Ability of native ectomycorrhizal fungi from northern Spain to colonize Douglas-fir and other introduced conifers

 Thirty-six isolates from 27 species of native ectomycorrhizal fungi collected in northern Spain were tested for ectomycorrhiza formation with Pseudotsuga menziesii seedlings in pure culture syntheses. Thirteen of those species were also tested for ectomycorrhiza formation with six other species of conifers (two native and four introduced) to compare their colonization potential. Twenty-three fungal isolates from 18 species formed ectomycorrhizas with Pseudotsuga menziesii. The colonization level of the root system varied markedly among the different fungal species. Eight fungi colonized over 50% of the short roots. Nine fungi did not form ectomycorrhizas even though some of them were collected in pure stands of Pseudotsuga menziesii. Laccaria laccata, Lyophyllum decastes, Pisolithus tinctorius, and Scleroderma citrinum formed abundant ectomycorrhizas on all the conifers tested. Lactarius deliciosus, Rhizopogon spp., and Suillus luteus showed the greatest host specificity. The success in the introduction of some exotic conifers for reforestation in northern Spain is discussed in relation to their compatibility with native ectomycorrhizal fungi. Accepted: 28 August 1995     

Simplified and improved somatic embryogenesis for clonal propagation of Pinus pinaster (Ait.)

In this study, several improvements and simplifications of SE protocols in Pinus pinaster (Ait.), a species of economic importance in the regions of Western Europe, are described. These improvements pertained to all stages of SE including high initiation frequencies in eight control pollinated seed families, relatively high somatic embryo maturation yield when cells were coated with particles of activated charcoal and a rapid production of plants directly in a shade house. The SE initiation frequency from isolated zygotic embryos was high (up to 100%) and plants were produced from 11 embryogenic lines representing all crosses. Based on these results, the estimated number of somatic embryos required to produce 1,000 plants varied from slightly more than the required number of plants to more than double this number depending on the line. Such an estimate is critical in developing plant production strategy when a number of embryogenic lines are considered for production of clonal plants.     

Establishment and characterization of Pinus pinaster suspension cell cultures

We report the establishment of a Pinus pinaster (Ait.) cell suspension culture in a modified MS medium supplemented with 2 mg ml⁻¹ 2,4-D and 1 mg ml⁻¹ BA. Calli were obtained from seedling root segments and established a friable isodiametric cell suspension, suitable for in vitro studies of maritime pine at the cellular level. Growth (dry weight), cell viability, pH, and nutrient consumption: carbon source (sucrose, fructose and glucose), nitrogen source (ammonia and nitrate) and phosphate were monitored over 24 h. Suspension cells exhibited a 15-day exponential growth stage, during which a biphasic consumption profile was observed for all nutrients. Phosphate was the first limiting nutrient and preferable consumption was observed for glucose over fructose and nitrate over ammonium.     

Ectomycorrhizal fungi of Pinus pinea L. in northeastern Spain

 Although Pinus pinea L. is an important forest species in the Mediterranean region, few reports exist on its ectomycorrhizal associates. Sixty isolates, obtained from fungal sporocarps collected in mixed forests of P. pinea in Catalonia (northeastern Spain), were tested for ectomycorrhiza formation on containerized P. pinea seedlings when applied as mycelial inoculum produced in peat-vermiculite. A total of 17 isolates, in 8 genera (Amanita, Hebeloma, Laccaria, Lactarius, Pisolithus, Rhizopogon, Scleroderma and Suillus), formed ectomycorrhizas and the percentages of mycorrhizal short roots varied among isolates and species from 13% to 89%. Some of these fungi are cited for the first time in association with P. pinea. The results indicate further fungal candidates for controlled inoculation of P. pinea seedlings in the nursery. Accepted: 29 October 1998     

Organisation of the variability of abundant proteins in seven geographical origins of maritime pine (Pinus pinaster Ait.)

