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.     

Competition between Tuber melanosporum and other ectomycorrhizal fungi under two irrigation regimes

A glasshouse experiment was conducted to simulate the competition between artificially introduced Tuber melanosporum (Vitt.) and other symbionts, occurring on outplanted truffle-producing trees. Hazel (Corylus avellana L.) seedlings, previously inoculated with Tuber melanosporum, were rapidly infected with the competing truffle Tuber brumale (Vitt.), added to the soil as spores. Coexistence of both species on the root system was observed. Tuber melanosporum survived and continued to spread. Tuber brumale, which is naturally present in many truffle-orchard soils, protects the roots from other ectomycorrhizal symbionts. Although high water content is unfavourable for the development of T. melanosporum in the absence of other Tube species, Tuber brumale stimulates the development of T. melanosporum under these conditions.     

Synthesis and establishment of <Emphasis Type="Italic">Tuber melanosporum</Emphasis> Vitt. ectomycorrhizae on two <Emphasis Type="Italic">Nothofagus</Emphasis> species in Chile

Axenically germinated seedlings of two species of Southern beech (Nothofagus obliqua, N. glauca) from Chile were inoculated with spores of the Périgord black truffle (Tuber melanosporum). Ectomycorrhizal development was monitored for 6 months in the greenhouse and compared to the performance of the natural host species Quercus ilex and Quercus robur. Seedling survival and mycorrhization showed major differences in both Nothofagus species: T. melanosporum readily formed ectomycorrhizae with seedlings of N. obliqua, although at a lower rate than with Q. ilex but at a proportion very similar to Q. robur; survival and colonization rates were high, and seedling growth was not visibly affected by the high soil pH required by T. melanosporum. In contrast, more than 50% of N. glauca seedlings died after inoculation, and mycorrhiza formation was very sparse. In both species, no colonization by adventive ectomycorrhizal fungi could be observed, whereas both species of Quercus showed minor colonization by another fungus, probably Inocybe or Hebeloma. Our results show that it is possible to infect N. obliqua with the Périgord black truffle under greenhouse conditions, which opens up the possibility of cultivating this truffle as a secondary crop during reforestation with N. obliqua in Chile.     

Afforestation of abandoned farmland with conifer seedlings inoculated with three ectomycorrhizal fungi—impact on plant performance and ectomycorrhizal community

The aim of a 3-year study was to investigate whether inoculation of Pinus sylvestris L. and Picea abies (L.) Karst. seedlings with mycorrhizas of Cenococcum geophilum Fr., Piceirhiza bicolorata, and Hebeloma crustuliniforme (Bull.) Quel. has any impact on: 1) survival and growth of outplanted seedlings on abandoned agricultural land, and 2) subsequent mycorrhizal community development. For inoculation, the root system of each plant was wrapped in a filter paper containing mycelium, overlaid with damp peat–sand mixture and wrapped in a paper towel. In total, 8,000 pine and 8,000 spruce seedlings were planted on 4-ha of poor sandy soil in randomized blocks. Already after the first year natural mycorrhizal infections prevailed in the inoculated root systems, and introduced mycorrhizas were seldom found. Yet, the seedlings that had been pre-inoculated with C. geophilum and the P. bicolorata during the whole 3-year period showed significantly higher survival and growth as compared to controls. Moreover, the independent colonization of roots by C. geophilum and the P. bicolorata from natural sources was also observed. A diverse mycorrhizal community was detected over two growing seasons in all treatments, showing low impact of inoculation on subsequent fungal community development. A total of 19 additional ectomycorrhizal morphotypes was observed, which clustered into two well-separated groups, according to host tree species (pine and spruce). In conclusion, the results showed limited ability to increase tree survival and growth, and to manipulate the mycorrhizal community even by extensive pre-inoculations, indicating that fungal community formation in root systems is governed mainly by environmental factors.     

