Enhancement of the Tuscan grapevine germplasm: Nocchianello Bianco and Nocchianello Nero cultivars

As part of a decades-long project to safeguard the viticultural biodiversity in the wine-growing district of Pitigliano (Tuscany, Italy), the research activity carried out made it possible to rediscover two valuable autochthonous grapevine varieties, otherwise doomed to extinction: Nocchianello Bianco (whiteberried) and Nocchianello Nero (black-berried). Both the cultivars were thoroughly characterized at the genetic, phenological, and ampelographic levels, as well as the yield and grape quality were evaluated. Thanks to the results obtained, Nocchianello Bianco and Nocchianello Nero were officially reintroduced in the local and regional winemaking scenario, following the registration in the Italian Catalogue of Grapevine Varieties and the addition to the list of grapevine varieties suitable for cultivation in Tuscany.


Introduction
Nocchianello Bianco and Nocchianello Nero are two old autochthonous grapevine varieties recovered in the area around Pitigliano (Tuscany, Italy), where an important project to preserve the viticultural genetic heritage was carried out in the last decades of the past century [1]. The vine-growing area included in the renowned "Maremma Toscana" territory has a very remote tradition that dates to the Etruscan period [2] and has not undergone substantial changes until the phylloxera invasion at the end of 1800. Subsequently, in the 1960s, the renewing process of the vineyards with allochthonous international cultivars and the transition to specialized cultivations led to a further reduction of the viticultural biodiversity. Even the production regulation of the Bianco di Pitigliano denomination of controlled origin wine, widely used in this wine district, has further helped reduce the cultivated varieties, limiting them mainly to Trebbiano Toscano, Verdello, and Malvasia Bianca Lunga [3].
The information about Nocchianello Bianco and Nocchianello Nero in the literature is very scarce; according to the opinion of elderly local winegrowers, the etymology of both cultivar names could derive from the Italian word "nocca" or "noccola" (meaning knuckle), probably referring to the bunch shape, as it resembles a hand closed in a fist.
Exploiting the existing grapevine germplasm collection created as part of the previous recovery project [1], we recently pursued the research activity to confirm the genetic fingerprinting of Nocchianello Bianco and Nocchianello Nero grape cultivars and to evaluate their ampelographic, phenological, productive, and qualitative characteristics ahead of the safeguard from extinction and a relaunch on the local winemaking scenario.

Experimental vineyard characteristics
Nocchianello Bianco and Nocchianello Nero vines were planted in 1995 in a vineyard collection set up in the municipality of Pitigliano (Grosseto, Italy).
The vineyard had a medium dough of volcanic origin soil (tuff and pumice) with a sandy texture and no active limestone. The vines were grafted on SO4 rootstock and were grown with a single high wire trellis system.
In this area, the climate is characterized by low minimum temperatures during winter and intense heat with low relative humidity in summer. Pest management was normally scheduled with calendar sprays at 10-day intervals.
The ampelographic characteristics of the main vine organs were described according to the recommended OIV -International Organisation of Vine and Wine methodology [9], and the ampelometric measurements on adult leaves were carried out using the software SuperAmpelo [10].

Yield and grape quality
Yields and grape qualitative data were evaluated for three consecutive years, corresponding to 2015-2017 vintages. At the harvest, the main production traits were determined: yield per vine (kg), clusters number per vine, cluster weight (g), and berry weight (g). Then, the technological maturity of grapes (sugar content -°Brix; total acidity -g/L tartaric acid; pH) was assessed. The parameters observed were compared with Trebbiano Toscano and Sangiovese, two Tuscan (white-berried and red-berried, respectively) grape varieties present as reference cultivars in the same experimental vineyard.
Lastly, Nocchianello Nero berry skins extracts were used to analyse the anthocyanin profile by HPLC following the method of [11]. Also in this case, a comparison with Sangiovese was carried out to check the typical differences of the variety under observation.

