Competition between ectomycorrhizal fungi colonizing Pinus densiflora

 Interactive competition of Pisolithus tinctorius (Pers.) Coker et Couch with an unidentified species Tanashi 01 and Suillus luteus (L.: Fr.) S. F. Gray was investigated using a rhizobox. Pinus densiflora Sieb. et Zucc. was used as the host plant and mycelia were distinguished by hyphal color. The speed of mycelial spread differed between the fungi;P. tinctorius and Tanashi 01 grew faster than S. luteus. A P. tinctorius mycorrhizal seedling and a Tanashi 01 mycorrhizal seedling were transplanted on opposite sides of the rhizobox. The mycelia and mycorrhizae of P. tinctorius were overgrown by Tanashi 01 hyphae and development of P. tinctorius was gradually inhibited. The areas occupied by mycelia and mycorrhiza of P. tinctorius decreased by 52% and 37%, respectively, 154 days after transplantation relative to that at 91 days. In the overlap area of P. tinctorius and Tanashi 01, the latter fungus infected new root tips emerging from P. tinctorius mycorrhiza, which lacked a mantle of P. tinctorius hyphae, and formed a composite mycorrhizal structure. P. tinctorius mycorrhizae were progressively replaced by Tanashi 01 mycorrhizae. Mycelial spread of P. tinctorius and S. luteus were naturally inhibited but there was no interaction in mycorrhizal formation. Accepted: 18 June 1999     

Mycorrhizal Fungi Prevent Disease in Stressed Pine Seedlings

The common rhizospheric fungus Cylindrocarpon destructans caused severe root damage leading to mortality in light-starved, stunted seedlings of Pinus sylvestris. This effect was, however, almost fully prevented by the presence of mycorrhizal fungi; i.e. Laccaria laccata, Pisolithus tinctorius, or unidentified species present in small amounts of conifer soil. Protection took place even before visible mycorrhizae were found on the roots. This is the first time that protection ofstressed plants against non-parasitic (non-invasive) pathogens has been reported. The results indicate that root protection by mycorrhizal fungi may not only be effective inside the root but probably also extends out into the rhizoplane or rhizosphere.     

Structure and histochemistry of mycorrhizae synthesized between Arbutus menziesii (Ericaceae) and two basidiomycetes,Pisolithus tinctorius (Pisolithaceae) and Piloderma bicolor (Corticiaceae)

Arbutoid mycorrhizae were synthesized in growth pouches between Arbutus menziesii Pursch. (Pacific madrone) and two broad host range basidiomycete fungi, Pisolithus tinctorius (Pers.) Coker and Couch and Piloderma bicolor (Peck) Jülich. P. tinctorius induced the formation of dense, pinnate mycorrhizal root clusters enveloped by a thick fungal mantle. P. bicolor mycorrhizae were usually unbranched, and had a thin or non-existent mantle. Both associations had the well-developed para-epidermal Hartig nets and intracellular penetration of host epidermal cells by hyphae typical of arbutoid interactions. A. menziesii roots developed a suberized exodermis which acted as a barrier to cortical cell penetration by the fungi. Ultrastructurally, the suberin appeared non-lamellar, but this may have been due to the imbedding resin. Histochemical analyses indicated that phenolic substances present in epidermal cells may be an important factor in mycorrhiza establishment. Analyses with X-ray energy dispersive spectroscopy showed that some of the granular inclusions present in fungal hyphae of the mantle and Hartig net were polyphosphate. Other inclusions were either protein or polysaccharides.     

Aluminum-mycorrhizal interactions in the physiology of pitch pine seedlings

Aluminum (Al) in the rhizosphere adversely affects plant nutrition and growth. Although many conifer species, and pitch pine (Pinus rigida) in particular, have evolved on acidic soils where soluble Al is often high, controlled environment studies often indicate that Al interferes with seedling growth and nutrient relations. Under normal field conditions, conifer roots grow in a symbiotic relationship with ectomycorrhizal fungi, and this association may modulate the effects of Al on root physiology. To investigate the influence of mycorrhizal infection on Al toxicity, pitch pine seedlings were grown with or without the ectomycorrhizal symbiont Pisolithus tinctorius and were exposed to low levels of Al in sand culture. Aluminum at 50 μM reduced nonmycorrhizal seedling growth and increased foliar Al concentrations, but did not alter photosynthetic gas exchange or other aspects of seedling nutrition. Nonmycorrhizal seedlings exposed to 200 μM Al exhibited decreased growth, increased transpiration rates, decreased water use efficiency, increased foliar Al and Na levels, and reduced foliar P concentrations. Seedlings inoculated with P. tinctorius exhibited unaltered growth, physiological function, and ionic relations when exposed to Al. The fungal symbiont evidently modulated ionic relations in the rhizosphere, reducing Al-P precipitation reactions, Al uptake, and subsequent root and shoot tissue Al exposure.     

