Mycorrhiza formation and growth of Eucalyptus globulus seedlings inoculated with spores of various ectomycorrhizal fungi

 As many eucalypts in commercial plantations are poorly ectomycorrhizal there is a need to develop inoculation programs for forest nurseries. The use of fungal spores as inoculum is a viable proposition for low technology nurseries currently producing eucalypts for outplanting in developing countries. Forty-three collections of ectomycorrhizal fungi from southwestern Australia and two from China, representing 18 genera, were tested for their effectiveness as spore inoculum on Eucalyptus globulus Labill. seedlings. Seven-day-old seedlings were inoculated with 25 mg air-dry spores in a water suspension. Ectomycorrhizal development was assessed in soil cores 65 and 110 days after inoculation. By day 65, about 50% of the treatments had formed ectomycorrhizas. By day 110, inoculated seedlings were generally ectomycorrhizal, but in many cases the percentage of roots colonized was low (<10%). Species of Laccaria, Hydnangium, Descolea, Descomyces, Scleroderma and Pisolithus formed more ectomycorrhizas than the other fungi. Species of Russula, Boletus, Lactarius and Hysterangium did not form ectomycorrhizas. The dry weights of inoculated seedlings ranged from 90% to 225% of the uninoculated seedlings by day 110. Although plants with extensively colonized roots generally had increased seedling growth, the overall mycorrhizal colonization levels were poorly correlated to seedling growth. Species of Laccaria, Descolea, Scleroderma and Pisolithus are proposed as potential candidate fungi for nursery inoculation programs for eucalypts. Accepted: 7 May 1998     

Comparative effects of four mycorrhizal fungi on loblolly pine seedlings growing in a greenhouse in a Piedmont soil

Summary A greenhouse study was conducted to evaluate the effect of ectomycorrhizae on loblolly pine (Pinus taeda L.) growing in a Piedmont soil. Pine seedlings were inoculated with one of four species of fungi (Scleroderma aurantium, Pisolithus tinctorius, Thelophora terrestris, andRhizopogon roseolus). The seedlings were grown in pots containing a Cecil sandy clay loam amended to create a gradient of extractable P ranging from 5.9 to 52.5 g/g. After ten months, all colonized seedlings were significantly larger than control seedlings. However, of the four fungi,Scleroderma aurantium mediated a far superior shoot growth response to increasing levels of soil P; the seedlings were significantly larger than those colonized by any other fungus and also had the largest root systems and greatest degree of mycorrhizal colonization.     

Arbuscular mycorrhizal fungi associated with common pteridophytes in Dujiangyan,southwest China

The colonization and diversity of arbuscular mycorrhizal (AM) fungi associated with common pteridophytes were investigated in Dujiangyan, southwest China. Of the 34 species of ferns from 16 families collected, 31 were colonized by AM fungi. The mean percentage root length colonized was 15%, ranging from 0 to 47%. Nineteen species formed Paris-type and 10 intermediate-type AM. In two ferns, only rare intercellular non-septate hyphae or vesicles were observed in the roots and AM type could not be determined. Of the 40 AM fungal taxa belonging to five genera isolated from rooting-zone soils, 32 belonged to Glomus, five to Acaulospora, one to Archaeospora, one to Entrophospora, and one to Gigaspora. Acaulospora and Glomus were the dominant genera and Glomus versiforme was the most common species. The average AM spore density was 213 per 100 g air-dried soil and the average species richness was 3.7 AM species per soil sample. There was no correlation between spore density and percentage root length colonized by AM fungi.     

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.     

Field response of wheat to arbuscular mycorrhizal fungi and drought stress

Mycorrhizal plants often have greater tolerance to drought than nonmycorrhizal plants. This study was conducted to determine the effects of arbuscular mycorrhizal (AM) fungi inoculation on growth, grain yield and mineral acquisition of two winter wheat (Triticum aestivum L.) cultivars grown in the field under well-watered and water-stressed conditions. Wheat seeds were planted in furrows after treatment with or without the AM fungi Glomus mosseae or G. etunicatum. Roots were sampled at four growth stages (leaf, tillering, heading and grain-filling) to quantify AM fungi. There was negligible AM fungi colonization during winter months following seeding (leaf sampling in February), when soil temperature was low. During the spring, AM fungi colonization increased gradually. Mycorrhizal colonization was higher in well-watered plants colonized with AM fungi isolates than water-stressed plants. Plants inoculated with G. etunicatum generally had higher colonization than plants colonized with G. mosseae under both soil moisture conditions. Biomass and grain yields were higher in mycorrhizal than nonmycorrhizal plots irrespective of soil moisture, and G. etunicatum inoculated plants generally had higher biomass and grain yields than those colonized by G. mosseae under either soil moisture condition. The mycorrhizal plants had higher shoot P and Fe concentrations than nonmycorrhizal plants at all samplings regardless of soil moisture conditions. The improved growth, yield and nutrient uptake in wheat plants reported here demonstrate the potential of mycorrhizal inoculation to reduce the effects of drought stress on wheat grown under field conditions in semiarid areas of the world.     

