Quality assessment of truffle-inoculated seedlings in Italy: proposing revised parameters for certification

Aim of study: The main aims of this study were to evaluate the quality of truffle-inoculated seedlings produced by commercial nurseries in Italy and to identify their minimum requisites in terms of plant age, health, homogeneity, and cut-off percentage of inoculated Tuber and non- Tuber ectomycorrhizae, based on the analysis of an extensive sample of seedlings subjected to quality control and certification. Truffle-inoculated Italian commercial nurseries. Material and methods : Analysis of truffle-inoculated seedlings for health and quality standards; recording of presence of inoculated Tuber spp. and other concurrent fungi according to the official Italian method for certification; selective amplification of ectomycorrhizal DNA by PCR species-specific primers. Main results: We showed that mycorrhization levels in truffle-inoculated seedlings increased with time after truffle-spore inoculation. The highest mean percentage of the inoculated Tuber spp., but also the highest presence of contaminants, were recorded after three years. The mycorrhization level of Tuber melanosporum and T. aestivum was higher in Corylus and Ostrya seedlings than in Q. ilex and Q. pubescens , but the latter two host species showed the lowest presence of other ectomycorrhizal fungi. Mycorrhization level distribution in truffle-inoculated seedlings of suitable batches differed very little from the distribution in only all suitable seedlings. Truffle seedlings with other Tuber spp. were very few and even absent after three years. The general quality of Italian truffle-inoculated seedlings is high but can be improved even further by revising the parameters used for their certification. Research highlights : Mycorrhization assessment in truffle-inoculated seedlings produced by commercial nurseries and a revision of the parameters of quality standards following several years of certification in Italy.

truffle-inoculated seedlings based on both morphological and molecular analyses (Govi et al., 1995;Fischer and Colinas, 1996;Reyna et al., 2002;Miko and GaÏo, 2007;Bach et al., 2010;Alvarado and Manjon, 2013). Each of these methods has prioritized specific issues, making them all different from each other. None of the existing methods establish quality standards specif ic to the host partners or to the Tuber spp. spore inocula, even though this inoculum can unexpectedly represent a source of contamination due to the unintentional inclusion of other Tuber species.
In Italy, as a result of the high economic importance of truffle-inoculated seedlings and their cultivation, some regional governments created specific certifications of mycorrhization levels and plant quality for marketing, out-planting and the establishment of truffle plantations (DGR n. 13429 of 24 May 1996). Since the late 1980s, the research group of the Laboratory of Applied Mycology at the University of Perugia, has carried out analysis and certification of truffle-inoculated seedlings following the off icial national method (Govi et al., 1995). On the basis of acquired knowledge and experience after several years of truffle-inoculated seedling quality control, this method was revised to take into account not only mycorrhization levels but also the plant host and inoculum quality (Donnini, 2005).
The analysis of a large amount of truffle-inoculated seedlings allowed us to: i) have an overview of the overall quality and mycorrhization levels of the seedlings produced by Italian nurseries; ii) evaluate the mycorrhization levels of the inoculated Tuber and different mycorrhizal contaminants (including other Tuber spp.) of suitable and unsuitable seedlings and homogeneous batches following the current methodology; iii) propose new and improved parameters for a fast, standardized and reproducible method for seedling quality certification.

Sample collection
In seven Italian commercial nurseries truffle-inoculated seedlings were sampled from 2006 to 2012 among different batches for quality control and mycorrhization level evaluation according to Donnini (2005). Seedling samples belonged to different plant species: Quercus pubescens Willd., Quercus ilex L., Quercus cerris L., Quercus robur L., Corylus avellana L., Tilia cordata Mill., Ostrya carpinifolia Scop., Pinus pinea L., Pinus halepensis L., Cistus incanus L. and were mycorrhized with Tuber melanospourm Vittad., Tuber magnatum Pico, Tuber aestivum Vittad., Tuber brumale Vittad., Tuber macrosporum Vittad., and Tuber borchii Vittad. A total of 4,737 truffleseedlings (2,986 being one year old, 1,371 two years, and 380 three years) were sampled and included in the analyses. Truffle-inoculated seedling samples differed according to plant species and inoculated truffles as well as pot type and dimension, potting mixes and watering regime. Detailed data on the mycorrhization level of the inoculated Tuber species and concurrent fungi were analyzed only for the most important truffle/host combinations. In particular C. avellana, O. carpinifolia, Q. ilex and Q. pubescens in association with T. melanosporum (2,189 truffle-seedlings) and T. aestivum (1,662 truffle-seedlings) respectively were included. Since a certification method must be standard, collected data were also analyzed all together regardless of the truffle/plant combination and the growth and cultivation conditions.

