Introduction

Nursery production of seedlings inoculated with Tuber melanosporum Vittad. is one of the keystones of modern black truffle cultivation. Since the 1960s, joint efforts between the University of Turin and the Institute for Woody Plants and Environment (IPLA, Italy), with the French National Institute for Agricultural Research (INRA) from Clermont-Ferrand (Chevalier and Grente 1973) allowed the establishment of a reliable method for the production of seedlings colonized by this ascomycete. The amount of seedlings produced by nurseries is proportional to the increase of land use for black truffle cultivation. Cocina et al. (2013) report that currently dozens of nurseries specialize on production of seedlings colonized by T. melanosporum in Spain, France, and Italy. Furthermore, they note at least 29 additional nurseries in the rest of the world.

Successful truffle cultivation involves a long-term process, so starting with high quality plant material for plantation establishment is crucial. This is especially important regarding the percentage of root tips colonized by the black truffle; a high level of truffle mycorrhizal colonization will limit colonization by other ectomycorrhizal fungi that exist in the soil and, thus, a potential decrease in truffle yields (Pruett et al. 2008; Iotti et al. 2012).

Seedling quality evaluation is performed on a sample of seedlings from each batch. A batch consists of plants that have the same seed provenance, sowing date, truffle inoculum, and matching nursery management practices (Palazón et al. 1999). Nursery seedlings must meet forestry plant quality requirements and achieve a specific level of truffle fungus mycorrhiza colonization as determined by a particular evaluation method. Colonized seedlings must also be free from contaminant mycorrhizae belonging to nontarget Tuber species, and specific levels of mycorrhizal colonization by other competing fungi cannot be exceeded (see Table 1).

Table 1 Summary of the methodologies used for mycorrhizal seedling evaluation
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Detecting mycorrhizae belonging to Tuber species other than the targeted truffle species on inoculated seedlings is unusual. Black truffle mycorrhizae can be confused mainly with those of Tuber indicum Cooke and Massee and Tuber brumale Vittad. Importation of fresh T. indicum fruiting bodies creates a high ecological risk. Mycorrhizae of T. indicum have been detected in at least one Italian T. melanosporum plantation (Murat et al. 2008) and have produced fruiting bodies in a truffle plantation from the USA (Bonito et al. 2011). The morphological resemblance between T. indicum and T. melanosporum mycorrhizae makes it difficult to differentiate them without a molecular analysis.

Although the use of well-colonized truffle-inoculated seedlings is critical to plantation success, we lack common criteria for evaluating the quality of T. melanosporum-colonized seedlings. Five methods for quality evaluation have been published and commonly used in Europe: Chevalier and Grente (1978) INRA-ANVAR, Govi et al. (1995) University of Perugia, Fischer and Colinas (1996) University of Lérida, Palazón et al. (1999) INIA-Aragón, and Reyna et al. (2000) CEAM-Valencia. Most methods examine the whole root system and then estimate the percentage of root tips colonized by T. melanosporum. However, the method used by Reyna et al. (2000) only analyzes a portion of the root system by extracting a cylindrical sample of the potting mix in the seedling container. All methods also examine and estimate the presence or absence of other contaminating ectomycorrhizal fungi and discard those batches with root tips colonized by other Tuber species. In Tables 1 and 2, the most important characteristics of each evaluation method are described.

Table 2 Summary of the protocols used by each tested method for seedling quality evaluation
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Given the lack of consensus on how best to evaluate the quality of truffle-inoculated seedlings, we tested the five most used methods to address the following objectives: (1) assess their ability to estimate root colonization by T. melanosporum in Quercus ilex subsp. ballota (Desf.) Samp. (holm oak) seedlings and (2) determine the similarity in the ability for the different methods to estimate root colonization by T. melanosporum.

Material and methods

Sampling of colonized seedlings in nurseries

In Spain, Q. ilex is the most common host seedling inoculated with T. melanosporum for truffle plantations. This is explained by its greater hardiness, longevity, and higher truffle production compared to other host species under Spanish environmental and edaphic conditions.

Ten batches of holm oak seedlings inoculated with black truffle were examined for mycorrhizal colonization. The batches belonged to two different nurseries and each batch had 1,000 seedlings. A random sample of 12 seedlings was selected from each batch; 12 seedlings are required by the two evaluation methods with the highest number of analyzed samples per batch, Fischer and Colinas (1996) and Reyna et al. (2000). Inoculation in both nurseries took place in April 2010. Sample collection took place in November 2011, thus allowing generous time to achieve an optimum mycorrhizal status for outplanting.