The differential behavior of arbuscular mycorrhizal fungi in interaction with Astragalus sinicus L. under salt stress

Three arbuscular mycorrhizal (AM) fungi (Glomus mosseae, Glomus claroideum, and Glomus intraradices) were compared for their root colonizing ability and activity in the root of Astragalus sinicus L. under salt-stressed soil conditions. Mycorrhizal formation, activity of fungal succinate dehydrogenase, and alkaline phosphatase, as well as plant biomass, were evaluated after 7 weeks of plant growth. Increasing the concentration of NaCl in soil generally decreased the dry weight of shoots and roots. Inoculation with AM fungi significantly alleviated inhibitory effect of salt stress. G. intraradices was the most efficient AM fungus compared with the other two fungi in terms of root colonization and enzyme activity. Nested PCR revealed that in root system of plants inoculated with a mix of the three AM fungi and grown under salt stress, the majority of mycorrhizal root fragments were colonized by one or two AM fungi, and some roots were colonized by all the three. Compared to inoculation alone, the frequency of G. mosseae in roots increased in the presence of the other two fungal species and highest level of NaCl, suggesting a synergistic interaction between these fungi under salt stress.     

Arbuscular mycorrhizal fungi and plant symbiosis in a saline-sodic soil

The seasonality of arbuscular mycorrhizal (AM) fungi–plant symbiosis in Lotus glaber Mill. and Stenotaphrum secundatum (Walt.) O.K. and the association with phosphorus (P) plant nutrition were studied in a saline-sodic soil at the four seasons during a year. Plant roots of both species were densely colonized by AM fungi (90 and 73%, respectively in L. glaber and S. secundatum) at high values of soil pH (9.2) and exchangeable sodium percentage (65%). The percentage of colonized root length differed between species and showed seasonality. The morphology of root colonization had a similar pattern in both species. The arbuscular colonization fraction increased at the beginning of the growing season and was positively associated with increased P concentration in both shoot and root tissue. The vesicular colonization fraction was high in summer when plants suffer from stress imposed by high temperatures and drought periods, and negatively associated with P in plant tissue. Spore and hyphal densities in soil were not associated with AM root colonization and did not show seasonality. Our results suggest that AM fungi can survive and colonize L. glaber and S. secundatum roots adapted to extreme saline-sodic soil condition. The symbiosis responds to seasonality and P uptake by the host altering the morphology of root colonization.     

Role of the modification in root exudation induced by arbuscular mycorrhizal colonization on the intraradical growth of Phytophthora nicotianae in tomato

We studied the role of modification in root exudation induced by colonization with Glomus intraradices and Glomus mosseae in the growth of Phytophthora nicotianae in tomato roots. Plants were grown in a compartmentalized plant growth system and were either inoculated with the AM fungi or received exudates from mycorrhizal plants, with the corresponding controls. Three weeks after planting, the plants were inoculated or not with P. nicotianae growing from an adjacent compartment. At harvest, P. nicotianae biomass was significantly reduced in roots colonized with G. intraradices or G. mosseae in comparison to non-colonized roots. Conversely, pathogen biomass was similar in non-colonized roots supplied with exudates collected from mycorrhizal or non-mycorrhizal roots, or with water. We cannot rule out that a mycorrhiza-mediated modification in root exudation may take place, but our results did not support that a change in pathogen chemotactic responses to host root exudates may be involved in the inhibition of P. nicotianae.     

