Greenhouse production of Burgundy truffle mycorrhizae on oak roots

Truffles, subterranean fruit-bodies of the ectomycorrhizal genus Tuber, are currently cultivated on host trees in plantations, both to offset declining wild production and to extend their geographic distribution. We report the effects of the potting medium and potting method components of two very different seedling production systems on the colonization of Quercus bicolor × Q. robur seedlings by T. aestivum (the Burgundy truffle). Seedlings grown in book planter containers using a peat-based medium were smaller yet much better colonized by T. aestivum than seedlings produced in RPM^® (Root Production Method) containers using a ground bark-based RPM medium. In either container method, the peat-based medium supported development of more T. aestivum mycorrhizae than did the RPM medium. Seedlings grown in book planters developed significantly more root tips l^?1 of medium than did seedlings grown in RPM containers. The optimum pH for T. aestivum mycorrhiza development in the peat-based medium is between 6.7 and 7.5.     

Monitoring the fate of a 30-year-old truffle orchard in Burgundy: from Tuber melanosporum to Tuber aestivum

Truffles, i.e. tree root-associated fungal fruiting bodies, clearly range among the world’s most exclusive delicacies. Despite the quite restricted natural geographic occurrence of one of the most renowned fungal species, namely Tuber melanosporum, the development of inoculation procedures in the late 1960s made it possible to enlarge its production area in different countries. This was achieved by planting orchards with host tree seedlings colonized by the fungus. In the present work, we investigate the behavior of one of the earliest T. melanosporum orchards planted in Burgundy (France) over a long-term scale (more than 30 years). A picture of the orchard evolution was obtained by recording truffle yields and fungal morphotypes over the seasons and relating them to host-tree development and climate data. The most relevant results include the time-delayed, but rather fast replacement of inoculated T. melanosporum by naturally occurring T. aestivum and the key role of climate in the inter-annual variability of truffle production.     

Arbuscular mycorrhizal association of indigenous agroforestry tree species and their infective potential with maize in the rift valley, Ethiopia

Tree species in agroforestry are important source of inoculum for companion agricultural crops. Agroforestry trees can serve as a source of Arbuscular mycorrhiza (AM) inoculants to intercropped annuals. We studied spore abundance, root colonization of Albizia gummifera (J.F. Gmel.) and Croton macrostachyus (Hochst Ex Del.) trees and their effect on colonization of maize. Soil and root samples were collected from field standing trees from under and outside the canopy of trees and maize crops in the main rainy season. The number of spore count was significantly higher under the canopy of A. gummifera (791/100 g of dry soil) and C. macrostachyus (877/100 g of dry soil) trees than outside the canopy (547 and 588/100 g of dry soil, respectively). The level of root colonization of C. macrostachyus (45 %) was higher than A. gummifera (41 %). Root colonization of maize crops grown under the canopy of A. gummifera and C. macrostachyus trees was significantly higher than outside the canopy (P < 0.001). Maize seedlings grown on non-sterilized soils collected under and outside the canopy of A. gummifera and C. macrostachyus trees recorded higher root colonization, plant height, shoot and root dry weight than grown on sterilized soils (P < 0.001). The percentage of AM colonized roots of Zea mays seedlings was significantly positively correlated with the number of spore counts for field soils. The rhizospheres of indigenous agroforestry perennial species are important source of inoculum for annuals. The integration of perennials and annuals in an agroforestry system enhances the maintenance of soil quality in the tropics.     

Weed control modifies Tuber melanosporum mycelial expansion in young oak plantations

Context

Black truffle (Tuber melanosporum) cultivation is a promising agro-forestry alternative for Mediterranean rural areas, but adequate weed control at seedling establishment still remains a challenge in black truffle plantations.

Aims

The aim of this study is to evaluate the effects of several weed control strategies on early development of Quercus ilex seedlings and the symbiotic T. melanosporum.

