Introduction 

The European black truffle (Tuber melanosporum Vittad.) is an ectomycorrhizal (EM) fungus extensively cultivated due to its gastronomic value and decline of wild production ([25]). Each year, more than 2.000 ha of agricultural lands are afforested in southern Europe (mainly France, Italy and Spain) by entrepreneurs to produce black truffle, most of them with native Quercus as host plants ([25]). Modern truffle cultivation is based on planting nursery-inoculated seedlings on suitable lands, with appropriate edaphoclimatic environment for the fungus to complete its life cycle and with low EM inoculum potential ([30]).

High quality inoculated seedlings must exhibit a root system abundantly colonised by T. melanosporum ([1], [19]). The commercial production of these seedlings is customarily done with spore inoculum, either concentrating inoculum onto fine roots or incorporating it into substrate ([9], [22]). With the high price of sporocarps luring nurserymen into reducing inoculum application rates, selection of carrier materials providing close contact with fine roots and even distribution of inoculum is critical, especially when thousands of seedlings are produced ([2]). For Tuber species, little scientific information is publicly available on efficiency of the various inoculation methods or on their interaction with other nursery practices, often because of patents and confidentiality agreements ([7], [23]).

The quality of inoculated seedlings is determined not only by abundance of mycorrhizas but also by vegetative quality of seedlings ([14]). Large, nutrient-rich seedlings with high growth potential are likely to perform better in drylands with deep soils ([10]), such as the agricultural lands where truffle plantations are usually established in Spain ([16]). However, problems of scarce shoot development and stunted lateral root growth have been frequent in the commercial production of Quercus seedlings inoculated with T. melanosporum ([9], [2]). Some inoculation methods seem to exacerbate this problem ([6], [23]).

In forest nurseries a common solution for low vegetative quality of seedlings is fertilisation, which increases size, nutrient storage and root growth potential ([34]). However, Treseder ([32]) showed that nitrogen (N) and phosphorus (P) fertilisations decrease mycorrhizal abundance in the field. In nursery experiments, high fertilisation levels have been found to inhibit the formation of ectomycorrhizas of many fungal species including T. melanosporum ([13], [3], [37], [11]), although Castellano & Molina ([8]) noticed that some species are much more tolerant and that fertilisation effect was dependent on the application system, fertiliser type, dose and form. On the other side, Quoreshi & Timmer ([24]) and Rincon et al. ([26]) found fertiliser doses that increased growth of containerised conifer seedling without decreasing colonisation levels of inoculated fungi from various EM species.

In this study we inoculated Quercus ilex L. and Quercus faginea Lam. with T. melanosporum. We hypothesised that a moderate fertilisation regime could improve morphological quality of seedlings while maintaining EM colonisation levels suitable for commercial purposes. We assess the effect of fertilisation on growth and EM status of seedlings, applying a dose similar to that used by Rincon et al. ([26]). We compare the effect of fertilisation on Q. ilex and Q. faginea, a faster-growing species ([28]), and we evaluate the use of fertilisation in two inoculation methods, root-dipping and root-powdering, both applying inoculum onto roots, although with different carrier materials. We hypothesised that host species and inoculation method would affect fine root traits, thus influencing EM status and ability of seedlings to respond to fertilisation.

  Materials and methods 

Fungal inoculum, plant material and inoculation

The sporocarps used as inoculum were acquired fresh and mature from plantations in Sistema Ibérico mountain range (eastern Spain). For each experiment, at least 20 sporocarps from five plantations were used in order to minimise spore germinability differences. They were surface sterilised with sodium hypochlorite solution, sliced thin, air dried under room conditions and homogenised with a coffee grinder ([22]).

We selected Q. ilex as host plant because it is the most used species in Spanish truffle plantations. It was compared to Q. faginea, which also produces truffles in the wild and is frequently used in Spanish truffle plantations. While having biological features similar to Q. ilex, Q. faginea is a faster-growing species whose nursery seedlings show more extensive lateral roots ([29], [28]). Acorns from the provenance regions Sistema Ibérico (for Q. ilex) and Sistema Ibérico Levantino (for Q. faginea) were acquired. They were surface sterilised with a 20% sodium hypochlorite solution for five minutes, and germinated in January in a tray with perlite and vermiculite. When seedlings had 6-8 leaves and had formed lateral roots (12 weeks after seedling emergence, in late April), they were removed from the tray, mechanically root-pruned at the tap root end to eliminate its defects ([22]), inoculated, and transplanted to Quick-pot^® containers (650 ml, 18 cm deep).