Detecting the heavy metal tolerance level in ectomycorrhizal fungi <Emphasis Type="Italic">in vitro</Emphasis>

Summary Eight isolates of ectomycorrhizal fungi namely, Laccaria fraterna (EM-1083),Laccaria laccata (EM-1191), Pisolithus tinctorius (EM-1081),Pisolithus tinctorius (EM-1293), Scleroderma cepa (EM-1233),Scleroderma flavidum (EM-1235),Scleroderma verucosum, (EM-1283) and Hysterangium incarceratum (EM-1185) were grown on specially designed cocktail media prepared by adding various concentrations of different heavy metals namely Al, As, Cd, Cr, Ni and Pb. The heavy metals were selected keeping in view their relative abundance in coal ash and potential toxicity. The fungal isolates were grown on such designed cocktail media. The colony diameter was used for the measurement of the fungal growth. Total heavy metal accumulated in the mycelia was assayed by atomic absorption spectrophotometry.In relation to metal tolerance ability in general,Hysterangium incarceratum (EM-1185) showed maximum tolerance with respect to growth, Laccaria fraterna (EM-1083) and Pisolithus tinctorius (EM-1293) also showed considerable tolerance to the heavy metals tested. In relation to metal uptake in particular, Pisolithus tinctorius (EM-1293), has reported maximum uptake of Al (34642.58 ppm), Cd (302.12 ppm) and Pb (3501.96 ppm). In Laccaria fraterna (EM-1083), As (130.57 ppm) and Cr (402.38 ppm) uptake was recorded maximum; and Hysterangium incarceratum (EM-1185) has recorded maximum Ni (2648.59 ppm) uptake among the three suitable isolates documented here.     

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.     

An in vitro method for establishing mycorrhizae on coniferous tree seedlings

Summary A method for in vitro synthesis of mycorrhizae on coniferous tree seedlings is described. Tree seedlings (Larix decidua Mill., Picea abies (L.) Karst, and Pinus sylvestris L.) and fungi Amanita muscaria (L. ex Fr.) Hooker, Piloderma croceum Erikss. et Hjorst., Pisolithus tinctorius (Pers.) Coker et Couch, and Suillus grevillei (Klotzsch) Singer were maintained under sterile conditions in petri dishes. Typical ectomycorrhizae were established within 2–3 weeks after inoculation and within 2 months after germination of seedlings. Eventually a high percentage of mycorrhizal root tips was obtained.     

Dynamics of lead accumulation in mycorrhizal and non-mycorrhizal Norway spruce (Picea abies (L.) Karst.)

Twelve-week-old seedlings of Norway spruce (Picea abies (L.) Karst), non-mycorrhizal or mycorrhizal with Laccaria laccata, Paxillus involutus or Pisolithus tinctorius were exposed to 5 M Pb for either 32 or 42 days in a quartz sand-nutrient solution system. Ultrathin sections of mycorrhizal and non-mycorrhizal short roots were examined by X-ray microanalysis. After 42 days Pb treatment, the Pb content of the cortex cell walls was lower in the non-mycorrhizal short roots and in the P. involutus mycorrhizae than in the mycorrhizae of L. laccata or P. tinctorius. The Pb content of the cell walls of the hyphal mantle was higher in P. involutus than in L. laccata or P. tinctorius. The short term experiment over 32 days showed that the Pb content of the cortex cell walls strongly increased during the first 16 days in the non-mycorrhizal roots and the L. laccata mycorrhizae, whereas it increased more slowly in the P. involutus mycorrhizae. After 32 days Pb treatment, the Pb content in the cortex cell walls in the P. involutus mycorrhizae was similar to that in the non-mycorrhizal roots. P. involutus also decreased Pb translocation from the roots to the stems. Mycorrhizal infection was not affected by Pb but with P. involutus, the amount of extramatrical mycelium was reduced by 50% on day 32 compared to day 16. The extramatrical mycelium of L. laccata was not reduced by Pb. It is concluded that ectomycorrhizal fungi differ in their effect on Pb accumulation in the roots of Norway spruce. The binding capacity of the extramatrical mycelium seems to be an important factor.     