Materials and methods

In a young black truffle-inoculated holm oak plantation, we assessed for 3 years the effects of two types of mechanical weed control and five mulches in a young Q. ilex plantation inoculated with T. melanosporum. Herbaceous cover, seedling growth and abundance of T. melanosporum mycelium, based on PCR analysis of soil DNA extracts using T. melanosporum-specific primers, were estimated to determine the effectiveness of these treatments in controlling weeds and supporting the growth of both the host tree and the target fungus.

Results

The amount of T. melanosporum mycelium in the soil 30 cm around the seedlings was larger under double-layer white mulch than in the rest of treatments tested. Under the white colour mulches, which had the largest light reflection, we registered the cooler soil temperature, and the best weed control was observed on the single- and double-layer black truffles and double-layer white mulch.

Conclusion

The effects of double-layer white mulch on herbaceous cover, soil temperature, reflected light, and the expansion of T. melanosporum bring us closer to being able to substitute traditional tilling of truffle orchards for the less expensive mulching treatments.     

Weed management and irrigation are key treatments in emerging black truffle (Tuber melanosporum) cultivation

Interest in conversion of marginal agricultural lands to small oak woodlands for the production of black truffles (Tuber melanosporum Vittad.) is increasing in the native black truffle areas of France, Spain and Italy as well as suitable or amended sites throughout the world due to high economic returns for gastronomically valuable truffles. Successful long-term management of a perennial plantation replacing an annual crop requires understanding the system in all phases of the life cycle, and in the case of truffles this involves understanding the interactive growth of the aboveground host and the belowground symbiotic ectomycorrhizal fungus whose fruitbody is the desired truffle. Here we focus on the pre-production phase, 4 years after establishing the truffle-oak plantation and prior to truffle production. We tested the influence of weed control, irrigation and fertilizer, each at 3 levels, on plant growth and ectomycorrhizal proliferation in three truffle-oaks plantations in northeast Spain. Results show that adequate weed control improves root and shoot dry weight while fertilizer and irrigation treatments did not influence plant growth. The low dose of irrigation (50% of the estimated water deficit for the site) and glyphosate weed control both increased total root tips/plant and T. melanosporum colonized tips (ectomycorrhizae) by approximately two-fold, compared to control treatments. Distribution of the ectomycorrhizae within the soil profile was significantly influenced by the low dose irrigation treatment, with increases observed in the 10?C20 cm and the 20?C30 cm deep layers compared to the control and high irrigation treatments. Four years after planting, T. melanosporum remained the dominant fungal symbiont, despite the presence of 14 other ectomycorrhizal morphotypes from these sites.     

Colonization of Pedunculate oak by the Burgundy truffle fungus is greater with natural than with pelletized lime

European black truffles can be profitable agroforestry crops outside their native ranges. Truffle fungi grow symbiotically as ectomycorrhizae on the roots of host trees, notably hazels and oaks. Conditions in the central USA appear conducive to cultivation of the Burgundy truffle (Tuber aestivum Vitt. syn. T. uncinatum Chatin), but research is needed to determine effects of management practices on truffle establishment and fruiting. In a greenhouse study we tested the effect of lime type, inoculation technique, and two truffle sources on Pedunculate oak (Quercus robur L.) growth and mycorrhizal colonization. We found that the type of lime used to raise potting mix pH can differentially affect the growth rate of root systems inoculated with different selections of Burgundy truffle inoculum. Seedlings inoculated with one selection of the truffle and grown in potting mixes amended with natural crushed dolomitic limestone developed larger root systems with more truffle mycorrhizae compared with potting mix amended with high-calcium pelletized quick-release lime. Seedlings inoculated with a second truffle selection were not affected by lime source and developed root systems as large as those developed with the first truffle source grown with natural lime. Supplemental root dip inoculation did not improve levels of colonization beyond those accomplished by potting mix infestation with truffle ascospores. Use of a hygroscopic polymer to maintain ascospore suspension in the inoculum slurry used to infest the potting mix had no effect on root system development or mycorrhiza formation.     

