Inhibition of Phytophthora species by secondary metabolites produced by the dark septate endophyte Phialocephala europaea

Dark septate fungal root endophytes of the Phialocephala fortinii s.l.–Acephala applanata species complex (PAC) are widely distributed throughout the temperate and subtropical regions of the Northern Hemisphere. Previous studies have shown that some PAC members are pathogenic, others suppress oomycete root pathogens and some have no obvious effect on their Norway spruce (Picea abies) host. The activity of 85 PAC isolates against Phytophthora citricola s.l. was investigated by co-culture on plates. We identified a strain of Phialocephala europaea that significantly reduced the growth of P. citricola in vitro. Characterization of its extracellular metabolites resulted in the identification of four major compounds, sclerin, sclerolide, sclerotinin A, and sclerotinin B. These compounds are known for their positive as well as negative effects on plant growth. We found that sclerin and sclerotinin inhibited the growth of P. citricola in vitro at 150 μg ml^?1 (～1 mM). This is the first report of their production by Phialocephala and of activity of these compounds against an oomycete. Therefore, our data suggest that some PAC might reduce disease resulting from P. citricola by the production of antibiotics and plant growth promoting metabolites.     

Monitoring the spatial and temporal dynamics of a community of the tree-root endophyte Phialocephala fortinii s.l

Phialocephala fortinii sensu lato was isolated from Picea abies roots that had been collected from the same 3 x 3-m forest plot in 2001 and 2004, to examine the spatial and temporal dynamics of this fungal community. RFLP analysis was used to define the multilocus haplotype (MLH) of each isolate. Pielou's measure of association and chi(2) tests of independence were employed to examine the randomness of patterns of spatial distribution of MLH observed in 2001 and 2004. Population differentiation between the two samplings was tested using the G(ST) statistic. In 2001, 144 strains of 28 MLH were isolated; in 2004, 139 strains of 29 MLH. Abundant MLH in 2001 also prevailed in 2004, and the same two cryptic species were dominant in both collections. The probability of being isolated in both years increased with increasing frequency of an MLH. The patterns of spatial distribution of most MLH did not differ between years. The G(ST) values indicated identity of the two collections. Communities of P. fortinii sensu lato remain spatially and genetically stable for at least 3 yr.     

Seasonal variation in the tissue water relations of Picea glauca

Seasonal variation in water relations of 3-yearold white spruce (Picea glauca (Moench) Voss) shoots, monitored with pressure-volume curves over 28 months, was closely related to shoot phenology and was sensitive to environmental fluctuations during both summer growth and winter dormancy. Turgor maintenance capacity was lowest during rapid shoot elongation from late May to early July; this was indicated by the lowest total turgor pressures, the highest (least negative) osmotic potentials at full turgor and the turgor loss point, the smallest differences between osmotic potentials at full turgor and the turgor loss point, the highest relative water contents at turgor loss and a linear decline in cell elasticity with decreasing turgor pressure. This suggests that the high susceptibility of white spruce seedlings to growth check after transplanting is largely attributable to the poor turgor maintenance capacity of this species in early summer.     

Proteome analysis of early somatic embryogenesis in Picea glauca

Forestry is a valuable natural resource for many countries. Rapid production of large quantities of genetically improved and uniform seedlings for restocking harvested lands is a key component of sustainable forest management programs. Clonal propagation through somatic embryogenesis has the potential to meet this need in conifers and can offer the added benefit of ensuring consistent seedling quality. Although in commercial use, mass production of conifers through somatic embryogenesis is relatively new and there are numerous biological unknowns regarding this complex developmental pathway. To aid in unravelling the embryo developmental process, two-dimensional electrophoresis was employed to quantitatively assess the expression levels of proteins across four stages of somatic embryo maturation in white spruce (0, 7, 21 and 35 days post abscisic acid treatment). Forty-eight differentially expressed proteins have been identified, which display a significant change in abundance as early as day 7 of embryo development. These proteins are involved in a variety of cellular processes, many of which have not previously been associated with embryo development. The identification of these proteins was greatly assisted by the availability of a substantial expressed sequence tag (EST) resource developed for white, sitka and interior spruce. The combined use of these spruce ESTs in conjunction with GenBank accessions for other plants improved the rate of protein identification from 38% to 62% when compared with GenBank alone using automated, high-throughput techniques. This underscores the utility of EST resources in a proteomic study of any species for which a genome sequence is unavailable.     

