allocated a greater percentage of assimilated¹⁴C to the root systems, and lost a greater percentage of¹⁴C by root respiration than did nonmycorrhizal plants. At 10 months, the quantity of¹⁴CO₂ respired by roots per unit root weight was 3.6-fold greater by mycorrhizal than nonmycorrhizal plants. Although the stimulation of photosynthesis and translocation of current photosynthate to the root system by mycorrhiza formation was consistent with the source-sink concept of sink demand, foliar N and P concentrations were also greater in mycorrhizal plants.Further studies examined Pn and dry matter production ofPinus contorta in response to various combinations of N fertilization (3, 62, 248 ppm), irradiance and mycorrhizal fungi inoculation. At 16 weeks of age, 6 weeks following inoculation with eitherPisolithus tinctorius orSuillus granulatus, Pn rates and biomass were significantly greater in mycorrhizal than nonmycorrhizal plants. Mycorrhizal plants had significantly greater foliar %P, but not %N, than did nonmycorrhizal plants. Fertilization with 62 ppm N resulted in greater mycorrhiza formation than either 3 or 248 ppm. Increased irradiance resulted in increased mycorrhiza formation.     

Changes in root growth patterns of (Picea abies) spruce roots by inoculation with an ectomycorrhizal fungusPisolithus tinctorius and jasmonic acid treatment

Symbiosis between fungi and plant roots forming a mycorrhiza involves extensive interactions at the molecular level between both partners. The role of plant hormones in the regulation of mycorrhizal infection is not known to involve jasmonates. Their endogenous levels increase during pathogen attack; however, little has been done on their involvement in mycorrhizae. In our recent work, root growth patterns of 2-month-old spruce seedlings after inoculation withPisolithus tinctorius and/or jasmonic acid (JA) treatment were studied using a paper-sandwich technique. Changes in root length, the degree of branching, presence and length of root hairs, and infection parameters were followed using a stereomicroscope. The first mycorrhizal contact of hyphae with roots was significantly accelerated upon treatment with 0.5 M JA. Interactions between root hairs and fungal hyphae were seen by scanning electron microscopy. The multiplication of root hairs of non-mycorrhized seedlings treated with 5.0 M JA and changes of the root surface were observed by the same technique.     

Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N

The development of an efficient transformation system is required to alter the expression of symbiosis-regulated genes and to develop insertional mutagenesis in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. Vegetative mycelium of this fungus was transformed by Agrobacterium tumefaciens-mediated gene transfer. The selection marker was the hygromycin resistance gene of Escherichia coli (hph) under the control of the gpd promoter from Agaricus bisporus and the CaMV 35S terminator as part of the T-DNA. PCR amplification of hph and Southern blot analyses showed that the genome of the hygromycin-resistant transformants contained the cassette. The latter proved mostly single copy and random integration of part of the transgene into the fungal genome. A. tumefaciens-mediated gene transfer should facilitate future development of insertional mutagenesis, targeted gene disruption and RNA interference technology in L. bicolor.     

Effect of time of inoculation on in vitro ectomycorrhizal colonization and nodule initiation in Acacia holosericea seedlings

The complex interactions that occur in systems with more than one type of symbiosis were studied using one isolate of Bradyrhizobium sp. and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch inoculated on to the roots of Acacia holosericea A. Cunn. ex G. Don in vitro. After a single inoculation with Bradyrhizobium sp., bacteria typically entered the roots by forming infection threads in the root hair cells via the curling point of the root hair and/ or after intercellular penetration. Sheath formation and intercellular penetration were observed on Acacia roots after a single inoculation with Pisolithus tinctorius but no radial elongation of epidermal cells. Simultaneous inoculation with both microorganisms resulted in nodules and ectomycorrhiza on the root system, occasionally on the same lateral root. On lateral roots bearing nodules and ectomycorrhiza, the nodulation site was characterized by the presence of a nodule meristem and the absence of an infection thread; sheath formation and Hartig net development occurred regularly in the region of the roots adjacent to nodules. Prior inoculation with Bradyrhizobium sp. did not inhibit ectomycorrhizal colonization in root segments adjacent to nodules in which nodule meristems and infection threads were clearly present. Conversely, in ectomycorrhizae inoculated by bacteria, the nodule meristem and the infection thread were typically absent. These results show that simultaneous inoculation with both microorganisms inhibits infection thread development, thus conferring an advantage on fungal hyphae in the competition for infection sites. This suggests that fungal hyphae can modify directly and/or indirectly the recognition factors leading to nodule meristem initiation and infection thread development.     