Truffle-seedling health and quality
The health and quality of truffle-seedlings were evaluated: the stem height, diameter, number of shot branches were measured, while lignification was assessed visually. Root system characteristics (e.g., length and abundance of fine roots, presence of circling roots), foliar health (e.g., leaf color and damage ) and presence of pathogens or pests were observed according to the most recent European rules (Council Directive 1999/105/EC of 22 December 1999).

Morphological and molecular analyses of Tuber spp. ectomycorrhizae
Ectomycorrhizae were identified morphologically following the key of Agerer (1987Agerer ( -2012 and the online key DEEMY (http://www.deemy.de) as guide references. Samples of ectomycorrhizae were randomly collected from the root system of each seedling analyzed, placed in 1.5-mL microfuge tubes containing deionized water, and stored at -20°C for subsequent molecular analysis.
The amplifications were carried out in a final volume of 50-µL containing 10 × PCR buffer (Invitrogen), 200 µM dNTPs, 20 mg of bovine serum albumin, 10 pmol of forward and reverse primer, and 2 U of Taq DNA polymerase (Invitrogen). PCR amplifications were carried out with a first denaturation of 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 60°C annealing for 30 s for UNCI/UNCII, TBA/TBB and TMAGN/TBACK3 (62°C for ITSML and ITSB and ITSCHCH/ITS4) with an extension of 1 min, and with a final extension step at 72°C for 10 min. Negative (no genomic DNA added) and positive (Genomic DNA extracted from different Tuber spp.) controls were added to PCR reactions. Ten Ìl of PCR products were run on 1.8% (w/v) agarose gel stained with ethidium bromide for evaluation of the presence/absence of bands.

Evaluation of mycorrhization levels, suitable and unsuitable truffle seedlings and batches
Differences in the percentage of mycorrhization were evaluated in 1) inoculated Tuber species, 2) other EM fungi (including other Tuber spp.) and 3) other Tuber spp. only, in batches of seedlings 1 year, 2 years and 3 years after truffle-spore inoculation. The analyses were carried out using the official method (DGR n. 13429 of 24 May 1996) and subsequent modifications (Donnini, 2005) which included biomolecular analysis. This procedure abandons the classical counts of ectomycorrhizae in the root system or portions of it and relies on assessment through a visual evaluation of the mycorrhization level in the whole root system, using a stereomicroscope. The estimated percentage of mycorrhization is evaluated with intervals of 5%. The distribution of mycorrhization levels was analyzed combining: 1) all truffle-inoculated seedling samples; 2) only suitable and unsuitable truffle-inoculated seedling samples (A truffle seedling is suitable when it has ≥30% Tuber mycorrhization, ≤ 5% of other EM fungi mycorrhization and the difference between these two percents is ≥20); 3) only truffle-inoculated seedling samples of suitable and unsuitable batches, one, two and three years after spore inoculation. A batch of seedlings is suitable when it has ≥ 80% of suitable samples. The frequency of suitability/unsuitability was calculated by dividing the number of suitable or unsuitable batches by the total number of seedlings. Frequency was also calculated in the following three types of batches: 1) batches with only suitable seedlings, 2) batches with 1 unsuitable seedling, and 3) batches with 2 unsuitable seedlings. Mean mycorrhization values and the frequency of the inoculated Tuber spp., concurrent fungi (including other Tuber spp.), and other Tuber spp., were also calculated for seedlings belonging to suitable and unsuitable batches. No relation between the number of ectomycorrhizae and total number of tips was recorded.

Forest quality
Truffle-inoculated seedling samples belonging to Quercus pubescens Willd., Quercus ilex L., Quercus cerris L., Quercus robur L., Corylus avellana L., Tilia cordata Mill., Ostrya carpinifolia Scop., Pinus pinea L., Pinus halepensis L., Cistus incanus L. species were of good quality with regard to health, stem height and diameter, lignification and number of shots and branches. Detailed data of Q. pubescens × T. melanosporum measurements are reported as a reference in Table 1. In some samples the root system was coiled, but only in those analyzed three years after truffle-spore inoculation (Fig. 1).

Molecular analyses of the inoculated
Tuber spp. ectomycorrhizae spp. used for the mycorrhization declared by commercial nurseries. A single case of contamination with T. brumale on seedlings mycorrhized by T. melanosporum was detected, the entire batch was therefore rejected and declared unsuitable for commercialization.

