Mycorrhization , Establishment and Growth of Outplanted Picea abies Seedlings Produced under Different Cultivation Systems

In forest nurseries seedlings are commonly produced using different cultivation systems which may have a profound effect on the extent of root colonisation by ectomycorrhizal (ECM) fungi and, consequently, on subsequent performance of the outplanted seedlings under field conditions. In this study, we compare effects of bare-root and containerised cultivation systems on mycorrhization, establishment and growth of nursery-produced Picea abies. One hectare experimental plantation was established on poor fertility sandy site. In total, 1250 seedlings of each treatment were planted in rows as ten individual replicates. The results of this study showed that production of P. abies seedlings using containerised system can yield abundant ECM colonisation of seedling roots and significantly improve seedling survival in the field. Some reduction in height increment may occur during the first years as a possible cost for support of ECMs. Study demonstrated that selection of proper cultivation system might result in similar or higher mycorrhization and survival rates of outplanted seedlings than achieved by expensive and laborious artificial mycorrhization.


Introduction
In forest nurseries seedlings are commonly produced using standardised cultivation systems which can be divided into two major types: i) bare-root systems -seedlings are grown in an open field beds; ii) containerised systems -seedlings are grown (in the greenhouse or outdoor) in different types of interconnected plastic pots or other specialised vessels filled with the growth substrate.It has been demonstrated that cultivation system may have a significant effect on the extent of root colonisation by beneficial ectomycorrhizal (ECM) fungi (Menkis et al. 2005).ECM are known to provide nutritional benefits for their hosts and protection against unfavourable abiotic and biotic stress factors (Smith and Read 1997), and therefore they may be of considerable importance for seedling performance in the field.A number of studies has reported that colonisation of roots by particular ECM fungi may significantly promote survival, establishment and growth of tree seedlings in the forest plantations (Garbaye and Churin 1997, Pera et al. 1999, Ortega et al. 2004, Menkis et al. 2007).The main mechanisms behind those effects are thought to be enhanced uptake of water and nutrients through a greatly increased root-absorbing surface (Smith and Read, 1997), increased longevity and growth of roots (Chilvers andGust 1982, Wilcox 1996), and protection of roots against drought, pathogens and heavy metal pollution (Morin et al. 1999, Van Tichelen et al. 2001, Ortega et al. 2004).
Intensive nursery cultivation (use of fertilisation, irrigation, mechanical and chemical weed, and pest control) may adversely affect ECM colonisation of seedling roots (Väre 1990, O'Neill and Mitchell 2000, Laatikainen and Heinonen-Tanski 2002).To overcome deficiency of ECM fungi in roots of nursery cultivated seedlings, artificial ECM inoculation was often suggested (Trappe 1977, Dunabeitia et al. 2004, Menkis et al. 2007) but this is associated with a large investment of resources.An alternative to this would be to explore an existing natural mycorrhization in forest nurseries by selecting a cultivation system that may yield seedling material with a high extent of ECMs.
The main aim of this study was to evaluate whether production of Picea abies (L.) H. Karst.seedlings using bare-root and containerised cultivation systems has any impact on mycorrhization, survival and growth of the seedlings following their outplanting in the field.Consequently, we assessed the possibility to avoid expensive and laborious artificial ECM inoculation, studying specifically field performance of the seedlings produced under two different cultivation systems with respectively lowest and highest ECM colonisation of seedling roots, showing that selection of seedlings produced under particular cultivation system which provides conditions for abundant natural ECM colonisation of seedling roots in the nursery may result in better performance of those seedlings after their transfer to the field, which is of considerable practical importance to nursery managers and foresters.