Changes in ectomycorrhizal community structure on two containerized oak hosts across an experimental hydrologic gradient

Shifts in ectomycorrhizal (ECM) community structure were examined across an experimental hydrologic gradient on containerized seedlings of two oak species, Quercus montana and Quercus palustris, inoculated from a homogenate of roots from mature oak trees. At the end of one growing season, seedlings were harvested, roots were sorted by morphotype, and proportional colonization of each type was determined. DNA was subsequently extracted from individual root tips for polymerase chain reaction, restriction fragment length polymorphism, and rDNA sequencing of the ITS1/5.8S/ITS2 region to determine identities of fungal morphotypes. Twelve distinct molecular types were identified. Analysis of similarity showed that ECM fungal assemblages shifted significantly in composition across the soil moisture gradient. Taxa within the genus Tuber and the family Thelephoraceae were largely responsible for the changes in fungal assemblages. There were also significant differences in ECM community assemblages between the two oak host species. These results demonstrate that the structure of ECM fungal communities depends on both the abiotic and biotic environments and can shift with changes in soil moisture as well as host plant, even within the same genus.     

Arbuscular mycorrhizal colonization and nodulation improve flooding tolerance in <Emphasis Type="Italic">Pterocarpus officinalis</Emphasis> Jacq. seedlings

Pterocarpus officinalis (Jacq.) seedlings inoculated with the arbuscular mycorrhizal fungus, Glomus intraradices, and the strain of Bradyrhizobium sp. (UAG 11A) were grown under stem-flooded or nonflooded conditions for 13 weeks after 4 weeks of nonflooded pretreatment under greenhouse conditions. Flooding of P. officinalis seedlings induced several morphological and physiological adaptive mechanisms, including formation of hypertrophied lenticels and aerenchyma tissue and production of adventitious roots on submerged portions of the stem. Flooding also resulted in an increase in collar diameter and leaf, stem, root, and total dry weights, regardless of inoculation. Under flooding, arbuscular mycorrhizas were well developed on root systems and adventitious roots compared with inoculated root systems under nonflooding condition. Arbuscular mycorrhizas made noteworthy contributions to the flood tolerance of P. officinalis seedlings by improving plant growth and P acquisition in leaves. We report in this study the novel occurrence of nodules connected vascularly to the stem and nodule and arbuscular mycorrhizas on adventitious roots of P. officinalis seedlings. Root nodules appeared more efficient fixing N₂ than stem nodules were. Beneficial effect of nodulation in terms of total dry weight and N acquisition in leaves was particularly noted in seedlings growing under flooding conditions. There was no additive effect of arbuscular mycorrhizas and nodulation on plant growth and nutrition in either flooding treatment. The results suggest that the development of adventitious roots, aerenchyma tissue, and hypertrophied lenticels may play a major role in flooded tolerance of P. officinalis symbiosis by increasing oxygen diffusion to the submerged part of the stem and root zone, and therefore contribute to plant growth and nutrition.     

Effect of soil temperature on nutrient allocation and mycorrhizas in Scots pine seedlings

We studied the effect of soil temperature on nutrient allocation and mycorrhizal development in seedlings of Scots pine (Pinus sylvestris L.) during the first 9 weeks of the growing season. One-year-old seedlings were grown in Carex-peat from a drained and forested peatland at soil temperatures of 5, 9, 13 and 17 °C under controlled environmental conditions. Fourteen seedlings from each temperature treatment were harvested at intervals of three weeks and the current and previous year's parts of the roots, stems and needles were separated. Mineral nutrient and Al contents in all plant parts were determined and the tips and mycorrhizas of the new roots were counted. Microbial biomass C and N in the growth medium were determined at the end of the experiment. None of the elements studied, except Fe, were taken up from the soil by the seedlings during the first three weeks. Thereafter, the contents of all the elements increased at all soil temperatures except 5 °C. Element concentrations in needles, stems and roots increased with soil temperature. Higher soil temperature greatly increased the number of root tips and mycorrhizas, and the numbers of mycorrhizas increased more than did the length of new roots. Cenococcum geophilum was relatively more abundant at lower soil temperatures (5 and 9 °C) than at higher ones (13 and 17 °C). A trend was observed for decreased microbial biomass C and N in the peat soil at higher soil temperatures at the end of the experiment.     