Solution pH modifies the response of Norway spruce seedlings to aluminium

Norway spruce (Picea abies) was exposed to nutrient solutions containing a range of aluminium (Al) concentrations at several pH levels (3.2, 4 and 5). Root growth was reduced by 100 µM and 400 µM Al at pH 4 and 5, but at pH 3.2 only by 400 µM Al. The Al content of the roots increased with increasing pH. The Al content of the roots was higher at the root tips than at the older root parts at all pH values. Using X-ray microanalysis it could be shown that higher levels of Al at increased pH were mainly due to increased Al contents in root cortex cell walls. In seedlings, mycorrhizal with Pisolithus tinctorius or Lactarius rufus, the Al concentration of cortex cell walls was higher when nitrate (NO₃) rather than ammonium (NH₄) was the nitrogen (N) source.     

Heavy metal tolerance by ectomycorrhizal fungi and metal amelioration by Pisolithus tinctorius

Five ectomycorrhizal fungi, Pisolithus tinctorius, Thelephora terrestris, Cenococcum geophilum, Hymenogaster sp. and Scleroderma sp., which were demonstrated previously to be capable of forming ectomycorrhizas with some pine, eucalypt and fagaceous tree species were grown in vitro in liquid cultures for 3 weeks at six different concentrations of nine heavy metals, aluminium, iron, copper, zinc, nickel, cadmium, chromium, lead and mercury. Measurements of mean mycelial dry weight yields indicated that the local isolates of Hymenogaster sp. and Scleroderma sp., as well as the introduced fungal species P. tinctorius, were able to withstand high concentrations of Al, Fe, Cu and Zn and might, therefore, have potential for revegetation schemes in metal-contaminated soils. The metal amelioration mechanism in the metal-tolerant fungal species P. tinctorius was observed to involve extrahyphal slime and, as demonstrated by energy-dispersive X-ray spectrometry, was achieved by polyphosphate linkage of Cu and Zn.     

The influence of mycorrhizal inoculation and paclobutrazol on water and nutritional status of Arbutus unedo L.

The purpose of this study was to analyze morphological and physiological aspects of Arbutus unedo L. plants treated with paclobutrazol (PAC), compounds characterized by their double activity as plant growth regulators and fungicides, and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch, which forms a special type of mycorrhizal colonization called arbutoid mycorrhiza. Native A. unedo L. seedlings were grown in a greenhouse and subjected to four treatments for 4 months: 0 or 60 mg of PAC and inoculated or not with P. tinctorius (Pers.). The arbutoid mycorrhizal inoculation increased in plants treated with PAC. Paclobutrazol reduced shoot and root biomass, plant height, internode length, stem diameter, leaf area, total root length and number of tips. P. tinctorius increased plant height and had a beneficial effect on the root system (increasing root diameter and the number of tips). PAC treatment led to an increase in ion levels in the leaf tissue, while mycorrhizal inoculation induced lower K and higher P contents in the roots. Leaf water potentials (at predawn and at midday) increased with the combined treatment. The absence of water deficit conditions meant there was no osmotic adjustment. Higher photosynthesis (P_n) values were associated with higher stomatal conductance (g_s) values in the mycorrhizal plants, which influenced water uptake from the roots. However, g_s decreased in the PAC-treated plants, reducing photosynthesis and, as a consequence, growth. The higher hydraulic conductivity (L_p) in the plants treated with PAC may have induced a better water energy status and good water transport. The combined treatment produced beneficial effects in the plants, improving their water and nutritional status.     