The inoculation was performed by root-dipping, following a traditional method described by Hall et al. ([17]) as frequently used in Spain. The bare roots were dipped in a slurry of homogenised sporocarps in a sucrose solution (2:1 water: sucrose v/w) aimed to produce a high viscosity suspension. The spore concentration in the slurry was adjusted to obtain an application rate of 2.0 g fresh truffle per seedling (6×10⁵ spores per seedling), although some variability between seedlings is impossible to avoid due to differing levels of fine root development. This method is compared to a root-powdering inoculation, also concentrating spores onto roots but with a solid carrier instead of a liquid one ([7]). A mix of talcum powder (hydrated magnesium silicate) and homogenised sporocarps was applied onto seedling bare roots, with spore concentration adjusted to obtain an inoculum rate of 2.0 g fresh truffle per seedling.

The potting substrate consisted of 12:6:1 (v/v) calcareous sandy loam soil, base-fertilised Sphagnum white peat (Kekkila^® White 420 W), and limestone coarse sand. It was solarised during summer, and subsequently presented a pH of 7.9, conductivity (1:5) of 418 mS m^-1, 1390 ppm N (Kjeldahl), 32 ppm P (Olsen) and 337 ppm K (ammonium acetate extraction), with pH and nutrient levels falling within the common range in Spanish wild truffle soils ([16]). A soil-based potting mix was selected because these are still used with good results in truffle nurseries and research ([7], [4]). Seedlings were cultivated in a greenhouse and sprinkle irrigated to saturation 2-3 times per week during summer and once each 7-14 days during winter.

Following the first shoot flush after inoculation (seven weeks after inoculating, in mid June), slow-release fertiliser Osmocote Exact Mini^® (NPK 16-8-11, with a longevity of 3-4 months at 21 °C) was added in the surface of substrate at a dose 0.5 g L^-1, providing 52 mg N, 26 mg P, 36 mg K, 6.5 mg Mg, 1.3 mg chelated Fe, 0.16 mg Mn and Cu, 0.07 mg B and Mo and 0.06 mg Zn per seedling. We selected a slow-release fertiliser because it is the most common in Spanish forest nurseries. The dose was selected following that used by Rincon et al. ([26]) for avoiding inhibition of EM formation in containerised Pinus seedlings.

Experimental design

Two experiments were done independently. In 2009 an experiment to compare the effect of fertilisation on two host species (Q. ilex and Q. faginea) was conducted. Four treatments were established in a 2×2 factorial design, with 12 replicates per treatment: unfertilised Q. ilex, fertilised Q. ilex, unfertilised Q. faginea and fertilised Q. faginea. All seedlings were inoculated with root-dipping.

In 2010 an experiment to compare the effect of fertilisation on two inoculation methods was conducted. Four treatments were established in a 2×2 factorial design, with six replicates per treatment: unfertilised root-dipping, fertilised root-dipping, unfertilised root-powdering and fertilised root-powdering. The experiment was conducted with Q. ilex as plant host.

Data collection and analysis

The seedlings of the fertilisation and host species experiment were analysed 12 months after inoculation (in April-May), whereas the fertilisation and inoculation method experiment was analysed 11 months after inoculation (in March). The mycorrhizal status was assessed through a volumetric sampling. In each seedling a sample with 9% of the substrate volume (57 ml) was taken. To cope with heterogeneity across soil depth, every sample consisted of three subsamples: the depth of the container was divided into three equal parts, and in the centre of each third (4, 9 and 13.5 cm depth) a horizontal core (2 cm diameter) across the container was taken.

Samples were kept in water at 4°C. Length of fine roots (diameter <2 mm) was measured according to Tennant ([31]). Root tips were counted and classified as non-mycorrhizal, T. melanosporum ectomycorrhizas or contaminant ectomycorrhizas. Shoot dry weight, root dry weight, fine root dry weight, stem height and root collar diameter were measured after drying to constant weight at 80 °C.

Plant dry weight, shoot and root dry weight, stem height, root collar diameter, specific root length (SRL, ratio of root length to dry weight of fine roots), number of root tips and number of T. melanosporum tips per seedling were analysed by conventional ANOVA. Proportion of root tips colonised by T. melanosporum and frequency of occurrence of contaminants in seedlings were analysed through generalised (binomial) linear models. When model assumptions were violated, the response variable was transformed. In the model for proportion of roots colonised by T. melanosporum, fine root length was included as a covariate to account for within-treatment variability in fine root development.

  Results 

Experiment 1: fertilisation/host species

Total dry weight of seedlings was positively affected by fertilisation (P<0.001), with no significant differences between host species (P=0.50 - Tab. 1). Both shoot and root dry weight followed the same pattern, with a positive effect of fertilisation (P<0.001 in both cases) and no significant differences between host species (P=0.59 and P=0.46, respectively - Tab. 1). Stem height was positively affected by fertilisation (P<0.001), with no significant differences between host species (P=0.69 - Tab. 1). Root collar diameter was positively affected by fertilisation (P<0.001) and higher in Q. faginea than in Q. ilex (P<0.001 - Tab. 1). SRL was higher in Q. faginea (P=0.001), with no significant effect of fertilisation (P=0.22 - Tab. 1). The number of root tips per seedling was higher in Q. faginea (P<0.001) and in fertilised seedlings (P=0.04 - Tab. 1).