Vacuolar localization of phosphorus in hyphae of Phialocephala fortinii, a dark septate fungal root endophyte

Phialocephala fortinii is a dark septate fungal endophyte that colonizes roots of many host species. Its effect on plant growth varies from being pathogenic to beneficial. The basic biology of this species has received little research, and thus the main objectives of this study were to determine cytological features of hyphae, including the nature of the vacuolar system, and whether polyphosphate was present in vacuoles. Both living hyphae and hyphae that had been rapidly frozen and freeze substituted before embedding were studied. A complex system of vacuoles, including a motile tubular vacuolar system, elongated vacuoles, and spherical vacuoles, was demonstrated in living hyphae by the fluorescent probe Oregon Green 488 carboxylic acid diacetate, using laser scanning confocal microscopy. The motile tubular vacuolar system was more prevalent at the hyphal tip than in more distal regions, whereas elongated vacuoles and spherical vacuoles were more abundant distal to the tip. All vacuoles contained polyphosphate as shown by labelling embedded samples with recombinant polyphosphate binding domain of Escherichia coli exopolyphosphatase, containing Xpress tag at the N-terminal end, followed by anti-Xpress antibody and a secondary antibody conjugated either to a fluorescent probe for laser scanning confocal microscopy or colloidal gold for transmission electron microscopy. The polyphosphate was dispersed in vacuoles. This was confirmed by staining embedded samples with 4',6-diamidino-2-phenylindole and viewing with UV light using epifluorescence microscopy. These cytological methods showed that the tubular vacuolar system had lower concentrations of polyphosphate than the spherical vacuoles. Lipid bodies were present around vacuoles.     

Some aspects of differentiation in cells of Picea glauca cultivated in vitro

Microscopic observation of cells of Picea glauca, grown in tissue culture under a variety of cultural conditions, reveals a series of phenomena which are believed to be associated with the development of specific types of cells recognizable in intact plants. Characteristically patterned deposits of resin drops, and strands, furrows, and channels in the cytoplasm appear to be capable of serving as templates around which cellulose and lignin may later be deposited. The cytoplasm is extruded into characteristic papillae, and cell content may be extruded slowly or ejected with force through the cell walls, through multiple pores of characteristic distribution. Material ejected from such cells condenses in characteristic extracellular patterns. All of these processes may contribute to the differentiation of recognizable distinct cell types.     

An embryogenic cell suspension culture of Picea glauca (White spruce)

A cell suspension culture of Picea glauca (White spruce) which continuously produces somatic embryos has been established. Embryogenic callus derived from cultured zygotic embryos was used to initiate the culture. Numerous embryos at various early stages of development were recognized; they exhibited a meristematic embryonic region and suspensor consisting of elongate, vacuolated cells. The culture also contained clumps of meristematic cells and large irregular — shaped cells. The culture could be readily re-established on solid medium.     

Cryopreservation of interior spruce (Picea glauca engelmanni complex) embryogenic cultures

Summary Embryogenic cultures of interior spruce derived from 12 full-sib families were subjected to cryopreservation, with a 97 % success rate for 357 genotypes. Analyses suggested that cryotolerance was not related to family ranking (height increment), embryogenic potential or culture dispersability in suspension, and long-term storage in or above liquid nitrogen did not affect regenerative potential. By contrast, differences in cryotolerance among cell lines appeared to be prevalent in certain families. Analysis with a DNA fingerprinting probe used for clonal identification demonstrated no evidence of somaclonal variation as a result of cryopreservation. The results of this work indicate the applicability of cryopreservation as a long-term storage strategy for spruce embryogenic cultures from a wide genetic background.Abbreviations ABA ± abscisic acid - BA N⁶-benzyladenine - BSA bovine serum albumin - CTAB cetyldimethylethyl-ammonium bromide - DMSO dimethylsulfoxide - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid disodium salt - IBA indole-3-butyric acid - LN liquid nitrogen - PEG polyethylene glycol - SLS N-lauroyl sarcosine - Tris tris[hydroxymethylamino] methane - 2,4-D 2,4-dichlorophenoxyacetic acid     