Seedling and batch evaluation

As each seedling had to be evaluated by five methods, the sequence of analyses was arranged such that each method would not affect the following one. First, a cylindrical sample of the root system was extracted from each of the 120 seedlings by means of a punch tool and stored. This procedure follows the method of Reyna et al. (2000) and was done first because it is the least destructive. Next, roots were washed free of potting mix, and forestry plant quality was evaluated by measuring height and root collar diameter (according to Council Directive 1999/105/CE). Additional morphological quality observations related to root health and root system architecture were recorded (Peñuelas 1993). Samples were frozen together with the cylinders at −20 °C until processed. Next, the method of Chevalier and Grente (1978) was performed by rating seedling mycorrhizal quality on a 1 to 5 scale. For statistical purposes, this scale was converted to percentages according to Trouvelot et al. (1986). We followed the procedure by Govi et al. (1995), which extracts root fragments by spreading the root system onto a numbered grid. This procedure was followed by the method of Palazón et al. (1999), which divides the root system into three sections and takes a fragment from each. Finally, the method of Fischer and Colinas (1996) was performed. It requires the root system to be cut into 2 − 3 cm fragments for mycorrhizal root tip analysis. The time needed for evaluating each seedling by each method was also recorded. Following the guidelines of each method, which are summarized in Table 1, batches were sorted by their suitability. Batch suitability can be understood as the final diagnosis of the quality of the batch given by each method.

Morphological and anatomical identification of T. melanosporum ectomycorrhizae were carried out following the descriptions of Zambonelli et al. (1993) and Rauscher et al. (1995). Other nursery contaminants were described according to the studies of Agerer (2006) and Agerer and Rambold (2004–2013).

Statistical analysis

Each variable was tested for normality (Kolmogorov–Smirnov test; p > 0.05), and each analysis checked for homoscedasticity (Levene’s test; p > 0.05) to comply with the premises of parametric analyses. Colonization percentage was transformed by raising it to a positive power, but the means shown are not transformed. A Pearson correlation matrix was constructed to examine the relationship between the colonization percentages obtained by the different methods. The differences in the colonization percentage obtained by the different methods and in both nurseries were tested by ANOVA (general linear model) with two fixed factors (method and nursery) and their interaction. However, for the representation of confidence intervals for colonization percentages obtained by the different methods, a one-factor ANOVA was performed in order to differentiate between them. In both cases, post hoc Tukey’s test was used to discriminate between methods.

The time needed for estimating colonization percentage by each method was analyzed using nonparametric methods. Their differences were evaluated by the Kruskall-Wallis test and the Mann-Whitney U test was used to compare the means in pairs as independent samples. The design and basic information about the tests used can be found in Montgomery (2001). Their implementation was carried out using SPSS (2012).

Results

The data on batch suitability in relation with colonization levels and in relation to the evaluation method used for each of the ten batches selected are shown in Table 3. The average percentages of colonization by T. melanosporum mycorrhizae obtained for each batch by taking the mean of the five methods are also shown in Table 3. Consensus among methods, understood as agreement in evaluating each batch suitability, only happened in the batches with the highest (batches N2B6 and N1B3, with 42.3 and 35.0 % average colonization, respectively) and the lowest (batch N1B2, 17.6 %) colonization levels. In the other seven batches, results differ depending on the evaluation method used. According to our data, the most liberal method seems to be that of Chevalier and Grente (1978), with nine out of ten batches considered acceptable. The most conservative method was that of Govi et al. (1995), which found only three batches acceptable.

Table 3 Batch averages and standard deviations of the percentages of colonization by T. melanosporum estimated by each method and the grand mean for the methods
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In nursery 1, mycorrhizae from Sphaerosporella brunnea (Alb. and Schwein.) Svrček and Kubička were found on some seedlings. No ectomycorrhizae belonging to the genus Tuber (other than T. melanosporum species) were found in any of the two nurseries. Furthermore, forestry plant quality of all seedlings met the basic standards required by the Council Directive 1999/105/CE. Thus, both the presence of contaminant mycorrhizal fungi and plant quality did not lead to the rejection of any batch evaluated by any of the methods.

The correlation analysis between evaluation methods concerning the colonization percentage obtained for each seedling (Table 4) shows the existence of a significant (p < 0.05) and positive correlation between all methods, with the exception of the method of Reyna et al. (2000), which only had a significant correlation with the method of Govi et al. (1995). The highest correlation coefficient (0.662) was obtained between the method of Palazón et al. (1999) and the method of Fischer and Colinas (1996). The lowest coefficient in a significant correlation (0.399) was found between the methods of Chevalier and Grente (1978) and Govi et al. (1995).