Economically profitable post fire restoration with black truffle (Tuber melanosporum) producing plantations

Cultivating black truffle (Tuber melanosporum Vittad.) is highly profitable in sites suitable for its development. Land use history is an important factor when choosing suitable habitat, and lands with non-ectomycorrhizal host species are recommended when introducing T. melanosporum-inoculated seedlings in order to reduce competition from native, soil-borne ectomycorrhizal (ECM) inoculum. Nearly every year Mediterranean wildfires cause the loss of forested lands where soil and climatic characteristics are suitable for truffle cultivation. Introducing T. melanosporum-inoculated seedlings in these sites could promote reforestation with a native ECM fungus and, in addition, may provide economic incentives. We tested the viability of burnt forest lands for truffle cultivation by introducing T. melanosporum-inoculated Quercus ilex seedlings in burnt forest sites with and without resprouting forest species which act as repositories for native ECM fungi. We also evaluated the role of the ECM host plants, which resprout after a forest fire, in the maintenance and dynamic processes of the ECM fungal community. The study followed a factorial design with two levels: (1) T. melanosporum-inoculated and non-inoculated Q. ilex seedlings and (2) presence and absence of ECM host plants resprouting after the fire. We established 10 experimental plots with 360 holm oaks. After four and a half years, the truffle-inoculated holm oaks maintained 36?% of their root tips colonized with T. melanosporum, and 10?years after plantations, 26.2?% of the surviving inoculated holm oaks displayed a “truffle burn” area. A greater ECM morphotype richness was associated with seedlings planted in plots with the presence of ECM host plants, whereas the T. melanosporum mycorrhizal ratio was constant. These results suggest that reforestation with T. melanosporum-inoculated seedlings can be successful following forest fires and highlights the competitiveness of this fungus within the ECM community in these soils.     

Regeneration patterns of Eastern white pine (Pinus strobus L.) in hardwood-dominated forests in Virginia, USA

Eastern white pine (Pinus strobus L.) is a moderately shade-tolerant species that co-occurs with hardwood tree species in many forests of the eastern United States, as well as in pure stands. The species is valued for its timber, as well as for wildlife and recreation. Regeneration of this species is somewhat unpredictable and often occurs in patches of similarly-aged cohorts. We described the regeneration patterns of this species and examined their relation to environmental variables within hardwood forests of southwestern Virginia, USA. An average of 5.3 white pine patches per ha were observed in this study. The majority of patches consisted of saplings (85%), with 9% of patches in pole size classes, and 6% in seedling size classes. The average density of patches was 43.5 stems with an average age of 20 years. The size of patches averaged 80.6 m². The total density of seedlings and the number of regeneration patches of all sizes of regeneration (seedlings, saplings, and poles) in plots was related to the surrounding density of large white pine trees (potential seed trees). The density of seedlings or patches was not significantly related to current vegetation cover or soil surface cover variables, but more than half of regeneration patches were located in or adjacent to old canopy gaps, most of which were old logging gaps. While seedling regeneration may occur within the understory of these forests near seed trees, advancement to the sapling and pole stage appears to be associated with canopy gap formation.     

Effects of two Glomus species on the growth and physiological performance of Sophora davidii seedlings under water stress

Sophora davidii is an important leguminous scrub that is widely used for revegetation in the semiarid Loess Plateau and other arid valley areas of China, where it usually suffers drought stress. This study investigated the effects of arbuscular mycorrhizal (AM) fungi (Glomus mosseae and Glomus constrictum) and water stress on the growth and physiological performance of S. davidii seedlings under greenhouse pot conditions. Two soil water availability treatments (well-watered (WW) ?0.10 MPa; water-stressed (WS) ?0.86 MPa) were applied for 61 days. At the end of this experiment, G. mosseae and G. constrictum had colonized the roots of S. davidii seedlings. Water stress inhibited AM colonization, plant growth, chlorophyll concentration, gas exchange and chlorophyll fluorescence of S. davidii seedlings. Mycorrhizal seedlings had greater shoot dry weight, root dry weight, plant height, root length, instantaneous water use efficiency (iWUE), net photosynthetic rate (Pn), stomatal conductance (g_s), maximal photochemical efficiency of PSII photochemistry (Fv/Fm), lower intercellular CO₂ concentration and photochemical quenching values (qP), when compared with non-mycorrhizal seedlings under both WW and WS conditions. Furthermore, G. constrictum was found to be more efficient at improving the shoot and root mass, plant height, iWUE, Pn, g_s, qP, and ΦPSII of S. davidii seedlings, when compared with G. mosseae under both WW and WS conditions. Our results demonstrate that AM Glomus symbiosis enhanced S. davidii seedling resistance by improving its growth and physiological performance under water stress conditions. This suggests that Glomus inoculation is a potential tool for enhancing outplanting performance of S. davidii in semiarid areas of China.     