Mycorrhizas on nursery and field seedlings of Quercus garryana

Oak woodland regeneration and restoration requires that seedlings develop mycorrhizas, yet the need for this mutualistic association is often overlooked. In this study, we asked whether Quercus garryana seedlings in nursery beds acquire mycorrhizas without artificial inoculation or access to a mycorrhizal network of other ectomycorrhizal hosts. We also assessed the relationship between mycorrhizal infection and seedling growth in a nursery. Further, we compared the mycorrhizal assemblage of oak nursery seedlings to that of conifer seedlings in the nursery and to that of oak seedlings in nearby oak woodlands. Seedlings were excavated and the roots washed and examined microscopically. Mycorrhizas were identified by DNA sequences of the internal transcribed spacer region and by morphotype. On oak nursery seedlings, predominant mycorrhizas were species of Laccaria and Tuber with single occurrences of Entoloma and Peziza. In adjacent beds, seedlings of Pseudotsuga menziesii were mycorrhizal with Hysterangium and a different species of Laccaria; seedlings of Pinus monticola were mycorrhizal with Geneabea, Tarzetta, and Thelephora. Height of Q. garryana seedlings correlated with root biomass and mycorrhizal abundance. Total mycorrhizal abundance and abundance of Laccaria mycorrhizas significantly predicted seedling height in the nursery. Native oak seedlings from nearby Q. garryana woodlands were mycorrhizal with 13 fungal symbionts, none of which occurred on the nursery seedlings. These results demonstrate the value of mycorrhizas to the growth of oak seedlings. Although seedlings in nursery beds developed mycorrhizas without intentional inoculation, their mycorrhizas differed from and were less species rich than those on native seedlings.     

Field performance in northern Spain of Douglas-fir seedlings Inoculated with ectomycorrhizal fungi

 Experimental plantations were established in northern Spain to determine the effects of different ectomycorrhizal fungi on growth and survival of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) under field conditions. Douglas-fir seedlings were inoculated with Laccaria bicolor S238 mycelia in two bareroot nurseries in central France or with spore suspensions of three hypogeous ectomycorrhizal species: Melanogaster ambiguus, Rhizopogon colossus and R. subareolatus, in a Spanish containerised nursery. The effects of ectomycorrhizal inoculation on plant survival after outplanting were limited, being only significant at the Guipuzkoan (Spain) site, when plants inoculated with L. bicolor S238 were compared to non-inoculated plants grown in non-fumigated soil. L. bicolor S238 had a significant effect on plant growth during the phase of bareroot nursery growth and this difference was maintained after field outplanting. Nursery inoculations with M. ambiguus, R. colossus and R. subareolatus improved plant growth during the first 2 and 3 years after field outplanting. The positive effects of the inoculation treatment on seedling height, root collar diameter and stem volume persisted after 5 years of field growth. Inoculation with these ectomycorrhizal fungi may improve the field performance of Douglas-fir seedlings in northern Spain. Accepted: 12 February 1999     

The co-occurrence of ectomycorrhizal,arbuscular mycorrhizal,and dark septate fungi in seedlings of four members of the Pinaceae

Although roots of species in the Pinaceae are usually colonized by ectomycorrhizal (EM) fungi, there are increasing reports of the presence of arbuscular mycorrhizal (AM) and dark septate endophytic (DSE) fungi in these species. The objective of this study was to determine the colonization patterns in seedlings of three Pinus (pine) species (Pinus banksiana, Pinus strobus, Pinus contorta) and Picea glauca x Picea engelmannii (hybrid spruce) grown in soil collected from a disturbed forest site. Seedlings of all three pine species and hybrid spruce became colonized by EM, AM, and DSE fungi. The dominant EM morphotype belonged to the E-strain category; limited colonization by a Tuber sp. was found on roots of Pinus strobus and an unknown morphotype (cf. Suillus–Rhizopogon group) with thick, cottony white mycelium was present on short roots of all species. The three fungal categories tended to occupy different niches in a single root system. No correlation was found between the percent root colonized by EM and percent colonization by either AM or DSE, although there was a positive correlation between percent root length colonized by AM and DSE. Hyphae and vesicles were the only AM intracellular structures found in roots of all species; arbuscules were not observed in any roots.     

Effects of manipulation of litter and humus layers on ectomycorrhizal colonization potential in Scots pine stands of different age

Effects of manipulation of litter and humus layers (removal, doubling and control treatments) on the colonization potential of ectomycorrhizal fungi were studied in two secondary stands of Pinus sylvestris (5 and 18 years old) in The Netherlands. Five-mont-hold, sterile-grown Scots pine seedlings, inoculated with Laccaria bicolor, Paxillus involutus or Rhizopogon luteolus and noninoculated seedlings were used as baits. The seedlings were harvested after one growing season. For comparison, sporocarps of ectomycorrhizal fungi were also investigated. Genus composition on the seedlings was independent of initial inoculum, but determined by both treatment and age of the stands. In both stands, removal of litter and humus layers increased, and addition of organic material decreased the number of ectomycorrhizal types on the seedlings. Not all indigenous genera were observed by either outplanting seedlings or sporocarp surveys.     