Effect of soil Pseudomonads on colonization of hazel roots by the ecto-mycorrhizal species Tuber melanosporum and its competitors

Hazel (Corylus avellana L.) seedlings, inoculated with the ecto-mycorrhizal fungus Tuber melanosporum (Vitt.) were grown in pots with soil previously inoculated with fluorescent Pseudomonads. These bacteria have been previously isolated from a typical truffle orchard soil. The effect of Pseudomonads on T. melanosporum synthesis proceeded by a two-stage process. Tuber melanosporum infection first declined and then resumed to almost the original level. Infection by competing mycorrhizal fungi declined after 6 months and was very low after 12 months, at the end of the experiment. The same dynamic process occurred with all bacterial strains tested, regardless of their taxonomical group or iron-chelating power. However, some isolates of Pseudomonads seemed to be more efficient than others. A protective effect of Pseudomonads against soil-borne competitors of mycorrhizae was postulated to occur in orchards, consequently favouring the continuation of the T. melanosporum symbiosis. Prospects for use of Pseudomonads in nurseries are discussed.     

Mycorrhizal associations in Pinus massoniana Lamb. and Pinus elliottii Engel. inoculated with Pisolithus tinctorius

Dichotomous mycorrhizas were induced in Pinus massoniana and Pinus elliottii seedlings inoculated with Pisolithus tinctorius growing under non-axenic conditions. Six months after inoculation, Pinus massoniana seedlings exhibited a higher degree of infection, bore more mycorrhizas and had developed more abundant extramatrical mycelium than seedlings of Pinus elliottii. Nevertheless, seedlings of Pinus massoniana were stunted and exhibited chorosis of the needles, indicating a possible nutrient deficiency. Histological examination of these pine mycorrhizas showed an ectomycorrhizal association typical of gymnosperms with an intercellular Harting net penetrating between several layers of cortical cells close to the endodermis. However, strong polyphenolic reactions, intracellular hyphae and wall modifications were occasionally observed, indicating that both host-tissue incompatibility and ectendomycorrhizal association can occur in pine species under stressed conditions.     

Selecting ectomycorrhizal fungi for inoculating plantations in south China: effect of Scleroderma on colonization and growth of exotic Eucalyptus globulus, E. urophylla, Pinus elliottii, and P. radiata

Plantation forestry with exotic trees in south China needs compatible symbionts to improve the growth of seedlings in nurseries and to enhance establishment and growth in the field. Scleroderma, a potentially suitable symbiont for inoculation, is not being used in containerized nurseries in the region due to poor knowledge of its host range. The ability of 15 collections of Scleroderma, nine from Australia and six from Asia, to colonize and promote growth of four important exotic plantation trees (Eucalyptus globulus Labill., Eucalyptus urophylla ST Blake, Pinus elliottii Engl., and Pinus radiata D. Don) was examined in a nursery potting mix. There was generally low host specificity of Scleroderma between tree genera. At 12 weeks after inoculation, 13 to 14 of the 15 spore collections formed ectomycorrhizas on seedlings of eucalypts or pines. The extent of colonization differed between spore treatments with two or four collections forming abundant mycorrhizas (>50% fine roots colonized) on E. globulus or E. urophylla, respectively, and three or five on P. radiata or P. elliottii, respectively. Three collections from Australia strongly colonized all hosts resulting in 26 to 100% of short roots being colonized. Chinese Scleroderma collections resulted in fewer mycorrhizas on eucalypts than on pines. Inoculation stimulated the growth (shoot height and dry weight) of eucalypt and pine seedlings by up to 105% where Scleroderma mycorrhizas developed. The results suggest that there is a need to source Scleroderma from outside China for inoculating eucalypts in Chinese nurseries whereas Chinese collections of Scleroderma could be used in pine nurseries. Further screening of Australian and Chinese Scleroderma should be performed in Chinese nurseries and in the field before final commercial decisions are made.     

Ectomycorrhizal and arbuscular mycorrhizal colonization of two species of floodplain willows

To understand the relationships between the distribution of Chosenia arbutifolia and Salix sachalinensis and their mycorrhizal colonization, changes in the quality and types of ectomycorrhizas and arbuscular mycorrhizas of the seedlings of two species were studied at five different sites with different soil conditions in the floodplain of the Satsunai River, Hokkaido. High ectomycorrhizal and low arbuscular mycorrhizal colonization were found in roots of both plants. Ectomycorrhizal colonization of S. sachalinensis in wet sandy or muddy soil conditions was at the same level as that in dry gravelly sites. In contrast, ectomycorrhizal colonization of C. arbutifolia seedlings was lower from wet sandy sites than that from dry gravelly sites. In all study sites, the same three morphological types of ectomycorrhizas were dominant.     

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.     