Postglacial range expansion and population growth of Picea mariana, Picea glauca and Pinus banksiana in the western interior of Canada

The fossil pollen records from the sediments of thirteen lakes are used to reconstruct the postglacial spread and population expansions of Picea mariana (Mill.) B.S.P., Picea glauca (Moench) Voss and Pinus banksiana Lamb. across the western interior of Canada. The objective of this study was to examine temporal and spatial patterns in the growth of the populations and to determine if intraspecific and interspecific variations exist across a range of latitudes and elevations. Pollen accumulation rates (PARs) are estimated from pollen grain counts and sedimentation rates throughout the early to mid-Holocene and are used to represent the population level at the time of pollen deposition. Exponential equations are used to calculate population growth rates. Three-dimensional diagrams (time, space, abundance) are constructed to illustrate range expansion and population growth.The rates of population growth varied from south to north for all three species. Pinus banksiana , on average, had slower population growth rates, and its expansion across the region began over 4000 years later and lasted approximately 1000 years longer than the two Picea species. All three tree taxa experienced reduced rates of population growth at northern high elevation sites. These variations are examined in light of the physical and biological environmental conditions which existed during postglacial range expansion and population growth.     

Nitrogen-15 NMR studies of nitrogen metabolism in Picea glauca buds.

In vivo (15)N nuclear magnetic resonance (NMR) as well as (15)N solid-state magic angle spinning (MAS) NMR spectroscopy were used to investigate nitrogen metabolism in cultured white spruce (Picea glauca) buds. Long-term as well as short-term experiments were carried out involving the use of inhibitors of the nitrogen pathways such as methionine sulfoximine (MSO), azaserine (AZA) and aminooxyacetate (AOA). Both in vivo and solid-state NMR showed that when MSO blocked glutamine synthetase (GS) no NH(4)(+) is incorporated. When glutamate synthase (GOGAT) is blocked by AZA there is some incorporation into glutamine (Gln), but very little into alpha-amino groups (glutamate, Glu). The transamination inhibitor AOA does not affect the metabolism of (15)NH(4)(+) into Gln and Glu, but blocks the production of arginine (Arg), as would be expected. Proline (Pro) and gamma-aminobutyric acid (GABA), which are produced directly from Glu without a transamination step, were not affected. The solid-state NMR experiments showed that protein synthesis occurred. Collectively, our results show that NH(4)(+) can only be assimilated through the GS/GOGAT pathway in P. glauca buds.     

The influence of cambial age on breeding for wood properties in Picea glauca

We investigated the influence of cambial age on correlations between different wood traits and the possibility of early selection in order to help decision-making for the improvement of juvenile wood in white spruce (Picea glauca (Moench) Voss). Increment cores were analysed from 375 trees covering 25 open-pollinated families from a 30-year-old provenance–progeny trial in Quebec, Canada. Genetic and phenotypic correlations between different mechanical and fibre anatomy-related wood traits were found to vary with cambial age. Most correlations became stronger in magnitude in rings closer to the bark. An exception is the correlation between microfibril angle (MFA) and the modulus of elasticity where correlations were strongly negative from the pith to the bark. Age–age correlations for different wood traits were found to be high and possible gains from early selection were estimated to be good in ring 8 and older for most traits. MFA was the trait with the strongest potential for selection as early as ring 4, but a detrimental correlation with wood density may represent a drawback of such a juvenile selection approach. Estimates showed that selection concentrated on a few easily measurable traits such as wood density and core length holds promise to obtain superior genetic gains for mechanical properties, but negative impacts would be expected on fibre anatomy traits related to pulp quality. These findings show the need for more carefully planned breeding and selection strategies if one wishes to improve several traits for different end uses.     

