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     

Coinoculation of containerized Douglas-fir (Pseudotsuga menziesii) and maritime pine (Pinus pinaster) seedlings with the ectomycorrhizal fungi Laccaria bicolor and Rhizopogon spp.

 Coinoculations with mycelium of Laccaria bicolor and spores of Rhizopogon spp. included in alginate gel have been carried out to determine: (1) the ability of the mixed inoculum to produce dual-colonized containerized Douglas-fir and maritime pine planting stocks and (2) the colonization pattern of the two fungi in individual root systems. For both tree species, the maximal proportion of dual-colonized seedlings obtained almost never exceeded 50%. The rest of the seedlings remained colonized by a single fungus or were non-colonized. In Douglas-fir inoculations, the relationship between the dual-colonized seedlings obtained and the initial dose of the two fungi was highly significant. The highest proportion of dual-colonized seedlings was obtained when the highest dose of R. subareolatus was used (10⁶ spores/seedling), regardless of the dose of L. bicolor. Among the treatments producing 25% or more dual-colonized seedlings, differences in the proportion of Laccaria/Rhizopogon mycorrhizas and total root colonization percentages were not clearly related to the initial combination of doses. The proportion of dual-colonized maritime pine seedlings was not significantly related to the initial inoculation doses of the two fungi. The proportion of Laccaria/Rhizopogon mycorrhizas was not significantly different among treatments with 25% or more dual-colonized seedlings, whereas total colonization percentages ranged from 37% with the combination 0.08/10⁴ (g L. bicolor / spores R. roseolus per seedling) to 74% with the combination 0.08/10⁶, this difference being statistically significant. Accepted: 15 September 1998     

Coinoculation efficacy of ectomycorrhizal fungi on Pinus patula seedlings in a nursery

 The coinoculation efficacy of the ectomycorrhizal fungi Laccaria laccata and Thelephora terrestris on the growth and mycorrhizal development of Pinus patula seedlings was studied and compared to individual inoculation of these fungi in a nursery. The total number of mycorrhizas was higher in seedlings inoculated with the combined inoculum than with the individual inocula. The colonization by T. terrestris was higher than L. laccata when the seedlings were inoculated with the two fungi simultaneously. Coinoculation significantly increased the height and dry weight of the seedlings compared with individual inoculation, both in steam-sterilized and unsterilized soil. Accepted: 12 May 1997     

Agrobacterium tumefaciens-mediated transformation of Pinus pinea L. cotyledons: an assessment of factors influencing the efficiency of uidA gene transfer

 This study is the first report of a protocol for transfer and expression of foreign chimeric genes into cotyledons excised from Pinus pinea L. embryos. Agrobacterium tumefaciens EHA105 harbouring the plasmid p35SGUSint was more infective than LBA4404 or C58 GV3850, as determined by the percentage of cotyledons showing uidA expression. Factors which significantly affected the T-DNA transfer included: (1) preinduction and concentration of bacteria, (2) days of coculture and (3) the wounding procedure applied. More efficient transfer of the uidA gene was achieved growing the bacteria in YEP medium at pH 7, infecting the cotyledons according to the sonication-assisted Agrobacterium-mediated transformation procedure with a bacterial density of 1 (OD_600 nm) for 5 min, and coculture for 72 h. Using this protocol, 49.7% of the cotyledons showed a diffuse blue staining 7 days after infection. However, all were necrotic 30 days after inoculation. Since a decrease in bacterial density to 0.01 allowed the recovery of about 4% of cotyledons forming buds 1 month after inoculation, we conclude that the high mortality associated with the infection may be related to the hypersensitive response of the plant to bacterial infection. Received: 19 October 1998 / Revision received: 18 February 1999 / Accepted: 24 February 1999     

Crown architecture of grafted Stone pine (Pinus pinea L.): shoot growth and bud differentiation

The singular umbrella-like crown shape of Stone pine can be interpreted as a consequence of primary shoot-growth patterns and posterior axis differentiation due to differential secondary growth and down-bending of branches. This paper centres on the first aspect, analysing the growth, branching and flowering behaviour of about 5,000 individual shoots on 27 grafted Stone pines. The data measurement on standing trees allowed to study correlations of topologic and geometric variables in the shoot and their ancestors. The only significant correlations were found with parameters of the mother shoot formed the previous year and with the number of cones born 3 years before by the respective ancestor. The fitted relationships between geometric and topologic shoot and branch variables are the first step of a structural model construction that can be completed with functional components like a radiation and a carbon allocation submodel, stressing the importance of the heavy Stone pine cones as carbon sinks, with a total annual allocation similar to stem wood. In conclusion, the Stone pine crown shape emerges as consequence of the lack of initial vigour differentiation between stem and main-branch apical meristems that favour the generalized sylleptic reiteration in the open-grown trees.     