The comparison of 42 two-dimensional protein patterns from megagametophytes of maritime pine from seven geographical origins enabled the analysis of the genetic variability of abundant proteins. More than 84% of the polypeptides were variable. The intra- and inter-origin variability levels were of a similar magnitude. Correspondence analysis and a dendrogram computed using a dissimilarity index between individuals showed three main groups. The first group included the individuals from Landes (France), Portugal, eastern Spain, and Corsica, without individualising the provenances. The second group was composed of accessions from Italy and Sardinia, and the individuals of each location were separated. The third group included all of the individuals of Moroccan origin. This clustering was in agreement with the Atlantic, Mediterranean and North African structuration of maritime pine established from terpene data.     

Ectomycorrhizal fungi diversity in a white sand forest in western Amazonia

The genera Dicymbe and Aldina (Fabaceae) host ectomycorrhizal fungi (EcM) and are common in white sand forests (WSFs), a highly specialized habitat with a high level of plant endemism compared with terra-firme forests. In this study, we visited four times a 1-ha permanent plot established in a small patch of a WSF in the south of Colombia Amazonia. Forty-eight species of EcM fungi were recovered from sporocarps and 15 ITS species-level were detected from root tips. Seventeen species were new reports to Colombia and seven corresponded to undescribed species. These results confirm that this WSF supports a significant EcM fungal diversity. Most of the species found in this study have been previously reported to be associated with other legume and/or dipterocarp species from geographically distant forests. The long-distance occurrence combined with low host specificity, suggest the possibility of gene flow between geographically distant populations of EcM fungi in neotropical lowland rainforests.     

Growth responses to climate and drought at the southernmost European limit of Mediterranean Pinus pinaster forests

Climate warming and increasing aridity may negatively impact forest productivity across southern Europe. A better understanding of growth responses to climate and drought in southernmost populations could provide insight on the vulnerability of those forests to aridification. Here we investigate growth responses to climate and drought in nine Pinus pinaster (maritime pine) stands situated in Andalusia, southern Europe. The effect of climatic variables (temperatures and precipitation) and drought on radial growth was studied using dendrochronology along biogeographic and ecological gradients. We analyzed old native stands with non-tapped and resin-tapped trees mixed, showing their usefulness in dendroclimatic studies. Our results indicate a high plasticity in the growth responses of maritime pine to climate and drought, suggesting that site aridity modulated these responses. The positive growth responses to spring precipitation and the negative responses to summer drought were stronger in the more xeric inland sites than in wet coastal ones, in particular from the 1980s onwards. The characterization of tree species’ responses to climate at the southern or dry limits in relation to site conditions allows improving conservation strategies in drought-prone forest ecosystems.     

Stable Agrobacterium-mediated transformation of embryogenic tissues from Pinus pinaster Portuguese genotypes

Protocols for genetic transformation of maritime pine (Pinus pinaster Sol. ex Aiton) embryogenic tissues were developed using the Agrobacterium C58pMP90/pPCV6NFGUS. This is the first report of Agrobacterium-mediated T-DNA integration in P. pinaster confirmed by Southern blot analysis. The omission of casein hydrolysate from culture medium during cocultivation and subsequent subculture was crucial to control Agrobacterium growth. Two different transformation protocols were compared: (1) bacterial drops were spread over embryogenic clumps; (2) a mixture of bacterial and embryogenic cell suspensions was plated on filter paper. The highest frequency of transformation (22 independent transformed lines per g fresh weight, for embryogenic clone 31/668/00) was obtained with Protocol 2. The same basic procedure allowed transformation of embryogenic cell suspensions, which was dependent on subculture age. From 52 hygromycin-resistant independent lines obtained, 47 showed stable uidA gene expression and were PCR-positive for uidA gene and 42 for hpt gene. No residual Agrobacterium was detected in the transformed lines. Transgene integration was achieved using both protocols, as confirmed by Southern hybridization. From 38 (90%) transformed lines successfully cryopreserved and recovered, 71% regrown replicates have maintained the frequency of cell aggregates and early-formed embryos with uidA expression. Maturation of 44 transformed lines gave rise to 3 mature somatic embryos, each one coming from a different transformed line. Our results show the high potential of Protocol 2 for application to different culture systems.     