Ectomycorrhizae formed in vitro by quaking aspen: including Inocybe lacera and Amanita pantherina

Ectomycorrhizae formed in synthesis tubes by aspen (Populus tremuloides) seedlings and each of seven fungal isolates are described. Isolates of Amanita muscaria v. formosa, A. pantherina, Inocybe lacera, and Paxillus vernalis, from sporocarps collected in aspen stands in southwestern Montana, developed mantles and Hartig nets on aspen roots, as did the broad-hostrange fungi Cenococcum geophilum and Pisolithus tinctorius from the VPI culture collection. Chalciporus piperatus failed to form mycorrhizae, and Piloderma croceum formed a mantle, but no Hartig net. The first syntheses of I. lacera and A. pantherina with aspen are reported.     

Differential Influence of Ectomycorrhizae on Plant Growth and Disease Resistance in Pinus sylvestris Seedlings

The influence of Laccaria laccata, Hebeloma crustuliniforme, and Pisolithus tinctorius mycorrhizae and a mixture of naturally occurring mycorrhizae on the resistance of Pinus sylvestris seedlings to attack by Fusarium moniliforme and Rhizoctonia solani was investigated periodically during an entire year under greenhouse conditions following inoculation with both mycobiont and pathogen. Using artificial root substrate the seedlings were provided with a balanced butlow nutrient supply. Seedling growth (dry weight) as well as short-root formation was stimulated to different extents depending on the mycobiont species; in addition, the degree of stimulation varied over the year. Parasitized plants were more stimulated, than nonparasitized ones. Disease severity and plant mortality caused by either pathogen were drastically reduced in the presence of the mycorrhizal fungi, even before mycorrhiza formation. All mycorrhizae, except the association with Hebeloma, remained highly protective throughout the year; with Hebeloma it remained protective for only about 3 months. Growth stimulation or mycorrhiza frequency was not always correlated with the level of protection; e.g. Hebeloma protected withoutstimulating growth. Two types of protection apparently exist, one related to and another unrelated to growth stimulation. Mycorrhizal fungi had little or no antagonistic effects on fungal pathogens in vitro.     

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.     

Mycorrhizal associations in Hong Kong Fagaceae

A rapid method of screening has been devised whereby the large seeds (acorns) of Castanopsis, Lithocarpus and Quercus growing in Hong Kong can be germinated in the presence of a variety of known mycorrhizal fungi and observations made on the initiation of host/fungal associations. Early stages of Hartig net formation could be detected in bleached roots, and an observation was made on the antibiotic activity of the mycorrhizal fungus, Pisolithus tinctorius. The method offers a simple and effective means of assessing the potential of fungi to form mycorrhizal associations under non-sterile conditions.     

Effects of sodium chloride on growth of ectomycorrhizal fungal isolates in culture

We studied the tolerance of ectomycorrhizal (ECM) fungi to sodium chloride (NaCl) to find the best fungus to aid growth of Pinus thunbergii. Four ECM fungi, Cenococcum geophilum, Pisolithus tinctorius, Rhizopogon rubescens, and Suillus luteus, were grown in liquid MMN media with five different concentrations of NaCl for 30 days, and their mycelial weights were determined. Mycelial weights of P. tinctorius and R. rubescens were not significantly different between 0 mM and 200 mM, whereas those of C. geophilum and S. luteus decreased with increasing NaCl concentration, indicating that the former two species were more tolerant to higher NaCl concentrations than the latter species. We further studied the intraspecific differences in NaCl tolerance of nine P. tinctorius isolates. They were grown on MMN agar media with six different concentrations of NaCl for 21 days, and their radial growth was measured. In total, the hyphal growth at 25 mM NaCl was significantly higher than those at the other NaCl concentrations, and EC₅₀ values were confirmed at between 50 mM and 200 mM. Among the isolates, Pt03 and Pt21 showed measurable growth at 200 mM; the growth of Pt03 was not significantly different between 0 mM and 200 mM. The results indicate that there are intraspecific variations in NaCl tolerance of Pisolithus species.     

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.     