Influence of ground flora on Fraxinus mandshurica seedling growth on abandoned land and beneath forest canopy

A better understanding of root/shoot interactions influencing seedling growth on abandoned land could yield insight into seedling regeneration and restoration of the abandoned lands. Field work had been conducted for 2 years (2008–2009) to investigate the impacts of neighbouring plants on Manchurian Ash (Fraxinus mandshurica) seedling growth under the canopy of an old secondary forest and on the abandoned land exposed to full solar radiation in North-eastern China. Four different interaction treatments were designed for the study: neither shoot nor root interaction, shoot interaction only, root interaction only, and both shoot and root interaction. The presence of either the shoots or roots of neighbours had a competitive effect, reducing the growth of the target seedlings at each site. The total competitive effect of roots and shoots of neighbouring plants was significantly less than the sum of root and shoot competition separately on the abandoned land, but this difference was not significant beneath the forest canopy. Root competition was more restraining than shoot competition on the abandoned land. Target seedlings adjusted their root morphology and growth rates in response to the competitive effects from different parts of the neighbouring plants. Our results indicated that the root:shoot ratios of the target seedlings at the end of the experiment were affected by neither initial tree size nor the competition from either above- or belowground at either site. The results also highlight the importance of reducing root competition in boosting seedling regeneration and forest rehabilitation on the abandoned land.     

Ectomycorrhizal inoculation of Douglas-fir transplanted container seedlings with commercially produced inoculum

Commercially produced vegetative inocula of Laccaria laccata and Hebeloma crustuliniforme successfully formed ectomycorrhizae with Douglas-fir transplanted container (plug+1) seedlings. After 4.5 months in containers, 83% and 90%, respectively, of short roots were mycorrhizal. L. laccata- or H. crustuliniforme-inoculated seedlings had significantly more mycorrhizal and total short roots than Pisolithus tinctorius-inoculated (4% mycorrhizal root tips) or uninoculated control seedlings. No significant differences were detected in seedling growth at the end of the container phase.After transplantation and growth in nursery beds for 17 months, mean new short root colonization of all seedlings was 80%. H. crustuliniforme persisted as a dominant mycorrhizal fungus on seedlings initially inoculated with this fungus. L. laccata-inoculated seedlings had 40% of their short roots colonized by L. laccata and another 40% by native fungi Rhizopogon and Thelephora spp. All mycorrhizae of control seedlings and those inoculated with P. tinctorius were formed by fungi native to the nusery beds. A significant fungal treatment effect was detected for shoot height only. Control seedlings were significantly taller than L. laccata-inoculated seedlings after transplanting.This article is part of senior author's thesis in partial fulfillment of the Ph.D. degree in the Department of Forest Science at Oregon State University, Corvallis.     