Mycorrhization standards of all the analyzed truffle-inoculated seedling samples
For 50% of all the truffle-inoculated seedling samples, the mycorrhization level distribution falls bet-ween 20% and 50% after one year (Y1) from inoculation, while it falls between 35% and 60% after two (Y2) and three (Y3) years from truffle-spore inoculation, respectively (Fig. 2). Fig. 3 shows that three years after spore inoculation there was a substantial increment of contamination by concurrent fungi in comparison to after only one and two years.
The mycorrhization level of the truffle-inoculated seedling samples with T. melanosporum was between 20% and 50% in C. avellana and between 35% and 60% in O. carpinifolia. For T. aestivum, the mycorrhization level was between 35% and 65% in C. avellana, and between 30% and 65% in O. carpinifolia. The distribution was therefore similar in Q. ilex and Q. pubescens, both with T. melanosporum and T. aestivum (Fig. 4). Observation of mycorrhization levels in truffle-inoculated seedling samples with concurrent fungi showed that C. avellana and O. carpinifolia were the most susceptible host species. In contrast, only outliers were detected in Q. ilex and Q. pubescens (Fig. 4).
The batches comparisons showed that suitable batches increased from 56.5% after one year to 82.3% after two and 66% after three years from inoculation. Similarly, batches with all suitable truffle-inoculated seedling samples as well as with one or two unsuitable ones increased ( Table 2). The inoculated Tuber spp. was always present in the suitable seedling samples regardless the year when they were analyzed.
The frequency of seedlings contaminated by concurrent fungi increased consistently from ≈12% to ≈13% and even to ≈25% after one, two and three years from spore inoculation, respectively (Table 3). Mean levels Quality assessment of truffle-inoculated seedlings in Italy 389 Figure 2. Box plots illustrate the medians, upper and lower quartiles, the range and extreme values of the mycorrhization level distribution in: all the truffle-inoculated seedling samples collected in the commercial nurseries after one (1Y), two (2Y) and three years (3Y) from spore inoculation; only the suitable truffle-seedling samples after one (1YS), two (2YS), three years (3YS) from spore inoculation; only the truffle-seedling samples of suitable batches after one (1YSB), two (2YSB), three years (3YSB) from spore inoculation; only the unsuitable truffle-seedling samples after one (1YU), two (2YU) and three years (3YU) from spore inoculation, and only the truffle-seedling samples of unsuitable batches after one (1YUB), two (2YUB) and three years (3YUB) from spore inoculation according to the official method of certification in Italy.   of mycorrhization in truffle-inoculated seedling samples belonged to suitable batches increased from ≈46% to ≈49% and even ≈54% after one, two and three years from spore inoculation, respectively (Table 3).
The mycorrhization levels of concurrent fungi was in general low, but lowest (≈ 1.5%) after two years from spore inoculation. Moreover, their frequency was similar after one (≈12%) and two (≈13%) years from 390 D. Donnini et al. / Forest Systems (2014) 23 (2): 385-393  spore inoculation it and doubled after three years (≈25%). It was worth nothing that in unsuitable truffle-inoculated seedling samples mean mycorrhization level with the inoculated Tuber spp. was ≈34% after three years from spore inoculation, even higher than the cut-off value of 30% identified for suitable seedlings. Anyway, for the same year, the mean mycorrhization level of concurrent fungi was ≈21% and their frequency ≈77%. In suitable batches other Tuber spp. mean mycorrhization level and frequency were very low and even absent after three year from spore inoculation. The highest presence of other Tuber spp. in suitable seedlings was recorded after two years from spore inoculation (≈2%) with a mean mycorrhization of 0.16% (Table 3).

Concerning the quality standards of truffle-inoculated seedlings produced in Italy
The forest quality of the seedlings was in general very good and in agreement with European law for forest propagation material (Council Directive 1999/105/EC of 22 December 1999). Nevertheless, the main problem detected was the presence of coiled root systems in truffle-seedlings after three years from spore inoculation. Plants with severe circling roots close to the trunk can eventually slow growth and girdle the trunk making the tree unstable to winds (Gilman and Sadowsky, 2007). Truffle-inoculated seedlings with malformed root, even with high mycorrhization level, were therefore discarded like unsuitable ones.
The mycorrhization level of the inoculated Tuber spp. increased with time from spore inoculation regardless of whether the truffle-inoculated seedling samples were analyzed altogether, divided in suitable and unsuitable or even if analyzed in suitable and unsuitable batches. Nevertheless, with age, the seedlings also increased their contamination with concurrent fungi: the highest frequency and mean mycorrhization levels were therefore registered after three years from spore inoculation. At this age more seedlings showed higher inoculated Tuber spp. mycorrhization level but most of them had also a percentage (even small) of concurrent fungi.
The mycorrhization level of suitable batches was not dramatically lower than that of all suitable truffleseedlings: less than the 25% of observations of suita-ble batches was less than the minimum value for the suitable truffle-inoculated seedlings.
Different host species have diverse responses to inoculation treatments in terms of truffle mycorrhization and presence of concurrent fungi. In particular the range of mycorrhization level distribution of C. avellana and O. carpinifolia is higher than of Q. ilex and Q. pubescens, but the distribution range of concurrent fungi is narrower in the last host species. This was also more evident if only truffle-inoculated seedling samples of suitable batches were included in the analysis, meaning that the quality standard adopted can upgrade the general truffle-inoculated seedlings quality.