Effects of Pseudomonas fluorescens DF57 on growth and P uptake of two arbuscular mycorrhizal fungi in symbiosis with cucumber

 The effect of Pseudomonas fluorescens DF57 on growth and P uptake of two arbuscular mycorrhizal (AM) fungi in symbiosis with cucumber plants was studied in compartmentalised growth systems. Hyphae of Glomus intraradices Schenck & Smith (BEG87) or G. caledonium (Nicol. & Gerd.) Trappe & Gerdeman (BEG15) grew into lateral root-free compartments. Non-mycorrhizal plants served as control. The soil in half of the growth units of each mycorrhizal treatment was inoculated with P. fluorescens DF57. P. fluorescens DF57 enhanced hyphal length density of one of the AM fungi, G. caledonium, but this was not reflected in a higher hyphal transport of P from the root-free soil to the plant. The total P content was higher in plants grown in symbiosis with G. intraradices than in plants in the other treatments. G. caledonium and P. fluorescens DF57 had a synergistic effect in that total P content in plants inoculated with G. caledonium was higher in the presence than in the absence of P. fluorescens DF57. Accepted: 7 January 1999     

The effects of Laccaria proxima and fibrous pulp waste on the growth of nine container-grown conifer seedling species

 Japanese larch (Larix kaempferi), white spruce (Picea glauca), black spruce (Picea mariana), red spruce (Picea rubens), jack pine (Pinus banksiana), mugo pine (Pinus mugo), red pine (Pinus resinosa), Japanese black pine (Pinus thunbergii) and Douglas-fir (Pseudotsuga menziesii var. menziesii), were inoculated to test the effective host range of the ectomycorrhizal fungus Laccaria proxima and the possibility of utilizing pulp waste as a potting medium for containerized seedling production. Laccaria proxima tended to improve the container growth of Japanese black pine and white spruce, and significantly improved that of jack pine, mugo pine, black spruce, red spruce and Douglasfir. The growth of red pine and Japanese larch were only slightly improved with L. proxima. Pulp waste (33% by volume) had negative effects on tree seedling growth, except for Douglasfir (no significant effect). The interactions of Laccaria proxima and pulp waste varied; the hosts were significantly positive (P<0.01) in the case of jack pine and black spruce, but there was no significant effect for the rest. Negative effects were found with Japanese black pine. Use of pulp waste in seedling production of jack pine, black spruce, mugo pine, red spruce and Douglasfir inoculated with L. proxima and of Japanese black pine both with and without L. proxima is feasible, but further research is necessary to determine the optimal percentage of pulp waste that can be utilized in seedling production of tree species and the field performance of these seedlings. Accepted: 30 August 1995     

Intraspecific physiological variation: implications for understanding functional diversity in ectomycorrhizal fungi

 Several thousand fungal species worlwide are thought to form ectomycorrhizas (ECM) with tree hosts and there is currently much interest in determining the functional significance of such diversity in natural and managed ecosystems. While only a few taxa have been investigated in detail, it is clear that ECM fungi display extensive intraspecific variation in a range of physiological and other life-history parameters. Thus, comparative investigations of single (or even a few) isolates of different species are unlikely to provide reliable information on functional capabilities. Extensive screening of taxonomically well-defined isolates is required. This must take into account spatial and temporal variation in gene expression in mycelia growing in axenic culture or in association with a host plant. Accepted: 29 June 1999     

Morphological and anatomical characterisation of black alder Alnus glutinosa (L.) Gaertn. ectomycorrhizas