The fungus-specificity of mycorrhization helper bacteria (MHBs) used as an alternative to soil fumigation for ectomycorrhizal inoculation of bare-root Douglas-fir planting stocks with Laccaria laccata

Mycorrhization helper bacteria (MHBs) isolated and selected from the Douglas fir-Laccaria laccata symbiotic system have previously been shown to be fungus-specific: they promote ectomycorrhizal establishment of Laccaria laccata but inhibit mycorrhiza formation by other fungi. In this paper, two experiments in a nursery producing two years-old bare-root Douglas-fir planting stocks confirm the specificity of MHBs under field conditions. They also show that, by selectively helping the introduced L. laccata against the resident symbionts, MHBs are an interesting alternative (safer and easier) to soil fumigation for the success of routine controlled mycorrhization of planting stocks in forest nurseries.     

Assimilation dynamics of soil carbon and nitrogen by wheat roots and Collembola

It has been demonstrated that plant roots can take up small amounts of low-molecular weight (LMW) compounds from the surrounding soil. Root uptake of LMW compounds have been investigated by applying isotopically labelled sugars or amino acids but not labelled organic matter. We tested whether wheat roots took up LMW compounds released from dual-labelled (¹³C and ¹⁵N) green manure by analysing for excess ¹³C in roots. To estimate the fraction of green manure C that potentially was available for root uptake, excess ¹³C and ¹⁵N in the primary decomposers was estimated by analysing soil dwelling Collembola that primarily feed on fungi or microfauna. The experimental setup consisted of soil microcosm with wheat and dual-labelled green manure additions. Plant growth, plant N and recoveries of ¹³C and ¹⁵N in soil, roots, shoots and Collembola were measured at 27, 56 and 84 days. We found a small (<1%) but significant uptake of green manure derived ¹³C in roots at the first but not the two last samplings. About 50% of green manure C was not recovered from the soil-plant system at 27 days and additional 8% was not recovered at 84 days. Up to 23% of C in collembolans derived from the green manure at 56 days (the 27 days sampling was lost). Using a linear mixing model we estimated that roots or root effluxes provided the main C source for collembolans (54−79%). We conclude that there is no solid support for claiming that roots assimilated green manure derived C due to very small or no recoveries of excess ¹³C in wheat roots. During the incubation the pool of green manure derived C available for root uptake decreased due to decomposition. However, the isotopic composition in Collembola indicated that there was a considerable fraction of green manure derived C in the decomposer system at 56 days thus supporting the premise that LMW compounds containing C from the green manure was released throughout the incubation. Responsible Editor: A. C. Borstlap.     

The effect of ectomycorrhizas on the phosphorus and dry weight acquisition of Eucalyptus seedlings

The inoculation ofEucalyptus pilularis seedlings withPisolithus tinctorius and the subsequent development of ectomycorrhizas, led to an improved acquisition of phosphorus (P) from three different sources of P. These sources included insoluble phytate. Dry weight gain of seedlings was increased by inoculation in those treatments where growth was limited by P supply.     

Induced hydrolytic enzymes of ectomycorrhizal fungi against pathogen Rhizoctonia solani

Interactions between ectomycorrhizal fungi (Suillus laricinus, S. tomentosus, Amanita vaginata and Gomphidius viscidus) and the pathogen Rhizoctonia solani in co-culture were studied using both light and scanning electron microscopy. S. laricinus, S. tomentosus and A. vaginata inhibited the growth of the pathogen. Moreover, A. vaginata exhibited coiling around and penetration of the hyphae into R. solani was observed in the interaction zone. Furthermore, the production of chitinases, beta-1,3-glucanases and beta-glucosidases by these ectomycorrhizal fungi on colloidal chitin or cell walls of R. solani was evaluated: chitinases were not induced by colloidal chitin but all three enzymes were induced by R. solani cell walls. No correlation between inhibition rate and production of lytic enzymes was found.