Genetic identification
According to the molecular results, Nocchianello Bianco and Nocchianello Nero were confirmed as two independent genotypes with unique microsatellite profiles (Table 1), perfectly matching the reference SSR profiles already present in the CREA-VE molecular database.
Despite the homonymy, Nochianello Bianco and Nocchianello Nero turned out to be genetically distant from each other; except for the identical allele lengths in the locus VVS2, they share only one allele in few other SSR loci (highlighted in bold in Table 1).
From further observations (data not shown), it was seen that Nocchianello Bianco has a higher number of alleles overlapping Trebbiano Toscano SSR profile, and the same happens between Nocchianello Nero and Sangiovese, suggesting a rather close relationship between the respective pairs of grapevine varieties.

Vine phenology
In both the cultivars considered, bud break (DOY range 91-100) and flowering (DOY range 145-161) dates occurred on average in overlapping periods (Fig. 1) In general, the trend of the phenological growth stages of Nocchianello Nero is similar to Sangiovese but with a delay of about ten days in reaching full ripeness. Nocchianello Bianco has phenological growth stages comparable to Trebbiano Toscano, but it completes grape maturation about ten days before (data not shown).

Ampelography
Nocchianello Bianco has a medium-sized, pentagonal, five-lobed leaf blade with strong blistering on the upper side and a twisted profile. The bunch is medium-long and narrow, with medium density and conical shape with 1-2 wings. The berry is globose, medium-short in length and width, and has yellow-green color. The pulp is juicy, colorless, and contains an average of 2 seeds. The skin has medium thickness and is slightly waxy (Fig. 2 A-C). Nocchianello Nero has a medium-large, pentagonal, five-lobed leaf blade with a wavy edge, strong blistering on the upper side, and twisted profile. The bunch has medium length, is narrow, dense, conical or funnelshaped with 1-2 wings. The berry is globose, with medium size and blue-black color. The pulp is colorless, juicy, and contains an average of 2 seeds. The skin is thick and very waxy (Fig. 2 D-F).

Yield and grape quality
Nocchianello Bianco and Nocchianello Nero have shown good fertility, with medium vigor and regular grape productions in the different vintages considered ( Table  2). Nocchianello Bianco was less productive than Trebbiano Toscano, with a lower yield per vine mainly due to a lower cluster weight (Table 2a). Nocchianello Nero also revealed a significantly lower grape production with respect to Sangiovese, which on average has a higher number of clusters per vine (Table 2b).
Both the cultivars have always reached appropriate sugar contents, maintaining good values of total acidity and pH, within an optimal range for winemaking. In detail, Nocchianello Bianco displayed must technological maturity characteristics similar to Trebbiano Toscano (Table 2a), while Nocchianello Nero had higher values of total acidity (with corresponding lower pH) than Sangiovese (Table 2b), an optimal feature given the growing environment, where the climate is abundantly warm during the ripening period.
Nocchianello Bianco has proven to be a variety exploitable for blending with local cultivars (for example, Trebbiano Toscano), perfectly suitable for the wines traditionally produced in the Pitigliano wine district.
On the contrary, Nocchianello Nero, given its interesting anthocyanin profile (Table 3) very different from Sangiovese and characterized by a prevalence of malvidin (64%) and a high content of trisubstituted anthocyanins (86%), turns out to be proper for monovarietal winemaking. These features enable predicting high color stability in wines and, hence, great aptitude for medium-long aging.

Conclusions
The research undertaken has allowed completing the recovery activity, reaching the formal recognition of two valuable grapevine cultivars belonging to the Tuscan viticultural heritage, otherwise doomed to extinction. As a result, in 2017 Nocchianello Bianco and Nocchianello Nero were officially added to the Italian Catalogue of Grapevine Varieties [12] with the codes 858 and 859. Then, since 2018, both the cultivars were included in the list of grapevine varieties suitable for cultivation in the Tuscany region. The reintroduction of these two ancient autochthonous cultivars can be considered an important achievement, not only for the protection of the local grapevine biodiversity but also for the enhancement of the viticultural tradition linked to the territory of origin.