Influence of sulfometuron methyl on conifer seedling root development

On three sites in coastal northwestern Oregon, USA, seedling root and shoot development were assessed for Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), western hemlock (Tsuga heterophylla (Raf.) Sarg.), and western red-cedar (Thuja plicata Donn ex D. Don) container seedlings under varying sulfometuron methyl (Oust XP^®) herbicide application treatments. Treatments consisted of application of 0.16 kg active ingredient (ai) ha^?1 as a site preparation in fall 2003, a release application in fall 2004, or a control treatment with no application. Seedlings were planted in winter 2004 and measurements recorded in summer 2004, winter 2005, and summer 2005. During first season growth, western red-cedar seedlings showed the greatest negative impact to site preparation compared to the control with overall average new root length outside the root plug reduced by 67%. Significant reductions in root length also occurred for western hemlock (47%) and Douglas-fir (40%) seedlings. About 9 months after the release treatment, and 21 months after the site preparation application, there were no significant differences between treatments for any measured parameter. These findings suggest that seedlings under the site preparation treatment recovered from initial damage incurred to the root system. Lack of seedling response under the release treatment may be the result of opposing influences from the herbicide application associated with seedling phytotoxicity and enhanced seedling development resulting from effective vegetation control. Although our study was limited to 21 months following planting, reduced vegetation cover in the site preparation and release treatments suggests that these treatments may benefit future seedling growth.     

Introduced <Emphasis Type="Italic">Tuber aestivum</Emphasis> replacing introduced <Emphasis Type="Italic">Tuber melanosporum</Emphasis>: a case study

A Tuber melanosporum plantation established in 1994/1995 on Kibbutz Bar’am (in the Upper Galilee, Israel) gradually lost its T. melanosporum mycorrhiza. In 1999, T. aestivum inoculated seedlings were inadvertently introduced into the plantation to fill the gaps between trees. A single T. melanosporum fruit body was found in 1999. Although no truffles were found after 1999 and until 2009, in that year and in 2010, truffles were collected. Morphological and molecular analyses proved these to be T. aestivum. Thus, the intentionally introduced T. melanosporum mycorrhiza was replaced by that of another introduced mycorrhizal fungus, T. aestivum. Local oak species produced higher yields compared to introduced host species known to be good T. melanosporum plant symbionts. The yield was comparable to that reported for young commercial orchards, but the fruiting season was earlier than in Europe.     

Heavy thinning in hinoki plantations in Shikoku (southwestern Japan) has limited effects on recruitment of seedlings of other tree species

Heavy thinning may provide a feasible means to convert single-species coniferous plantations to broadleaved or mixed forests. To assess this possibility, we monitored tree seedling dynamics of other tree species, the understory microenvironment, and seed availability following heavy thinning (50 % by volume) in two hinoki (Chamaecyparis obtusa) plantations in Shikoku, southwestern Japan. Conventional (35 % by volume) and row thinning (50 % by volume) treatments were also applied at one of the sites. Diffuse transmittance in the understory dramatically increased after all thinning treatments then rapidly decreased, except after row thinning. Heavy thinning accelerated recruitment of small seedlings (with stems <30 cm high) at one of the sites. However, the densities of these seedlings declined sharply, and increases in the densities of larger seedlings recorded in subsequent surveys were very small. Furthermore, at the other site, recruitment of small seedlings under heavy thinning was equivalent to that under conventional thinning and lower than that under row thinning. Abundant species in the recruited seedlings were pioneer or subcanopy species found in the soil seed bank. Low seed rain density at both sites appeared to be responsible for the poor recruitment of canopy tree species. Our results suggest that heavy thinning had limited effects on the seedling recruitment of other tree species in these hinoki plantations due to insufficient seed rain and rapid canopy closure.     

Differences in growth and nutrient uptake from a coarse-soil substrate by ectomycorrhizal- and fungicide-treated Picea abies seedlings

In order to quantify the importance of ectomycorrhizal fungi on nutrient uptake from the coarse-soil fraction of a haplic Cambisol (alumic), a microcosm study that allowed for nutrient budgets was designed. Ectomycorrhizal- and fungicide-treated spruce seedlings were grown on isolated and cleaned gneiss fragments (6.3 mm > Ø > 2 mm) from 90 cm soil depth. The substrate was the only source of Ca, K and Mg. Ectomycorrhizal seedlings showed no signs of nutrient deficiencies and biomass increased significantly compared to initial seedling biomass. Ectomycorrhizal seedlings seemed well adapted to survive on the coarse-soil substrate and acquired Ca, K and Mg from the coarse-soil substrate. Gneiss fragments of the ectomycorrhizal treatment were covered by fine roots and ectomycorrhizal hyphae, as observed microscopically. Fungicide-treated seedlings?? root development was retarded, and shoot biomass only increased from stored nutrient reserves of the seedlings. The suppression of EcM colonization by the fungicide Topas^® apparently caused a root growth inhibiting effect. Furthermore, the extinction of mycorrhiza initiated an increased nitrification and acidification and a consequent nutrient cation release triggered by nitrate in the drainage.     