Study Site and Experimental Design
The study site comprised one hectare of perennial meadow situated in the vicinity of the Baltic Sea coast in western Lithuania (N 55°57´, E 21°06´).The site is characterised by relatively poor fertility sandy soil.Within the area, mean annual precipitation is ca.720 mm and the length of the growing season is ca.195 days.Temperatures average ca.14 °C during the growth season.
Planting was carried out in spring 2006 with 4-year-old P. abies seedlings produced in the forest nurseries Kulautuva and Varena (Menkis et al. 2005) using two cultivation systems: i) bareroot in open field beds (BR); ii) containerised in polyethylene rolls (CR) (Fig. 1).The soil in which BR seedlings were grown was derived from a sandy loam podzol.The substrate in which CR seedlings were grown was low-humified Sphagnum peat.In nurseries, fertilizers were applied routinely at annual levels of 34-56 N kg/ha for BR seedlings and 0.11-0.15N kg/m 3 of substrate for CR seedlings (Juska et al. 1982), which were estimated to be the optimal N levels for seedling growth in each respective cultivation systems.Both BR and CR seedlings used in this study represented standard planting material of each respective cultivation system and at outplant-ing were visually similar in their morphological parameters.
In previous study, P. abies yielded highest ECM colonisation in CR and the lowest in BR cultivation system, and the detailed description of ECM species composition in seedling roots of each respective nursery and cultivation system is in Menkis et al. (2005).Before outplanting of seedlings, the site was ploughed with a forestry plough in 20 rows at 2 m intervals.CR seedlings were prepared using standard procedure as follows: polyethylene was removed and root systems of individual plants were gently separated from each other to preserve fine roots and attached substrate.Seedlings of both treatments were established in the bottom of prepared rows in ten replicates, arranging different treatments in every second row.In order to avoid human factor in planting quality, standardised planting was done using an automated seedling planter.In total, 1250 P. abies seedlings were planted in each at an initial density of 2500 seedlings per hectare.

Assessment of Root Mycorrhization
To assess the extent of root mycorrhization at outplanting, 40 seedlings were randomly sampled from the stock of each treatment.Assessment of roots was carried out using mycorrhizal mor-photyping as described by Menkis et al. (2005).Briefly, each root system was washed in the tap water to remove the soil and 20 single root tips from each plant were randomly collected from different parts of the root system using forceps.Sampled roots were assessed for ECM colonisation using dissection microscope.Mycorrhizal tips were identified by the presence of a mantle, external hyphae or rhizomorphs, and the absence of root hairs.

Field Measurements
Survival of plants was determined three times, at the end of first, second and third growing seasons (2006, 2007, and 2008), by counting all living trees.The height increment was measured for all living trees at the end of the second and third growing seasons (2007 and 2008).

Statistical Analyses
The impact of the treatment on seedling mycorrhization and survival was analysed using chisquare (χ 2 ) tests (Mead and Curnow 1983).The survival of the seedlings was evaluated separately for each respective year 2006, 2007 and 2008.Height increment of the seedlings was analysed Arrows show abundant colonisation of seedling roots and substrate by ECM basidiomycete Amphinema byssoides.
After the first growing season, seedling survival in the plantation was high in both treatments ± 0.91 SE in CR and 97.2% ± 0.96 SE in BR) and the differences were statistically insignificant (χ 2 test; p > 0.05) (Fig. 2).After the second season, seedling survival decreased in both treatments, but CR seedlings showed significantly better survival than BR seedlings (88.5% ± 1.13 SE and 83.8% ± 1.04 SE, respectively; p < 0.0001) (Fig. 2).After the third season, seedlings from CR showed even higher survival than seedlings from BR (88.2% ± 0.94 SE and 81.7% ± 1.07 SE, respectively; p < 0.0001).
Certain differences in height increment were observed between CR and BR treatments after second and third growing seasons (Fig. 3).After the second season, BR seedlings showed significantly higher growth increment than CR seedlings (21.6 cm ± 0.26 SE and 18.0 cm ± 0.23 SE, respectively; p < 0.0001).However, already during the third season CR seedlings demonstrated better growth, thus the gap in height increment between CR and BR seedlings after three years has decreased, yet the difference still remained significant (31.5 cm ± 0.32 SE in BR and 29.5 cm ± 0.37 SE in CR, p < 0.0001) (Fig. 3).