Proposing revised parameters for truffle-inoculated seedling certification
With this work new parameters for truffle-inoculated seedling quality as well as a modification of the current Italian method for their certification were proposed (Table 4). We believe that an ideal method must be fast and simple, and needs to reflect the effective quality of truffle seedling batches. The screening by visual estimation of the entire root system allowed us to identify contaminants that are not always detected by random sampling of root portions. Moreover, visual estimation of mycorrhization levels had already proven its worth: no statistical differences between visual and root tip counting-based assessments had been reported (Benucci et al., 2012a).
The taxonomic identification of truffle-inocula can help to avoid contamination with other Tuber spp. accidentally introduced in spore-slurries preparation. Although low percentages of other Tuber spp. were also detected in suitable batches, these were discarded to reduce possible co-generic competition after seedling outplanting. The pre-screening of the rootsystems of randomly selected young seedlings immediately before inoculation is recommended to reduce the presence of concurrent fungi [e.g., Sphaerosporella brunnea (Alb. & Schwein.) Svrcek and Kubicka, Pulvinula constellatio (Berk. & Broome) Boud.] that usually occurs during the first phase of growth in climatically controlled greenhouses Amicucci et al., 2001;Benucci et al., 2012c). The minimum limit of 30% mycorrhization with the inoculated truffle is confirmed for truffle-inoculated seedling suitability, as recommended by the official method (DGR n. 13429 of 24 May 1996).
The limited incidence of concurrent fungi and their low value of mean mycorrhization in both suitable and unsuitable truffle-inoculated seedling batches found in this study, demonstrated the careful work of the commercial nurseries, and allowed us to reduce their presence from 15% to < 10%.
Concerning batch suitability, in order to validate the batch examined only one unsuitable seedling at most is acceptable in the analyzed sample. Despite the fact that two years after the truffle-spore inoculation we had the best balance between the frequency and the mycorrhization levels of the concurrent fungi and the inoculated Tuber spp., no limitations of specific mycorrhization levels or ages were adopted. We believe that only the distinction between suitable and unsuitable truffle-inoculated seedlings is important for truffle cultivation, no concerns regarding higher or lower quality can therefore be contemplated. Similarly, within seedling root systems we did not foresee any minimum number of inoculated Tuber spp. mycorrhizae with respect to all the root tips present. Although non-mycorrhizal tips can easily be contaminated by soil-resident fungi, the real connection between the mycorrhization level and truffle production is as yet unknown (Horton and Bruns, 2001).
Finally, the biomolecular analysis for the identification of the inoculated Tuber spp. must be the fastest and cheapest possible. Reliable , simple techniques like the use of species-specific PCR primers for selective amplifications are recommended (Rubini et al., 1998;Mello et al., 2002;Benucci et al., 2011). We discourage laborious procedures like cloning previously amplif ied products and sequencing, as required by other methods of truffle-inoculated seedling certification (Alvarado and Manjon, 2013).
This experience is proposed as a stimulus for discussion on the potential establishment of an official European method for truffle plant certification which, as technicians and professionals have argued for many years, would be a way of protecting truffles and truffle-farmers.

Ripeness
Well ripe truffles have well-formed germinable spores.

Species
The correct taxonomic identification is essential to avoid contamination from other truffles.

Quality of the host plant
Habitus Size, stem height, diameter, number of shot branches need to be in proportion to age.

Lignification
Lignified stem and branches to resist the winter after planting.

Health
Good phytosanitary status, absence of pathogens or pests.
Root systems Well-formed, equipped with numerous lateral roots.

Quality of truffle-inoculated seedlings
Pre-screening Inspection of the root system before inoculation to evaluate the absence of concurrent fungi.
Identification of -Visually under stereomicroscope and light microscope according to their specific morectomycorrhizae phological characteristics. -Molecular analyses through DNA markers to verify consistency of the inoculated Tuber spp. and to detect contamination.
Suitability requirements -Visual evaluation of mycorrhization levels in the whole root system (or counting of for truffle-inoculated first 50 root-tips in 6 proximal and 6 distal root portions). seedlings -The percentage of Tuber ≥ 30%.
-Difference between percentage of inoculated Tuber spp. and concurrent fungi ≥ 30.
Suitability requirements -Homogeneous (same species, age, truffle inoculum and growth conditions) no more for batches than 500 seedlings. -Samples of 1% plus 5 truffle-inoculated seedlings randomly collected.
-Only one unsuitable truffle-inoculated seedling is allowed in the same sample.