Response of Acacia senegal (L.) Willd. to inoculation with arbuscular mycorrhizal fungi isolates in sterilized and unsterilized soils in Senegal

The benefits of inoculation with six arbuscular mycorrhizal fungi (AMF) isolates (Glomus aggregatum, G. fasciculatum, G. intraradices, G. manihotis, G. mosseae, and G. verriculosum) were investigated on seedlings of Acacia senegal (L.) Willd., a multipurpose tree legume highly valued for arabic gum production. Mycorrhizal root colonization, plant growth and relative mycorrhizal dependency (RMD) were measured in A. senegal seedlings growing in soils from three geographical sites in Senegal (Dahra, Bambey and Goudiry) and two soil conditions (sterilized vs unsterilized) in the glasshouse. The impact of inoculation on mycorrhizal root colonization and plant growth depended on AMF isolates, soil origins and soil conditions. Mycorrhizal root colonization and plant growth were increased in sterilized soils regardless of soil origin and AMF isolates. The degree of RMD of A. senegal seedlings varied with soil origin, soil condition and AMF isolates. A. senegal showed the highest RMD values, reaching a maximum of 45 %, when inoculated with G. manihotis. However, in unsterilized soils, no significant effect of AMF inoculation on plant growth was observed despite significant root colonization with certain AMF isolates in Dahra and Goudiry soils. This indicates that the most infective AMF isolates were not the most effective and unsterilized soils may contain effective mycorrhizal propagules. In conclusion, it is important to consider the native mycorrhizal component of the soils before harnessing mycorrhizal inoculation programs for sustainable agroforestry systems.     

Sustainable use of non-timber forest products: Impact of fruit harvesting on Pentadesma butyracea regeneration and financial analysis of its products trade in Benin

Pentadesma butyracea Sabine (Clusiaceae) is a multi-purpose tree that provides non-timber forest products (NTFPs). In particular, fruit almonds can be transformed into butter for cooking and cosmetics. During the present study, the following hypotheses were tested: (i) diameter structure of P. butyracea populations is independent of its fruit gathering intensity; (ii) P. butyracea seedling and sapling density and origin are independent of its fruit gathering intensity; (iii) P. butyracea fruit gathering and processing of its almonds are profitable activities and (iv) P. butyracea fruit collectors and almond transformers are receiving the lowest marketing margins in the commercial channel. The class distribution in the low-intensity harvesting sites showed a typical inverse J-shaped curve whereas the high-intensity harvesting ones showed an almost bell curve (G² = 23.93, p = 0.0008). After data analysis, all hypotheses turned out to be wrong except the hypothesis (iii). In order to assess the effects of fruit harvesting on natural regeneration of P. butyracea, we compared seedling and sapling density of regeneration originating from seeds and roots suckers in plots that had been differentiated according to fruit harvesting intensity. These plots were laid out in riparian forests, which are the natural habitats for the species in Benin. Observed seedling and sapling density was high (13,872 ± 7886 seedlings and saplings/ha) in low-intensity harvesting sites but very low (4200 ± 3810 seedlings and saplings/ha) in high-intensity harvesting sites (F = 17.16; p = 0.0006). However, there was no significant difference between root sucker density in either type of harvesting site (F = 0.79; p = 0.3861). Collection of P. butyracea fruits and subsequent processing of its almonds into butter is an important source of income for women involved in these activities. Commercial margin analysis showed that these women involved in almonds and butter trade, far from being exploited by traders, recuperate between 49% and 80% of the price paid by the consumer, depending on the quality of the product and the length of commercial channel used.     

Effects of shade on growth, biomass allocation and leaf morphology in European yew (Taxus baccata L.)