Discussion
The results of the present study on ECM colonisation of seedling roots appeared to be largely consistent with those from earlier study, where containerised P. abies seedlings showed 82.7% mycorrhization as compared to 44.1% observed in bare-root cultivation system (Menkis et al. 2005), confirming Rincón et al. (2005) that the growth environment may strongly influence intensity of ECM colonisation of seedling roots in the nursery.
We observed that in both treatments there was certain pattern in seedling survival rate during  three consecutive years: i) equally high survival rates in both CR and BR during the first year, ii) decreased in both but significantly higher survival in CR after the second year, and iii) even larger difference in survival after the third year.This could have been a consequence of annual fluctuations of environmental conditions in the plantation site, but seedling survival in CR was better, probably because ECMs are known to mitigate environmental stress (Rodriguez et al. 2004).Moreover, beneficial effects of ECM fungi are likely to be most pronounced on poor fertility planting sites under harsh environmental conditions (Smith and Read 1997).Vaario et al. (2009) suggested that ECMs on roots of containerised P. abies nursery seedlings was one of the best indicators of seedling performance after outplanting.In their study, A. byssoides has undergone a relatively rapid expansion from marginal levels of occurrence in the nursery seedlings to moderate levels in the third year seedlings after outplanting.Even more rapid, only during one growing season, change in ECM community structure towards dominance of A. byssoides in the root systems of outplanted seedlings was reported by Menkis et al. (2007).Results of those studies suggest that A. byssoides is an efficient root coloniser of P. abies seedlings.Its common occurrence in newly established forest plantations (Kranabetter J 2004, Menkis et al. 2007, Vaario et al. 2009), and better survival of abundantly colonised CR seedlings of the present study further suggest that A. byssoides may play an important role in seedlings survival and establishment following their outplanting.In support to this, Jones et al. (2009) showed that 15 N accumulation in shoots and roots of spruce seedlings colonised by A. byssoides was respectively moderate and high compared to the seedlings colonised by other ECMs.
Furthermore, we hypothesise that in addition to the benefits provided by ECMs, organic substrate attached to the root systems of outplanted CR seedlings has likely contributed to better protection of the roots against unfavourable environmental conditions of the site as e.g.frosts and droughts.In addition, this could also improve nutrient availability to the seedlings at early stage of their establishment.Such improved seedling survival during first plantation years is important for practical silviculture.
The observed reduction in height increment in CR seedlings is likely to be temporal, but to confirm this more continuous observations are needed.In a related Finnish study, Vaario et al. (2009) reported that containerised P. abies seedlings grown in a similar substrate and with similar mycorrhization levels as CR seedlings, although produced satisfactory shoot growth in the nursery showed reduced growth rates after transfer to the field.Similar growth patterns, when P. abies seedlings with most abundant ECM colonisation showed reduced rates of height increment were observed in the study conducted on replanted forest clear-cut (Menkis et al. 2010).As ECM fungi are exclusively dependent on their hosts for carbohydrates, we hypothesise that support of ECMs in CR seedlings was probably the reason for reduced height increment observed during the first two years of this study.In support to this, it was shown that under the field conditions up to 22% of plant-produced carbohydrates can be allocated to ECMs and that growth reduction in plants may be equivalent to such allocations (Hobbie 2006).
In conclusion, present study demonstrated that selection of proper cultivation system in forest nursery might result in similar or higher mycorrhization and survival rates of outplanted seedlings than achieved by expensive and laborious artificial mycorrhization.In order to obtain more comprehensive picture on how different cultivation systems affect natural mycorrhization of nursery-grown tree seedlings and their subsequent field performance, more related studies are needed, encompassing wider range of tree species and field conditions.

Fig. 3 .
Fig. 3. Height increment of Picea abies seedlings after the second (2007) and third (2008) growing seasons.Within the same year, statistically significant differences between the treatments (bare-root and containerised) in one-way ANOVA are designated by different letters.Error bars indicate standard error of the mean.

Fig. 2 .
Fig. 2. Survival of Picea abies seedlings during three consecutive growing seasons.Within the same year, statistically significant differences between the treatments (bare-root and containerised) in chisquare tests are designated by different letters.Error bars indicate standard error of the mean.