Establishment of Sweet Birch on Surface Mine Spoil as Influenced by Mycorrhizal Inoculation and Fertility

Induced mycorrhization of sweet birch (Betula lenta L.) by Pisolithus tinctorius (Pers.) Coker & Couch, as influenced by substrate fertility, was evaluated for its effects on seedling growth and physiology. Following a brief period in seed flats, seedlings were transplanted to mine spoil where they resided for 30 months, and three nutrition regimes were imposed throughout the study by application of differing nutrient solution concentrations. High fertility suppressed mycorrhizal formation by P. tinctorius but promoted that of other mycobionts. Pisolithus mycorrhization induced substantial aboveground and belowground growth as indicated by dimensions and mass for the former and mass and length for the latter but favoring root over shoot growth overall. Furthermore, these mycorrhizae were frequently able to compensate for the growth stimulation of higher nutrient additions. Measurements of xylem pressure potential and soil water potential indicated that water uptake was enhanced by P. tinctorius during simulated drought episodes of two durations and in subsequent recovery periods. Inoculated seedlings had higher foliar concentrations of critical nutrients, especially N, and lower concentrations of potentially phytotoxic metallic elements, particularly Mn, than uninoculated seedlings, although the latter response was absent in high fertility. Spoil analyses clearly revealed the influence of the nutrition regimes but also the effects of seedling uptake on substrate chemistry, and reinforced the findings of the foliar analysis concerning suppression of metal uptake by P. tinctorius. Collectively, these results suggest that P. tinctorius can provide sweet birch an array of physiological benefits that will permit this tree species to flourish on harsh substrates such as surface mine spoils without heavy application of chemical fertilizers.     

Shoots, roots and ectomycorrhiza formation of pine seedlings at elevated atmospheric carbon dioxide

The effect of elevated atmospheric CO₂ concentration on the growth of shoots, roots, mycorrhizas and extraradical mycorrhizal mycelia of pine (Pinus silvestris L.) was examined. Two and a half-month-old seedlings were inoculated axenically with the mycorrhizal fungus Pisolithus tincto-rius (Pers.) by a method allowing rapid mycorrhiza formation in Petri dishes. The plants were then cultivated for 3 months in growth chambers with daily concentrations of 350 and 600 μmol mol^?1 CO₂ during the day. Whereas plants harvested after 1 and 2 months did not differ appreciably between ambient and increased CO₂ concentrations, after 3 months they developed a considerably higher root biomass (%57%) at elevated CO₂, but did not increase significantly in root length. The mycorrhizal fungus Pisolithus tinctorius, which depended entirely on the plant assimilates in the model system, grew much faster at increased CO₂: 3 times more mycorrhizal root clusters were formed and the extraradical mycelium produced had twice the biomass at elevated as at ambient CO₂. No difference in shoot biomass was found between the two treatments after 91 d. However, since the total water consumption of seedlings was similar in the two treatments, the water use efficiency was appreciably higher for the seedlings at increased CO₂ because of the higher below-ground biomass.     

Effects of carbon dioxide enrichment on response of mycorrhizal pitch pine (Pinus rigida) to aluminum: growth and mineral nutrition

 Carbon dioxide enrichment may increase the Al tolerance of trees by increasing root growth, root exudation and/or mycorrhizal colonization. The effect of elevated CO₂ on the response of mycorrhizal pitch pine (Pinus rigida Mill.) seedlings to Al was determined in two experiments with different levels of nutrients, 0.1- or 0.2-strength Clark solution. During each experiment, seedlings inoculated with the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker & Couch were grown 13 weeks in sand irrigated with nutrient solution (pH 3.8) containing 0, 6.25, 12.5, or 25 mg/l Al (0, 232, 463, or 927 μM Al) in growth chambers fumigated with 350 (ambient) or 700 (elevated) μl/l CO₂. At ambient CO₂, in the absence of Al, mean total dry weights (DW) of seedlings at the high nutrient level were 164% higher than those at the low level. Total DW at elevated CO₂, in the absence of Al, was significantly greater than that in ambient CO₂ at the low (+34%) and high (+16%) nutrient levels. Root and shoot DW at both nutrient levels decreased with increasing Al concentrations with Al reducing root growth more than shoot growth. Although visible symptoms of Al toxicity in roots and needles were reduced by CO₂ enrichment, there were no significant CO₂ × Al interactions for shoot or root DW. The percentage of seedling roots that became mycorrhizal was negatively related to nutrient level and was greater at elevated than at ambient CO₂ levels. Generally, elevated CO₂ had little effect on concentration of mineral nutrients in roots and needles. Aluminum reduced concentrations of most nutrients by inhibiting uptake. Received: 18 June 1997 / Accepted: 8 December 1997