Calcareous amendments to soils to eradicate Tuber brumale from T. melanosporum cultivations: a multivariate statistical approach

Calcareous amendments are being used in Tuber melanosporum truffle plantations in attempts to eradicate Tuber brumale. However, there are no studies available which provide soil analysis and statistical data on this topic. We studied 77 soil samples to compare the values for carbonates, pH and total organic carbon in T. brumale truffières with the values for T. melanosporum truffières on contaminated farms and in natural areas. Statistical analyses indicate that the concentrations of active carbonate and total carbonate in the soil are significantly higher in T. brumale truffières than in T. melanosporum truffières, but that there are no significant differences in pH and total organic carbon. We conclude that liming would not suppress T. brumale ectomycorrhizas in contaminated T. melanosporum farms, and calcareous amendments do not therefore seem be a means of eradicating T. brumale in these farms.     

Can NPK fertilizers enhance seedling growth and mycorrhizal status of Tuber melanosporum-inoculated Quercus ilex seedlings?

Although successful cultivation of the black truffle (Tuber melanosporum) has inspired the establishment of widespread truffle orchards in agricultural lands throughout the world, there are many unknowns involved in proper management of orchards during the 6–10 years prior to truffle production, and there are conflicting results reported for fertilizer treatments. Here, we systematically evaluate the combined effects of nitrogen, phosphorous, and potassium with different doses of each element, applied to either foliage or roots, on plant growth parameters and the mycorrhizal status of outplanted 3-year-old seedlings in five experimental Quercus ilex–T. melanosporum orchards. Fertilization did not significantly improve seedling aboveground growth, but the plants treated with the fertilizer 12-7-7 applied to the roots (HNr) displayed longer field-developed roots. Only the fertilizer with the highest dose of K (10-6-28) applied to the foliage (HKf) increased the probability of fine root tip colonization by T. melanosporum in field-developed roots. However, the plants treated with the same fertilizer applied to the soil (HKr) presented the highest probability for colonization by other competing mycorrhizal soil fungi. Potassium seems to have an important role in mycorrhizal development in these soils. Apart from T. melanosporum, we found 14 ectomycorrhizal morphotypes, from which seven were identified to species level, three to genus, two to family, and two remained unidentified by their morphological characteristics and DNA analyses.     

Fungi associated with terrestrial orchid mycorrhizas,seeds and protocorms

The identity and ecological role of fungi in the mycorrhizal roots of 25 species of mature terrestrial orchids and in 17 species of field incubated orchid seedlings were examined. Isolates of symbiotic fungi from mature orchid mycorrhizas were basidiomycetes primarily in the generaCeratorhiza, Epulorhiza andMoniliopsis; a few unidentified taxa with clamped hyphae were also recovered. More than one taxon of peloton-forming fungus was often observed in the cleared and stained mycorrhizas. AlthoughCeratorhiza andEpulorhiza strains were isolated from the developing protocorms, pelotons of clamped hyphae were often presents in the cleared protocorms of several orchid species. These basidiomycetes are difficult to isolate and may be symbionts of ectotrophic plants. The higher proportion of endophytes bearing clamp connections in developing seeds than in the mycorrhizas is attributed to differences in the nutritional requirements of the fully mycotrophic protocorms and partially autotrophic plants. Most isolates ofCeratorhiza differed enzymatically fromEpulorhiza in producing polyphenol oxidases. Dual cultures with thirteen orchid isolates and five non-orchid hosts showed that some taxa can form harmless associations with non-orchid hosts. It is suggested that most terrestrial orchid mycorrhizas are relatively non-specific and that the mycobionts can be saprophytes, parasites or mycorrhizal associates of other plants.     

Influence of ectomycorrhizal fungi on the response of Sitka spruce and Japanese larch to forms of phosphorus