The inoculum of T. melanosporum formed mycorrhizas with all seedlings. The number of T. melanosporum mycorrhizas per seedling and the proportion of root tips colonised by T. melanosporum were higher in Q. faginea (P<0.001 and P=0.01, respectively), with no significant effect of fertilisation (P=0.59 and P=0.24, respectively - Tab. 1). The only contaminant EM species found in seedlings was Sphaerosporella brunnea Svrcek and Kubicka, showing a higher occurrence frequency on Q. ilex (P=0.02) and no significant effect of fertilisation (P=0.22 - Tab. 1). The mean proportion of root tips colonised by S. brunnea on Q. ilex was 2.5%.

The distribution of T. melanosporum colonisation levels along the depth profile did not show any significant (α=0.05) interaction with fertilisation.

Experiment 2: fertilisation/inoculation method

Total dry weight of seedlings, shoot dry weight and root dry weight were higher in root-powdering inoculation than in root-dipping (P=0.004, P=0.003 and P=0.02, respectively). No significant effect of fertilisation was found (P=0.08, P=0.14 and P=0.08, respectively), although the trend with respect to fertilisation was the same as in the fertilisation/host species experiment (Tab. 2). Stem height was not significantly affected by either fertilisation (P=0.28) or inoculation method (P=0.26 - Tab. 2). Root collar diameter was positively affected by fertilisation (P=0.02), with no significant differences between inoculation methods (P=0.16 - Tab. 2). Neither SRL nor number of root tips per seedling were significantly affected by fertilisation (P=0.54 and P=0.39, respectively) or inoculation method (P=0.59 and P=0.12, respectively - Tab. 2).

The inoculum of T. melanosporum formed mycorrhizas with all seedlings. The number of T. melanosporum mycorrhizas per seedling and the proportion of root tips colonised by T. melanosporum were higher in root-powdering inoculation (P=0.009 and P=0.049, respectively), with no significant effect of fertilisation (P=0.46 and P=0.68, respectively - Tab. 2). The only contaminant EM species found was S. brunnea, showing a higher occurrence frequency in root-dipping inoculation (P=0.004) and no significant effect of fertilisation (P=0.56 - Tab. 2). The mean proportion of root tips colonised by S. brunnea in root-dipping inoculation was 5.2%.

The distribution of T. melanosporum colonisation levels along the depth profile did not show any significant (α=0.05) interaction with fertilisation.

  Discussion 

In modern truffle cultivation the use of inoculated seedlings is fundamental ([17]). The abundance of T. melanosporum mycorrhizas in the early years after plantation establishment has been found positively related to mycorrhizal abundance in the nursery and to plant performance after planting ([5], [15]). Nursery practices must be fine-tuned to encourage colonisation by T. melanosporum, but also to improve vegetative quality of seedlings. However, in nursery fertilisation experiments a conflict between optimal seedling growth and EM colonisation has been reported for many EM fungi ([8], [37], [11]). Two mechanisms have been suggested to explain this conflict: (i) the host reducing carbon supply to the fungus due to a greater carbon demand by growing shoots, or (ii) the fungus requiring most carbon received from the plant to assimilation of the greater N uptake ([38]).

The fertilisation dose used in the present study increased growth as well as vegetative quality according to Spanish standards ([35], [36]), while maintaining T. melanosporum colonisation levels. In the fertilisation/host species experiment, fertilisation increased seedling biomass by 70%, stem height by 49% and root collar diameter by 22%, without significant detrimental effects on fine root traits or EM colonisation levels. In the fertilisation/inoculation method experiment, fertilisation increased Q. ilex root collar diameter by 13%. The lower magnitude of the fertilisation effect in the latter experiment could be due to differences in acorn size ([20]), spore germinability ([22]) or greenhouse climatic conditions from year to year, although it could also be related to the lower number of replicates decreasing the accuracy of estimation.

Our results disagree with previous experiments with containerised Quercus seedlings. Beckjord et al. ([3]) inoculated Quercus alba L. and Quercus rubra L. with Pisolithus tinctorius (Pers.) Coker & Couch and Scleroderma citrinum Pers. and found that N and P fertilisation doses suitable for mycorrhizal colonisation were much lower than those needed to promote seedling growth. Dupré et al. ([13]) inoculated Quercus humilis Mill. with T. melanosporum and found that P fertilisation doses with increased seedling growth did not correspond with higher mycorrhizal colonisation. In Q. ilex inoculated with Paxillus involutus (Batsch) Fr., Hebeloma mesophaeum (Pers.) Quél. and Cenococcum geophilum Fr., Oliveira et al. ([21]) found that a fertiliser dose moderately higher than ours decreased mycorrhizal colonisation, with no significant increase in plant biomass or shoot height. On the other hand, Rincon et al. ([26]), Quoreshi & Timmer ([24]) and Khasa et al. ([18]) found fertilisation levels that improved conifer growth without damaging colonisation levels by various inoculated EM fungi, supporting that at least in some cases they can be reconciled.