 Ectomycorrhizal types of black alder [Alnus glutinosa (L.) Gaertn.] collected over a 3-year period within an alder forest were characterised by morphological and anatomical features. Of the total of 16 types, 14 are described for the first time in this paper. Eight identified types belong to the genera Russula, Lactarius, Naucoria, and Cortinarius, while eight further types remained unidentified. In some cases, similarities of mantle features indicate relationships to identified mycorrhizas. Mycorrhizas of Naucoria escharoides and N. subconspersa were not distinguished. Two unidentified mycorrhizal types exhibited hyphal mantle structures very similar to these Naucoria species. Within the genus Cortinarius, mycorrhizas of C. cf. helvelloides were easily distinguished from all other Cortinarius-like mycorrhizas described on Alnus, which in general showed little anatomical variation. Two further unidentified mycorrhizas, "Alnirhiza lilacina" and "A. violacea", probably also belong to Cortinarius. The ectomycorrhiza of Russula pumila was the only identified type within the genus Russula, but the unidentified type "Alnirhiza cremicolor" also likely belongs to this genus. Three Lactarius species were present in the experimental plot. Two species (L. obscuratus and L. omphaliformis) had indistinguishable mycorrhizal types, but were easily differentiated from the mycorrhizas of L. lilacinus, which caused intracellular penetration of Hartig net hyphae into epidermal and cortical cells. All other mycorrhizal types of black alder exhibited a paraepidermal Hartig net without penetration of root cells. Two unidentified mycorrhizal types "Alnirhiza atroverrucosa" and "A. cystidiobrunnea", already described from North American Alnus rubra as unnamed morphotypes, showed no similarity to identified mycorrhizas. All 16 mycorrhizal types appeared to be specific or at least typical for alders, since they have not yet been reported from other tree species. Accepted: 29 August 1997     

Influence of soil impoverishment on the interaction between Glomus mosseae and saprobe fungi

 The effect of the saprobe fungi Wardomyces inflatus (Marchal) Hennebert, Paecilomyces farinosus (Holm & Gray) A. H. S. Brown & G. Sm., Gliocladium roseum Bain., Trichoderma pseudokoningii Rifai and T. harzianum Rifai, isolated from sporocarps of Glomus mosseae, on arbuscular mycorrhizal (AM) colonisation and plant dry matter of soybean was studied in 2/3 and 1/5 diluted soils in a greenhouse trial. Soil dilution to 1/5 had no effect on shoot dry matter of soybean but decreased AM colonisation and root dry weight of plants. CFU of saprobe fungi, except T. harzianum, were higher in 1/5 than in 2/3 diluted soils. W. inflatus and Gliocladium roseum decreased the shoot dry weight of soybean plant when inoculated together with Glomus mosseae. The saprobe fungi P. farinosus and T. pseudokoningii increased the shoot dry weights of plants grown in 1/5 diluted soil. The shoot dry weight and AM colonisation in 1/5 diluted soil were also increased when T. harzianum was inoculated together with Glomus mosseae. Thus, saprobe fungi increased AM colonisation of soybean plants by indigenous endophytes. The AM colonisation of plants at both soil dilutions was increased by Glomus mosseae. The highest level of AM colonisation was observed when P. farinosus and T. pseudokoningii were inoculated together Glomus mosseae. The dilution of soils influenced the interaction between inoculated microorganisms and their effect on plant growth. Accepted: 7 June 1999     

Specifying the introgressed regions from H. argophyllus in cultivated sunflower (Helianthus annuus L.) to mark Phomopsis resistance genes

A method based upon targetting of intro-gressed markers in a Phomopsis-resistant line (R) of cultivated sunflower, issuing from a H. argophyllus cross was used to mark the Phomopsis resistance regions. Our study was based upon 203 families derived from a cross between an inbred line susceptible to Phomopsis (S1) and the introgressed resistant line (R). Families were checked for Phomopsis resistance level in a design with replicated plots and natural infection was re-inforced by pieces of contaminated stems. Thirty four primers were employed for RAPD analysis. Out of 102 polymorphic fragments between (S1) and H. argophyllus, seven were still present in (R) suggesting that they marked introgressions of H. argophyllus into (R). The plants were scored for the presence or absence of 19 fragments obtained from five primers, and the relationships between the presence/absence of fragments in plants and Phomopsis resistance/susceptiblity in the progenies was determined by using an analysis of variance. We found that at least two introgressed regions, as well as favourable factors from sunflower, contributed to the level of Phomopsis resistance in cultivated sunflower. Received: 28 June 1996 / Accepted: 5 July 1996     