Colonization of Sitka spruce stumps by decay-causing hymenomycetes in paired inoculations

Sitka spruce stumps were inoculated with decay fungi using colonized sawdust or dowel inoculum to investigate colonization in paired combinations. Estimates of domain sizes were made in the top 15 cm of stump after 13–14 or 21–23 months with sawdust or dowel inoculations, respectively. None of the co-inoculated species prevented colonization by Heterobasidion annosum; sapwood colonization by Resinicium bicolor may limit growth of H. annosum colonies out of heartwood, reducing the incidence of disease transfer at root contacts. H. annosum colonized stumps despite the presence of competing inoculum. Reduced colonization occurred in paired inoculations with R. bicolor, but not with other fungi. Co-inoculations with Stereum sanguinolentum increased colonization by H. annosum. R. bicolor largely remained in the upper 3–4 cm of stumps and reduced colonization by Melanotus proteus; growth of S. sanguinolentum was completely prevented. The results are discussed in relation to the colonization strategies of the decay fungi, their ability to colonize stumps in the presence of competitors and factors influencing development of communities of decay fungi in stumps.     

Use of soil transfer for reforestation on abandoned mined lands in Alaska

Soil transfers from an intermediate successional site and a mature forest site were applied to Populus balsamifera L. cuttings and Alnus crispa (Ait.) Pursh seedlings placed on an abandoned mined site in south central Alaska to improve plant establishment. Mycorrhizal fungi in the soil transfers from the two successional stages were hypothesized to have different effects on plant species that colonize disturbed sites at different times or on different substrates. The site consisted of coarse, dry, low-nutrient spoils and was naturally colonized by scattered P. balsamifera but not A. crispa, although seed sources for both were adjacent to the site. Physical dimensions of the transplanted seedlings and cuttings were measured at the beginning and end of each growing season. Selected plants were harvested at the end of the 2-year study and examined for mycorrhizal formation, current growth, and leaf tissue nutrient concentrations. Both plant species were taller when treated with the soil transfers from the mature forest than with soils from the intermediate site although the increase for A. crispa was greater. Physical dimensions, current growth, and nutrient concentrations were greater when A. crispa was treated with the mature soil transfer compared with the intermediate soil transfer. Mycorrhizae which infected Alnus were predominantly a brown woody type, while other types accounted for greater relative mycorrhizal infection percentage on Populus. Insufficient quantities of mycorrhizal inoculum of suitable species, as well as low moisture and low nutrient conditions, may be factors limiting A. crispa colonization on primary disturbed sites in south central Alaska.     

Fungal associations of Danish Calluna vulgaris roots with special reference to ericoid mycorrhiza

Fungi were isolated from young, serial-washed roots of Calluna sampled from a Danish heathland, Hjelm Hede. Of the 626 isolates, those that were dark, sterile and septate were divided into 13 morphological groups based on their appearance in culture on malt agar. Mycorrhizal synthesis in vitro showed that several groups formed typical ericoid mycorrhiza with seedlings of Calluna; these ericoid mycorrhizal fungi were morphologically similar to Hymenoscyphus ericae. The identities of the other dark, septate fungi are uncertain. Oidiodendron spp. were isolated in a very low frequency; these fungi also formed typical ericoid mycorrhiza. The Calluna root system on Hjelm Hede demonstrated a high morphological diversity among the associated dark, septate fungi suggesting that more than one fungus could coexist in the same host root system.     

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.     

Interaction of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> and <Emphasis Type="Italic">Glomus intrarradix</Emphasis> in Sugar Cane Roots

Fifteen-day-old variety NA 56-79 sugar cane seedlings were inoculated with Azospirillum brasilense and Glomus intrarradix. This article aims at examining changes in sugar cane root seedlings inoculated with Glomus intrarradix and Azospirillum brasilense, the increase in microbial biomass and the acetylene reduction process as well. The internal root colonization was studied 20 days after inoculation using scanning and a transmission electron microscope. Both microorganisms entered the sugar cane root through the emergent lateral roots. The microorganisms were capable of coexisting both intra and intercellularly, producing changes in the cell wall, thus allowing colonization and interaction between the organisms. These changes increased the number of microorganisms inside the root as well as acetylene nitrogen reduction. Sugar cane plant biomass increased with joint-inoculation. The number of endophytic microorganisms and nitrogen fixing activity increased when they were colonized by Azospirillum and Glomus together.     