Ectomycorrhizal fungi (Paxillus involutus, Suillus grevillei and two unidentified basidiomycetes from excised Sitka spruce mycorrhizas) were isolated from stands of Sitka spruce either in monoculture or in a mixture with Japanese larch in an Irish conifer plantation. The growth of these fungi and their mycorrhizal formation in Sitka spruce and Japanese larch were examined after incubation in modified Melin-Norkrans medium containing either KH₂PO₄, Ca₃(PO₄)₂ or Fe phytate as the phosphorus (P) source. P. involutus and S. grevillei utilized all three P sources. The unidentified basidiomycetes had limited ability to utilize Fe phytate. Basidiomycete 1 showed poor growth on KH₂PO₄ whereas growth of basidiomycete 2 was low on Ca₃(PO₄)₂. Pure culture synthesis studies confirmed that P. involutus and the two basidiomycetes formed mycorrhizas with both tree species but S. grevillei was mycorrhizal only on Japanese larch. P. involutus formed more mycorrhizas in both conifers than the other fungi. Following inoculation with each of the four fungi, shoot and root dry mass of both Sitka spruce and Japanese larch seedlings was enhanced compared with uninoculated/nonmycorrhizal controls. On Fe phytate, Paxillus-inoculated Sitka spruce seedlings had the lowest primary root length and on KH₂PO₄, Suillus-inoculated Japanese larch had the greatest number of short roots. The only differences when Sitka spruce seedlings were grown in either monoculture or in a mixture with Japanese larch mycorrhizal with S. grevillei were primary root length and number of short roots after growth on media containing Fe phytate.     

Relationship of inoculum ofVerticillium dahliae to disease in tomato plants

Summary Tomato (Lycopersicon esculentum Mill.) seedlings were inoculated by dipping roots in suspensions of conidia ofVerticillium dahliae Kleb. There was a linear relationship between inoculum concentration and infection at 21 days after inoculation in steam-disinfested soil. Thereafter the number of infections per plant increased. At higher concentrations data indicated a synergistic interaction between conidia. Synergism was more pronounced and was detected earlier in soil not disinfested before inoculation. When conidia from 3-, 7-, and 14-day-old cultures were used, the greatest response was from 3-day-old cultures. A lower total response and lower rate of response to older inoculum indicated a decrease in aggressiveness of conidia with age. Older plants were less affected by the pathogen when plants inoculated at the fourth and sixth leaf stages with minimum root disturbances were compared. There was also an increase in infection with an increase in the volume of root zone infested. In field experiments infection reduced stand when roots were disturbed at inoculation. With minimum root disturbance yields were reduced without a reduction in stand. Part of thesis to be submitted by the senior author to the University of Stellenbosch in partial fulfilment of the Ph.D. (Agric.) degree.     

In situ and in vitro colonization of Cathaya argyrophylla (Pinaceae) by ectomycorrhizal fungi

Cathaya argyrophylla, a critically endangered conifer, is found to grow at four isolated areas located in subtropical mountains of China. To examine the involvement and usefulness of mycorrhizas for sustaining the population of this tree, we compared the root system, morphology, and structure of mycorrhizal roots of C. argyrophylla, which were collected from a natural stand and an artificial stand, each grown at a different location. More mycorrhizal roots were found for trees from an artificial stand. The presence of extramatrical mycelium, mantle, and Hartig net revealed that C. argyrophylla formed an ectomycorrhizal association in both sampling sites. Starch granules were found in mycorrhizal roots collected only from a natural stand. The aseptic synthesis of C. argyrophylla and Cenococcum geophilum was established for the first time in vitro. Typical ectomycorrhizas formed on seedlings on RM medium containing 0.1 g/l glucose, 5 weeks after inoculation. By light microscopy, the synthesized mycorrhizas showed a thin mantle from which emanated extramatrical hyphae and highly branched Hartig net. A simple, rapid, and convenient mycorrhiza synthesis system was developed, which facilitates further studies on ectomycorrhizal development of C. argyrophylla.     

Formation of structures resembling ericoid mycorrhizas by the root endophytic fungus Heteroconium chaetospira within roots of Rhododendron obtusum var. kaempferi

A resynthesis study was conducted to clarify the relationship between the root endophyte, Heteroconium chaetospira and the ericaceous plant, Rhododendron obtusum var. kaempferi. The host plant roots were recovered 2 months after inoculation, and the infection process and colonization pattern of the fungus were observed under a microscope. The hyphae of H. chaetospira developed structures resembling ericoid mycorrhizas, such as hyphal coils within the host epidermal cells. These structures were morphologically the same as previously reported ericoid mycorrhizal structures. The frequencies of hyphal coils within the epidermal cells of host roots ranged from 13 to 20%. H. chaetospira did not promote or reduce host plant growth. This is the first reported study that H. chaetospira is able to form structures resembling mycorrhizas within the roots of ericaceous plants.