Western white pine (Pinus monticola Dougl.) reproduction: I. Gametophyte development

Male and female gametophyte development are described from light and transmission electron microscope preparations of ovules from first and second year Pinus monticola Dougl. seed cones. In the first year of development, pollen tubes penetrate about one-third the distance through the nucellus. The generative cell and tube nucleus move into the pollen tube. The megagametophyte undergoes early free nuclear division. First-year seed cones and pollen tubes become dormant in mid-July. In the second year, seed cones and pollen tubes resume development in April and the pollen tubes grow to the megagametophyte by mid-June. Early in June the generative cell undergoes mitosis, forming two equal-size sperm nuclei that remain within the generative cell cytoplasm. The generative cell has many extensions and abundant mitochondria and plastids. The megagametophyte resumes free nuclear division, then cell wall formation begins in early July. Cell wall formation and megagametophyte development follow the pattern found in other Pinaceae. Three to five archegonial initials form. The primary neck cell divides, forming one tier of neck cells. Jacket cells differentiate around each central cell. The central cell enlarges and becomes vacuolate; then vacuoles decrease in size and the cell divides, forming a small ventral canal cell and a large egg. Plastids in the central cell engulf large amounts of cytoplasm and enlarge. This process continues in the egg, and the peripheral cytoplasm of the egg becomes filled with transformed plastids. Mitochondria migrate around the nucleus, forming a perinuclear zone. The wide area of egg cytoplasm between these two zones has few organelles. A modified terminology for cells involved in microgametophyte development is recommended. Received: 9 December 1999 / Revision accepted: 30 April 2000     

Characterization of spelt (Triticum spelta L.) forms by gel electrophoretic analyses of seed storage proteins. I. The gliadins

A comparison betweeen the electropherograms of the spelt and wheat cultivars showed specific differences in the gliadin band patterns which provided the possibility of a clear classification into spelt or wheat. A special nomenclature was developed to be able to improve the presentation of the gliadin band pattern of spelt, which is different from that of wheat. This nomenclature, however, has not yet been applied to other cereals. The gliadin band patterns were presented in a schematic form. As a parameter for comparison, idealized band patterns of both wheat and spelt were developed by comparing the proportions of the bands of all available types. When comparing the gliadin band patterns of the spelt cross-breeds with their corresponding parental generations, it was noted that the same parental bands were not always transmitted and that the cross-breeds showed differences in the intensity, mobility, occurrence, and the splitting of single bands. In general it can be said that the band pattern of the daughter generation – even in the examined and generations – is more similar to the band pattern of the mother than to that of the father, which proves a maternal effect. Received: 29 June 1996 / Accepted: 12 July 1996     

The mycorrhizal status of the woody Mediterranean shrub Myrtus communis L.

  Myrtus communis L. (myrtle), a typical Mediterranean plant species belonging to the family Myrtaceae, was shown to form arbuscular mycorrhizal symbioses in nature. Many different spore types were isolated from its rhizosphere and grown in pot cultures; six of them were identified as Glomus species. In the laboratory, the myrtle root system was colonized by indigenous endophytes as well as by an Italian isolate of Glomus intraradices. In greenhouse experiments, mycorrhizal inoculation reduced transplant stress in 60-day-old myrtle seedlings; their growth was renewed immediately after transplanting, whereas non-mycorrhizal plants stopped development. Significantly larger growth responses were obtained using indigenous fungi than the Italian isolate of Glomus intraradices. Accepted: 16 January 1997     

Chloroplast phylogeography and evolution of highly polymorphic microsatellites in lodgepole pine ( Pinus contorta)

We employed a novel set of six highly polymophic chloroplastic simple sequence repeat (cpSSR) loci to investigate the phylogeography of lodgepole pine (Pinus contorta Dougl. Ex. Loud.), and to examine aspects of the evolutionary process operating on these repetitive DNA sequences. Chloroplast haplotypes of 500 trees, sampled throughout the range of lodgepole pine, were determined. We found a marked association of genetic distance with physical distance within the scale of 0 to 1,000 km, but no association beyond that range. Likewise, geographic clustering was observed only among recent clades in a dendrogram. These phylogeographic patterns are consistant with a rapid rangewide expansion (”big-bang”) followed by recent, local population differentiation (”galaxy formation”). In support of this expansion, coalescent simulations of the genealogical process gave a long-term effective population size in the low thousands, and a time to common ancestry of about 1,500 generations (12,000 years), consistent with a post-Pleistocene population expansion as documented by previous pollen-sediment analyses. Two lines of evidence (mapping mutational events onto a phylogeny, and evaluation of observed versus expected gene diversity) suggest that five of the cpSSR loci evolve primarily by a stepwise model of evolution of single repeat changes (but with a small proportion of changes involving two or more repeats), and the coalescent simulations point to a mutation rate of about 10^–3. Received: 7 December 2000 / Accepted: 17 May 2001     