Initiation of somatic embryogenesis in Pinus banksiana, P. strobus, P. pinaster, and P. sylvestris at three laboratories in Canada and France

During 2002–2004, three laboratories in Canada and France collaborated to improve initiation of somatic embryogenesis (SE) in jack pine (Pinus banksiana Lamb.), eastern white pine (P. strobus L.), maritime pine (P. pinaster Ait.), and Scots pine (P.␣sylvestris L.), giving particular attention to the effects of (1) N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) versus various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA), (2) differences in basal nutrient media, i.e., macro- and microelements, and (3) gelling agent concentration. The work was carried out separately at␣each laboratory, but the details of media compositions were shared and tested on their respective species. Results indicate that the developmental stage of the zygotic embryo (ZE) and genotype effects had a large influence on SE initiation, and that genetic effects were consistent over time. Different species responded differently to PGR types and concentration, basal nutrient media, trace elements, and their combinations. Currently, our best initiation rates based on a selected group of genotypes, optimal development stage of ZE, and medium are 3.9% for jack pine, 54.6% for eastern white pine, 76.2% for maritime pine, and 19.7% for Scots pine.     

Cadmium and copper change root growth and morphology of Pinus pinea and Pinus pinaster seedlings

Heavy metal loads in forest soils have been increasing over time due to atmospheric inputs. Accumulation in the upper soil layers could affect establishment of seedlings and forest regeneration. Mediterranean species show a high initial root development, allowing seedlings to reach the moisture of deeper soil layers. In the present work seedlings of stone pine ( Pinus pinea L.) and maritime pine ( Pinus pinaster Ait.), were grown in culture solution supplied with 0.0, 0.1, 1 or 5 μ M CdSO₄ or with 1 μ M CdSO₄ and 1 μ M CuSO₄ combined. In both species tap-root elongation was drastically reduced in the 5 μ M Cd²⁺ and in the (Cd²⁺+ Cu²⁺) treatments. A supply of 0.1 or 1 μ M Cd²⁺, however, enhanced root elongation in Pinus pinea without significantly influencing root elongation in Pinus pinaster . In both species the root density (weight per unit length) and the width of the cortex increased in response to Cd²⁺ exposure. In Pinus pinaster the mitotic index decreased at the higher Cd²⁺ concentrations and when Cd²⁺ and Cu²⁺ were combined. The data suggest that cell elongation is more sensitive to Cd²⁺ than cell division. The number and length of the lateral roots were also affected by Cd²⁺ treatment to a higher degree in Pinus pinaster than in Pinus pinea, reflecting the different Cd- tolerance of the two species.     

Genetic polymorphism in maritime pine (Pinus pinaster Ait.) assessed by two-dimensional gel electrophoresis of needle,bud, and pollen proteins

Using two-dimensional polyacrylamide gel electrophoresis, the genetic variation of proteins was examined in three organs (needle, bud, and pollen) from 18 trees of maritime pine. Three types of variation were noted: presence/absence, staining intensity, and position variation of the spots. Of the 902 polypeptides scored in the three organs, 245 (27.2%) were polymorphic. Moreover, among these variable spots, 117 were found in a single organ, demonstrating an increased polymorphism of the organ-specific polypeptides (56.0% vs 18.4% for the organ-unspecific polypeptides). Finally, a positive correlation was found between variability level and subunit molecular weight for spots showing position variation but not for spots showing presence/absence or staining intensity variations. Possible explanations for this observation are discussed.Correspondence to: N. Bahrman     

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     

Heartwood and sapwood development within maritime pine (<Emphasis Type="Italic">Pinus pinaster </Emphasis>Ait.) stems