Chemical,physical and biological control of carrot seedling diseases

In naturally infested soil containingPythium ultimum, P. acanthicum andPhytophthora megasperma, onlyP. ultimum was associated with root rot and damped-off seedlings. Damping-off was promoted by low soil temperatures and by flooding. Seedling stands were markedly reduced when seed was pre-incubated in soil at 12°C but not at 25°C or 35°C. Dusting carrot seed with metalaxyl significantly increased seedling stands in the field at rates from 1.5–6 g kg⁻¹ seed and in both flooded and unflooded, naturally infested soil at 3.15 g kg⁻¹. In greenhouse experiments using artifically infested soil,P. ultimum andP. paroecandrum caused damping-off of carrot seedlings andRhizoctonia solani reduced root and shoot weights.R. solani caused damping-off in nutrient-enriched soil.P. acanthicum andP. megasperma were not pathogenic to seedlings, although both fungi colonized roots. Soil populations of allPythium spp., particularlyP. ultimum, increased during growth of seedlings and population growth ofP. megasperma was promoted by periodic flooding. Infestation of soil withP. acanthicum did not reduce damping-off of carrot seedlings byP. ultimum orP. paroecandrum, but significantly increased root and shoot weights and decreased root colonization byR. solani P. acanthicum has potential as a biocontrol agent againstR. solani.     

Interaction dynamics between saprobic lignicolous fungi and Armillaria in controlled environments: Exploring the potential for competitive exclusion of Armillaria on peach

Armillaria root rot, caused by Armillaria tabescens and Armillaria mellea, is a major cause of premature tree death in peach orchards in the southeastern United States. The root systems of infected trees can become entirely colonized by Armillaria, serving as an inoculum source for adjacent trees and providing massive inoculum levels in replant situations. If dead or dying trees could be colonized by an effective competitor of Armillaria before their removal, the extent of root colonization by the pathogen could be reduced, thus decreasing the threat to adjacent trees and/or subsequent plantings. Interactions between five species of saprobic lignicolous fungi (Ganoderma lucidum, Hypholoma fasciculare, Phanerochaete velutina, Schizophyllum commune, and Xylaria hypoxylon) and the two Armillaria species were examined in controlled conditions to provide proof of concept for competitive exclusion of Armillaria from peach roots. On agar-coated glass slides, all five potential antagonists induced detrimental reactions in >58% of the Armillaria hyphae observed, with the majority resulting in hyphal swelling or granulation. On poplar wood blocks, all antagonists consistently either overgrew Armillaria colonies or—in the case of S. commune—engaged in deadlock reactions; in all cases, the viability of Armillaria colonies was reduced to <30% of that of unchallenged controls. When inoculated simultaneously onto opposite ends of peach root segments, all antagonists consistently reduced growth and viability of Armillaria on and under the bark, whereby reduction of pathogen growth underneath the bark, Armillaria’s primary ecological niche, was most pronounced for G. lucidum, S. commune, and X. hypoxylon. When root segments were allowed to be colonized entirely by Armillaria before being inoculated with the antagonists, the latter were able to overgrow the pathogen on the root surface but unable to pre-empt it from underneath the bark. In summary,G. lucidum, S. commune, and X. hypoxylon caused strong hyphal and mycelial interference reactions and the most pronounced reductions in growth of Armillaria above and below the bark, indicating that they would be the most promising candidates for field-scale evaluations to restrict colonization of dead or dying peach trees by Armillaria in the orchard.     

Evaluation of mycelial inocula of edible<Emphasis Type="Italic"> Lactarius</Emphasis> species for the production of<Emphasis Type="Italic"> Pinus pinaster</Emphasis> and<Emphasis Type="Italic"> P. sylvestris</Emphasis> mycorrhizal seedlings under greenhouse conditions

Different methods to inoculate seedlings of Pinus pinaster and P. sylvestris with edible Lactarius species under standard greenhouse conditions were evaluated. Fungal inoculations were performed both under pure culture synthesis in vitro, followed by transplantation of acclimatized seedlings, and directly in the greenhouse using different techniques for inocula production (mycelial slurries, vegetative inoculum grown in peat-vermiculite and alginate-entrapped mycelium). In vitro inoculations with L. deliciosus produced thoroughly colonized seedlings. However, a sharp decrease in mycorrhizal colonization was detected on transplanted seedlings after 4 month's growth in the greenhouse. On the other hand, all the inocula applied directly in the greenhouse, except the alginate-entrapped mycelium, produced a variable number of mycorrhizal seedlings and colonization rates after the first growing season, depending on the plant-fungal combination and the inoculation method. Inoculations with vegetative inocula of the strain 178 of L. deliciosus were the most effective in producing mycorrhizal seedlings. All the seedlings inoculated with this strain were colonized although the colonization rates were relatively low. The commercial feasibility of the different inoculation methods for the production of seedlings colonized with edible Lactarius species is discussed.