Group coancestry-controlled selection in a Pinus sylvestris L. breeding program

 Integer Linear Programming was used to maximize genetic gain from selection at a given level of relatedness. Variances and breeding values for total height were available for 296 plus-trees of Pinus sylvestris which had been evaluated by open-pollinated progeny testing at a single test site in northern Sweden. Second-generation breeding and selection scenarios for this breeding population were evaluated using simulated data derived deterministically from normal distributions of estimated breeding values of progeny around mid-parent family means. The study considered two mating designs, assortative and non-assortative single-pair mating, and two selection criteria, individual phenotype and performance of half-sib progeny. Relatedness (group coancestry) was restricted to a level equivalent to that given by within-family selection of 2 trees per family from each of 25 families (the current standard in Sweden). Selection that allows the best-performing families to contribute a greater number of progeny was superior, both when the breeding population size was limited to 50 individuals and when it was allowed to be larger. The selected set giving the greatest average breeding value under restricted group coancestry included the best individual from families that would have been rejected under application of standard within-family selection. We made a comparison of the present value on retrieved gain between phenotypic selection and evaluation by progeny testing. Received: 24 November 1998 / Accepted: 14 December 1998     

Western white pine (Pinus monticola Dougl.) reproduction: II. Fertilisation and cytoplasmic inheritance

Fertilisation and proembryo development are described from transmission electron micrographs emphasising the origin and fate of the maternal and paternal mitochondria and plastids. During central cell and egg development mitochondria migrate toward the nuclei, forming a perinuclear zone consisting predominantly of maternal mitochondria and polysomes. At the same time, maternal plastids transformed and at fertilisation are excluded from the neocytoplasm. The pollen tube releases two sperm nuclei into the egg with cytoplasm from the generative cell and the tube cell. The leading sperm nucleus fuses with the egg nucleus and a small number of paternal mitochondria and plastids are taken into the perinuclear zone. The second sperm nucleus degenerates. As the zygote nucleus undergoes mitosis followed by free nuclear division and nuclear migration to the chalazal end of the archegonium, maternal and paternal organelles intermingle within the neocytoplasm. The result is paternal inheritance of plastids and biparental, but predominantly maternal, inheritance of mitochondria. This pattern is consistent within the Pinaceae but differs from some other conifer families. Received: 9 December 1999 / Revision accepted: 30 April 2000     

The role of temperature in the onset of the Olea europaea L. pollen season in southwestern Spain

Temperature is one of the main factors affecting the flowering of Mediterranean trees. In the case of Olea europaea L., a low-temperature period prior to bud development is essential to interrupt dormancy. After that, and once a base temperature is reached, the plant accumulates heat until flowering starts. Different methods of obtaining the best-forecast model for the onset date of the O. europaea pollen season, using temperature as the predictive parameter, are proposed in this paper. An 18-year pollen and climatic data series (1982–1999) from Cordoba (Spain) was used to perform the study. First a multiple-regression analysis using 15-day average temperatures from the period prior to flowering time was tested. Second, three heat-summation methods were used, determining the the quantities heat units (HU): accumulated daily mean temperature after deducting a threshold, growing degree-days (GDD): proposed by Snyder [J Agric Meteorol 35:353–358 (1985)] as a measure of physiological time, and accumulated maximum temperature. In the first two, the optimum base temperature selected for heat accumulation was 12.5°C. The multiple-regression equation for 1999 gives a 7-day delay from the observed date. The most accurate results were obtained with the GDD method, with a difference of only 4.7 days between predicted and observed dates. The average heat accumulation expressed as GDD was 209.9°C days. The HU method also gives good results, with no significant statistical differences between predictions and observations. Received: 18 April 2000 / Revised: 14 September 2000 / Accepted: 19 September 2000