Heartwood and sapwood development in maritime pine (Pinus pinaster Ait.) is reported based on 35 trees randomly sampled in four sites in Portugal. It was possible to model the number of heartwood rings with cambial age. The heartwood initiation age was estimated to be 13 years and the rate of sapwood transformation into heartwood was 0.5 and 0.7 rings year^–1 for ages below and above 55 years, respectively. Reconstruction of heartwood volume inside the tree stem was made by visual identification by image analysis in longitudinal boards along the sawn surfaces. This volume was integrated into the 3D models of logs and stems developed for this species representing the external shape and internal knots. Heartwood either follows the stem profile or shows a maximum value at 3.8 m in height, on average, while sapwood width is greater at the stem base and after 3 m remains almost constant up the stem. Up to 50% of tree height heartwood represents 17% of stem volume, in 83-year-old trees and 12–13% in 42 to 55-year-old trees. Tree variables such as stem diameter, DBH and tree total height were found to correlate significantly with the heartwood content.     

Relationship between climate and growth of two North African varieties of Pinus pinaster Arn.

Planting tree species that are well adapted to local ecological conditions guarantees the success and sustainability of forest restoration. The aim of this study was to investigate the acclimation of two varieties of Pinus pinaster (var. renoui from Tunisia and var. maghrebiana from Morocco), to the ecological conditions of the Kroumirie Mountains in northwest of Tunisia. Tree growth performance (diameter at 1.30 m [DBH], ring widths and total height) and climate–growth responses over the period 1970–2013 were evaluated for two varieties. The trees used in this study were from pine variety and provenance trials growing in common garden in Souiniet (21 trees per variety). Significant difference in height growth rate, DBH and ring widths was found between the two varieties. The Maghrebiana variety had the highest survival and mean radial growth rates. The mean sensitivity to climate was the same in two varieties. A significant negative correlation between May precipitation and radial growth was found for var. maghrebiana. Both varieties showed a significant negative correlation between May and June temperatures and radial growth. January–February temperatures had a positive influence on ring width. The Maghrebiana variety appears well acclimatised so it is expected to ensure more successful restoration of Kroumirie Mountains.     

Influence of high temperatures on seed germination of a special Pinus pinaster stand adapted to frequent fires

This study examines the effect of fire on the germination of Pinus pinaster seeds from a population with special adaptations to this type of disturbance, due to the high frequency of fires to which they have been subjected. The action of fire was simulated in the laboratory using thermal shocks. Temperatures of 60, 90, 120, 200 and 300 °C were used for exposure times of 1 and 5 min. The viability of seeds of this species from the soil seed bank of a population subjected to a wildfire was also evaluated. The results show that germination is not increase by a thermal treatment. The effect is negative at temperatures above 90 °C and exposure times of 1 min with a significant decrease in, or even no, germination. Therefore the seeds cannot stand high temperatures and reproductive effort is placed on serotinous cone development and the production of high numbers of seeds. The high viability of the seeds from the area burned by wildfire, immediately after the fire and 1 year later, show the high potential of the soil seed bank to contribute to the regeneration of the burned area.     

Diversity of fungi on rainforest litter in North Queensland, Australia

Ten leaves from each of 13 different tree types from two differentrainforest sites in North Queensland, Australia were examined in order toestablish the fungal diversity developing on these leaves. A total of 57microfungi were identified, most of which were mitosporic fungi. Speciesdiversity in terms of richness and evenness were compared and the Mt Lewis sitewas found to be richer as compared to the Butchers Creek site. Statisticalmeasurements of diversity indices, however, showed that the two forest siteswere of similar diversity. Thirty-six of the fungi identified occurred only onone leaf type, indicating possible host specificities or recurrences. The samplesize, however, is deemed to be insufficient, as a larger sample size may haveresulted in less of the fungi appearing to be host specific. It is recommendedthat future studies should include more leaf samples and less tree types. It isparticularly important that the same leaf species are collected within the samesite and at different sites in order to establish the effects of host on fungalcomposition.