The impact of shade on the growth of European yew (Taxus baccata L.) saplings was investigated over a three-year period using artificial shading to simulate four different light regimes (3, 7, 27 and 100 % relative photosynthetic photon flux density, RPPFD). There was no mortality attributable to shading even under the 3 % RPPFD treatment. Increasing shade was positively associated with specific leaf area, leaf length, leaf width and total chlorophyll content, but negatively associated with plant height, stem diameter, total dry weight and root to leaf and shoot ratio. Discoloration of the foliage occurred in plants grown in 100 % RPPFD conditions (resulting in reduced growth rates) and those transferred to 100 % RPPFD conditions after being shade-acclimated for 2 years. Evidence suggests that T. baccata has the ability to regenerate beneath a lighter canopy but beneath denser canopies gap dynamics will play an important role in facilitating successful regeneration and this needs to be reflected in management of natural populations of this declining species.     

Combined effects of pre-hardening and fall fertilization on nitrogen translocation and storage in Quercus variabilis seedlings

Maintaining proper seedling nitrogen status is important for outplanting success. Fall fertilization of evergreen conifer seedlings is a well-known technique for averting nitrogen (N) dilution caused by continued seedling growth during hardening. For deciduous seedlings, this technique is much less understood, and regardless of foliage type, the interaction of N status prior to fall fertilization and the rate of fall fertilization have yet to be fully explored. Therefore, we fertilized Quercus variabilis container seedlings with either 25, 100, or 150 mg total N seedling^?1, applied exponentially, during a 23-week pre-hardening regime, followed by either 0, 12, or 24 mg total N seedling^?1 applied during hardening (i.e., fall fertilization) in equal aliquots for 4 weeks. For seedlings without supplemental N during hardening, N concentration in stems and roots increased significantly despite substantial growth. The absence of N dilution was attributed to N translocation from foliage to these tissues, which was independent of pre-hardening N status. Overall, 32 % of foliar N was translocated and accounted for 75 % of the total N increase in stems and roots. Final stem N status was a function of pre-hardening fertilization, whereas root N concentration was affected by the interaction of pre-hardening and fall fertilization. Roots appear to be the main site of N storage, and root N content was significantly affected by pre-hardening and fall fertilization, but not their interaction. A combination of pre-hardening and fall fertilizer at a rate of 100 and 24 mg total N seedling^?1, respectively, yielded seedlings with the largest root systems.     

Verification of a phenotypic discrimination method for hybrid larch seedlings using DNA markers

The hybrid between Kuril larch (Larix gmelinii var. japonica) and Japanese larch (L. kaempferi) is an important afforestation tree species in Hokkaido, Japan, because of its rapid juvenile growth, straight stem, and resistance to bark gnawing by voles. To produce desirable hybrid seedlings, precise seedling discrimination is essential. However, continuous variations in morphological and phenological traits occur across L. gmelinii var. japonica × L. kaempferi and L. gmelinii var. japonica × L. gmelinii var. japonica seedlings. Therefore, we used DNA markers to verify and improve the morphological and phenological discrimination method. We collected seeds from an interspecific seed orchard and, using chloroplast DNA analysis, we showed that the hybridization rates of 1-year-old seedlings were different between years (2004, 23.2 %; 2005, 53.6 %) and between mother trees (2004, 5.8–39.4 %; 2005, 20.0–81.0 %). Discriminant analyses revealed that the root collar diameter of 2-year-old seedlings, number of sylleptic branches of 2-year-old seedlings, and day of terminal bud set in 1- and 2-year-old seedlings are traits that aid in discriminating hybrid seedlings. The proportions of correctly discriminated individuals were found to be 81.7–88.2 % when using improved phenotypic discrimination methods. These methods adopted traits with high discrimination abilities during discriminant analyses and determined the selection intensity according to the hybridization rate estimated from the DNA markers. In contrast, the proportions of correctly discriminated individuals obtained using the current method were lower, 72.7–78.5 %, because this method was based on seedling height and adopted a constant selection intensity.