Table 4 Correlation matrix (Pearson Product Moment) of the colonization percentages by T. melanosporum obtained with the five evaluation methods tested. Significant correlations are highlighted in bold
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The percentage of colonization, averaged for the five methods, significantly differed between nurseries (p < 0.001) and was highest for seedlings produced by nursery 2 (33 % compared to 27 % in nursery 1). The statistical model, however, only explained 3 % of the variability in the observations. Furthermore, the estimates of colonization percentages obtained by each method differed significantly (p < 0.001). These estimates can be subdivided into three groups (Fig. 1): Fischer and Colinas (1996) and Govi et al. (1995) with the lowest colonization percentages, Chevalier and Grente (1978) with an intermediate percentage, and Palazón et al. (1999) and Reyna et al. (2000) with the highest colonization percentages. Additionally, each method gave different values for the colonization percentages from each nursery (Fig. 2), with the exception of the method of Reyna et al. (2000) that did not detect differences between the nurseries. The interaction between nurseries and evaluation method was not significant (p = 0.056). The evaluation methods account for 10.4 % of the colonization percentage variance. On the other hand, if the variation due to nursery is included, the value rises to 14.2 %.

Fig. 1
figure 1

Least square means and graphical representation of the percentage of colonization by T. melanosporum (confidence interval 1 − α = 95 %) obtained by the five methods evaluated. The ANOVA analysis was based on a fixed factor method: R 2adj  = 0.104; SEM = 2.19; p < 0.001. Different letters show significant differences between the methods contrasted by post hoc Tukey’s test

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Fig. 2
figure 2

Graphical representation of the confidence intervals (1 − α = 95 %) of the percentage of colonization by T. melanosporum obtained by the different methods for each nursery

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The methods differ significantly (Kruskall-Wallis K statistic = 536.852; p < 0.001) in the mean time spent estimating the colonization percentage of individual seedlings (Fig. 3). The method of Chevalier and Grente (1978) was the quickest for plant evaluation with an average of 6 min per seedling. The most time-consuming method is Fischer and Colinas (1996), with about 34 min per seedling.

Fig. 3
figure 3

Means, standard deviations, and graphical representation of the confidence interval (1 − α = 95 %) of the time spent in estimating the percentage of seedling colonization by T. melanosporum for one seedling by the different methods for evaluating seedling quality. Different letters show significant differences according to Mann-Whitney U test (p < 0.05)

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Discussion

Many variables affect truffle fungus colonization of inoculated seedlings: nursery practices, inoculum (dose, format, pretreatment), potting mix, type of container, and seedling condition at the time of inoculation, among others (Hall et al. 2007). However, we have also observed clear differences between the mycorrhizal colonization evaluation methods used and the resulting batch suitability. One method may find a batch of T. melanosporum-colonized Q. ilex seedlings acceptable while another method may not. Thus, it is imperative that truffle seedling certifying groups reach agreement on the best certification method. Based on our observations, the various methods agree to accepting or rejecting a batch when seedlings have either very high or low colonization. However, the strongest lack of consensus among methods for accepting or rejecting batches occurred on batches with intermediate colonization levels, which seems to be the most usual situation.

The methods that achieve the lowest colonization values—Fischer and Colinas (1996) and Govi et al. (1995)—use grids wherein sampling selects random root segments. In the other three methods, estimated colonization levels are higher, either because they are slightly subjective (Chevalier and Grente 1978; Palazón et al. 1999) or because they take a root sample from a highly colonized area (Reyna et al. 2000). In this latter case, the internal variability of each sampling unit should be taken into account (Rocchi et al. 1999). Colonization percentages for each seedling and method are correlated in all cases except in the method of Reyna et al. (2000). It is important to note that only the methods of Reyna et al. (2000) and Fischer and Colinas (1996) estimate the total root tip number for each seedling in addition to colonization percentage. The remaining methods, with the exception of that of Chevalier and Grente (1978), only ensure that suitable seedlings have a minimum number of T. melanosporum mycorrhizae. It was noteworthy that the method of Reyna et al. (2000) did not detect the presence of any contaminant fungi while all the other methods did detect the presence of contaminant fungi. The method used by Fischer and Colinas (1996) was particularly efficient at detecting contaminant fungi (data not shown) because it includes a more exhaustive examination of the complete root system.