The contrast between our results and other Quercus experiments could be due to differences in sensitiveness of EM species to fertilisers ([8], [38]), differences in the response of host species to fertilisation, differences in fertilisation implementation or differences in other cultivation practices. The response of non-inoculated Q. ilex seedlings to fertilisation follows a pattern similar to other Mediterranean Quercus such as Q. faginea, but different from Quercus from other biomes which show lower sufficiency levels ([33]). Castellano & Molina ([8]) warned that response of EM fungi to fertilisation was influenced not only by the dose but also by fertiliser type and form, as well as application system (timing, rate and method).

In our study, Q. ilex and Q. faginea showed very similar biomass and response to fertilisation, as expected for species with similar biological features and agreeing with results of Sanz-Pérez et al. ([28]) for non-inoculated seedlings fertilised with 50 mg N. We found the main differences between these species in fine root traits and EM status, with Q. faginea showing higher SRL, number of root tips and number of mycorrhizas. This agrees with findings of Domínguez-Núñez et al. ([12]) and Silla & Escudero ([29]) in young plantations. The former found more root tips and EM tips in Q. faginea than in Q. ilex seedlings inoculated with T. melanosporum, whereas the latter found higher SRL in Q. faginea than in Q. ilex non-inoculated seedlings. Our results agree with the accepted view that Q. faginea root system is more branched and extensive.

The distinct fine root traits of Q. ilex and Q. faginea went along with differences in EM status. This could be due to the inoculation method delivering somewhat more spore inoculum to seedlings with higher SRL, to inoculum being more evenly distributed within seedling fine roots, or to seedlings recovering before from transplantation. However no interaction in the response of fine root traits to fertilisation was found, suggesting that differences between Q. ilex and Q. faginea did not affect ability of seedlings to respond to fertilisation.

The inoculation method influenced seedling weight and EM status, with root-dipping showing worse characteristics. Pruett et al. ([23]) found that adding hydrogel in a water slurry for root-dipping inoculation had negative effects on Quercus robur L. survival, root development and Tuber aestivum Vittad. colonisation. Cartié et al. ([6]) found that using an alginate solution for root-dipping inoculation provoked an important Q. ilex mortality and a low T. melanosporum colonisation. These results suggest that inoculant carriers forming a sticky coating around the complete root system are able to damage Quercus development and Tuber colonisation.

However, Cartié et al. ([7]) found no detrimental effects of alginate solution when the inoculant was applied in a bilayer, firstly dipping roots in alginate solution and then powdering them with a mixture of inoculum and talcum. Pruett et al. ([23]) found no detrimental effect of root-dipping inoculation when it was performed without hydrogel. All this suggests that the damage was due to the combination of sticky carrier and spores in close contact with roots.

Our results show that seedling characteristics and inoculation effectiveness can be affected not only by host species but also by inoculation method. Further research would help to know if fertilisation or inoculation method interact with other nursery practices.

The only EM fungi found in our study other than T. melanosporum was the pioneer, nursery adapted S. brunnea ([27]). As expected, its occurrence was higher in treatments with lower T. melanosporum colonisation levels, thus suggesting that it was related to gaps left by the latter.

The inoculation and fertilisation procedures used in the present study proved effective for obtaining EM seedlings with quality levels comparable to commercial standards. All seedlings bore T. melanosporum mycorrhizas, with all treatments showing mean colonisation levels analogous to those in commercial nurseries ([1]). All seedlings met the morphology standards established in relevant regulation on forest seedlings marketing (European Directive 1999/105/CE for Q. ilex and Spanish RD 289/2003 for Q. faginea).

  Conclusions 

This study showed that a dose of slow-release fertiliser is able to improve growth and morphological quality of Quercus seedlings while maintaining commercial T. melanosporum colonisation levels under greenhouse conditions. Quercus ilex and Q. faginea showed differences mainly in fine root traits and EM status, but in spite of them both species showed similar response to the fertilisation dose. The inoculation method proved able to influence not only EM status but also seedling growth. The study provided an important basis for fine-tuning use of fertilisation in commercial production of T. melanosporum-inoculated seedlings, but showed that inoculation effectiveness can be altered by other cultural practices, hence the improvement of these practices should be addressed jointly.

  Acknowledgements 

This work was funded by Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA, Government of Spain) project PET2007-13-C07-04.

  References