Transformation of white spruce (Picea glauca) somatic embryos by microprojectile bombardment

Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of 605 embryos (2.2%) remained GUS positive after two months in culture. Three of those thirteen (23%) embryo-derived tissues consistently showed GUS activity for eight months in culture. These putatively transfomed embryogenic tissues were subjected to Southern blot analysis and the results suggested integration of the gus::nptll gene expression cassette in the white spruce genome.Abbreviations ABA (±)abscisic acid - BA benzyladenine - bp base pair - 2,4-D 2,4-dichlorophenoxyacetic acid - kb kilobase - gus E. coli gene uid A for -glucuronidase - nptll neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronic acid     

Multivariate interpretation of the foliar chemical composition of Norway spruce (Picea abies)

Twenty-four chemical elements were analysed by INAA, ICP-AES and CN in needles of Norway spruce (Picea abies (L.) Karst.). Branches were sampled from 54 trees on eight sites in Switzerland and South Germany. From each tree, twigs were sorted into the most recent four or five age classes and their needles analysed separately. All measured concentrations could be considered as log-normally distributed and statistical analyses were, therefore, performed on logarithms. Variance components were estimated by maximum likelihood and compared between elements. Non-essential elements varied more than essential nutrients (Mn was an exception). The sites and the age of the needles were the most important sources of variance. The interaction between site and age, the individual tree, the sampled branch and the residual variance were usually much smaller sources of variance. The effects of the most significant factors – site and age – were further described by principal components and cluster analyses. Mineral elements either increased or decreased with the age of the needles according to their mobility in the phloem. Two different components were identified in the effect of the sites: a geochemical component linked to soil pH and a climate component linked to altitude, temperature and precipitation. Multivariate statistics are discussed as a tool for the interpretation of complex interaction patterns between element concentrations in plants.     

Characteristics of foliar fungal endophyte assemblages and host effective components in Salvia miltiorrhiza Bunge

Salvia miltiorrhiza Bunge, a well-known medicinal plant, has more than 20 effective components. The aim of this study was to comprehensively investigate foliar fungal endophyte communities of S. miltiorrhiza and explore the inside relationship between host-specific fungal endophytes and effective components accumulation. Five plant samples were collected from four geological different provinces in China. Foliar fungal endophyte communities were determined by terminal restriction fragment length polymorphism (T-RFLP) of the ITS region. Effective components were analyzed with high-performance liquid chromatography. The results showed that S. miltiorrhiza foliage harbored a large diversity of fungal endophytes. Principal component analysis revealed similar T-RFLP profiles and the characteristics of the 24 effective components among the five samples, which could be clustered into three groups. In foliar T-RFLP profiles derived from the restriction digestion by CfoI, HaeIII, MspI, or TaqI, there were identical 45, 42, 38, and 34 terminal restriction fragments (T-RFs) from the five samples. We consider these T-RFs as host-specific fungal endophytes. Correlation analysis of these T-RFs’ area and 24 effective components contents revealed a significant correlationship between some host-specific fungal endophytes and foliage or root effective components accumulation.     

A set of microsatellite markers for use in Sitka spruce (Picea sitchensis) developed from Picea glauca ESTs

Few microsatellite markers have been specifically developed for Picea sitchensis. In January 2004 the appearance of over 10 000 sequences of expressed regions of DNA from P. glauca in GenBank presented an opportunity for the development of additional microsatellite markers in Sitka spruce. Mono‐ and dinucleotide repeat sequences were located in these sequences and primers were designed around these regions. Amplification was attempted in Sitka material from a broad geographical range and the level of polymorphism was assessed. Primers were also tested in progeny of a controlled cross. Nine polymorphic loci that demonstrated Mendelian inheritance in Sitka were discovered in this study.