Sample freezing limits the effect of detecting variation in colonization percentage due to the time elapsed from the first to the last analysis. Freezing and thawing had little effect on the morphology of mycorrhizae and also enabled a single analyst to carry out all evaluations. This uniformity and the single analyst-based analysis allowed us to make a comparative judgment (in terms of advantages and disadvantages) of the different techniques.

The method of Reyna et al. (2000) is fast, easy, and nondestructive. For a given batch, it also enables analysts to estimate the mean number of root tips per plant, as data relate to a specific volume. The main disadvantage of this method is that it assumes a homogeneous mycorrhiza distribution throughout the container volume when actually a considerable amount of mycorrhizae accumulates on the surface of the container inner walls. Thus, it would be necessary to adjust the cylinder extrapolation to volume and surface. Furthermore, some forestry plant quality parameters cannot be assessed when using this method.

The method developed by Chevalier and Grente (1978) is also fast and easy to implement. However, it is highly subjective, so its validity is linked to the observer’s experience. Only seedling colonization and contaminant fungi percentages are estimated, while direct and objective quantitative data are not obtained.

The grid sampling method of Govi et al. (1995) might affect the quality of samples, since root extraction through the slots marked in the grid can destroy some root tips. Thus, this grid method is not currently used in Italy. It has been modified by Bencivenga (2013); six fragments each from the upper and bottom sections of the root system are collected and 50 root tips from each counted (a total of 600 root tips). Unfortunately, this modified method could not be included in this work.

The method of Palazón et al. (1999) is similar to that of Govi et al. (1995) as it extracts root fragments but without the use of a grid. Although it is easy and fast, it can involve some subjectivity when choosing which root fragments to evaluate. This may be the reason for the higher colonization percentages observed when using this method, surpassed only by those obtained by the method of Reyna et al. (2000). The number of seedlings sampled (0.5 %) also seems low, so this method is only reliable with very homogeneous or large batches.

The method of Fischer and Colinas (1996) appears to overcome all the disadvantages discussed above. It also uses an analysis with high statistical robustness implemented in an easy-to-use spreadsheet. It provides, among other values, an estimation of the total number of root tips for each seedling. Its major drawback is the time needed for seedling evaluation, which is an important factor as it significantly increases the economic costs of plant certification. However, if the seedlings from a given batch have homogeneous characteristics, good black truffle colonization levels, and few contaminant fungi, the number of replicates can be safely reduced to five instead of the 12 seedlings initially required, thereby reducing the time needed for processing a given batch.

Although there remains a lack of consensus on the best seedling evaluation method, we recommend that the method be objective, systematic, and use a well-defined and clear process as is the case for the method by Fischer and Colinas (1996). It should also be easy to implement, in time and equipment and, if possible, nondestructive. The methods of Bencivenga (2013) and Palazón et al. (1999) are relatively less time consuming. Sensitivity and specificity of subjective methods, such as by Chevalier and Grente (1978), improve as the analysts gain practical experience (Sisti et al. 2010).

With the objective of reaching an agreement between all certification organizations, perhaps the best option would be to combine two correlated methods. For example, analysts could use the method of Chevalier and Grente (1978) when the colonization levels are high and contaminant fungi are clearly absent. However, when colonization is low and contaminant fungi are present, a more accurate method should be used to ensure that the colonization percentage and minimum number of colonized root tips of the batch are correctly estimated.

The assessment of contamination by other ectomycorrhizal fungi needs further development. This is especially important to prevent the introduction of T. indicum because this fungus represents a serious ecological risk for European truffle culture (Murat et al. 2008). An ideal new method would also incorporate routine molecular analyses. Molecular techniques that discriminate between morphologically related species within the genus Tuber are reported in the literature. These techniques are based on the use of RFLP (Pérez-Collazos et al. 2010), specific PCR primers (Paolocci et al. 1997; Mabru et al. 2001; Suz et al. 2006), or real-time PCR (Sánchez 2012; Parladé et al. 2013) and analyze the DNA of mycorrhizal root tips or ascocarps. Another option would be the analysis of extraradical mycelium present in the potting mix (Suz et al. 2006; Parladé et al. 2013).

There is an obvious need in Europe and elsewhere to reach agreement on unifying the criteria for truffle seedling evaluation. Consideration of compulsory controls by the European Union to limit the potential invasion of troublesome mycorrhizal fungi such as T. indicum is also needed to protect the truffle industry. Our results provide an important basis for future decisions concerning the unification of methods to estimate T. melanosporum colonization on inoculated Q. ilex seedlings.