BIOSTIMULANTS AND INDOLEBUTYRIC ACID IMPROVE ROOTING OF WOOD CUTTINGS FROM DIFFERENT GRAPEVINE ROOTSTOCKS

The rooting of rootstocks is considered a critical point in the production of high-quality grapevine planting material. To maximize the cutting rhizogenesis process, plant hormones, such as auxins, are used for the development


INTRODUCTION
It is generally accepted that the phylloxera (Daktulosphaira vitifoliae) was unintentionally introduced into Europe at the end of the nineteenth century through infected plant material from North America to fight oidium (powdery mildew), the fungus that threatened European vineyards in the 1850s (Macedo, 2011). Grape phylloxera occurrence almost destroyed European viticulture, and it provoked the most radical switch in viticultural practices of the last two centuries, when grape growing changed from the use of own-rooted 'V. vinifera' plants to their grafting onto partially resistant American non-vinifera Vitis spp. or hybrids used as rootstocks (This et al., 2006). Therefore, resistance to phylloxera was the first selection criterion for grapevine rootstocks (Whiting, 2012).
Rootstocks influence water relations, phenology, vigor, production and quality of grapes and wines (Keller, 2020). The choice of rootstock is aimed at adaptation to abiotic stresses such as salinity, drought, acidic, alkaline, low-fertility and shallow soils, as well as to biotic stresses (Smith et al., 2017). It is appropriate that the rootstock has characteristics such as vigor, deep root system, resistance to pests (such as nematodes) and diseases (Waite et al., 2015). It must root and heal easily on grafting and present satisfactory development in adverse conditions. Rootstock for Brazilian conditions must have characteristics such as tolerance to acidic soils, to aluminum and excessive humidity, traits like resistance to fusariosis (Fusarium oxysporum Sch. f.sp. herbemontis) and Brazilian ground pearl (Eurhizococcus brasiliensis) are also desirable (Fráguas, 1999;Borghezan et al., 2003;Leão et al., 2020).
Before grafting, the rootstock is propagated by cuttings. Cutting is one of the most important methods of propagating fruit planting material and is based on the principle that it is possible to regenerate a plant from a part of the mother plant, through tissue dedifferentiation (which means to return to an undifferentiated state); the root formation process in cuttings is affected by several factors, which can act alone or together. Among the main ones, the following stand out: genetic variability, physiological condition of mother plant, plant age, cutting type, time of year, environmental conditions and substrate (Pires and Biasi, 2003).
The rooting of rootstocks is considered a critical point in the production of high-quality grapevine planting material, but some substances produced by plants can help this process to occur in the best way. Among the different compounds produced by plants, hormones can be highlighted, which are naturally occurring organic and non-nutrient compounds, which are produced by the plant itself, and which, in low concentrations, promote, inhibit or modifies plant morphological and physiological processes (Vieira, 2010). Therefore, to maximize the cutting rhizogenesis process, plant hormones, such as auxins (indolebutyric acid), are used for the development of adventitious roots and, above all, to increase the percentage of rooting, through accelerating the emission of uniform and quality roots (Baldotto and Baldotto, 2014).
The indolebutyric acid (IBA) works to stimulate the formation of root primordia; its characteristics are low mobility, low photosensitivity, high chemical stability and low toxicity for most plants (Pires and Biasi, 2003). Its action results in a high percentage of rooting of cuttings and shortens the period of cuttings in the nursery (Bastos et al., 2009).
In addition to the plant growth regulators, biostimulants can also be used for this purpose. They are defined by many authors as natural or synthetic substances, arising from the mixture of two or more plant bioregulators or with other substances such as amino acids, nutrients and vitamins. Substances and microorganisms capable of altering plant metabolism, favoring its development, can also be included (Calvo, et al., 2014;Du Jardin, 2015).
Among the viticultural management processes, the application of biostimulants to grapevines to avoid specific diseases and to improve grape and wine quality is one of the most common practices performed by wine-growers that provides several benefits to the grapevines. These substances can stimulate growth and the synthesis of secondary metabolites, which in turn can provide protection against biotic and abiotic stresses (Gutiérrez-Gamboa et al., 2018a). Some biostimulant groups have as their main role the development of root system, ensuring good growth of plant aerial part and ensuring satisfactory yield; generally, its composition includes natural phytohormones, such as auxins, cytokinins and gibberellins, amino acids, zinc and magnesium sulfates, iron citrate, boric acid, ammonium molybdate, among other macro and micronutrients (Santos et al., 2013;Tecchio et al., 2015).
Among the natural substances to which biostimulant effects have been ascribed, the use of algae extract in agriculture is being described in several studies . Seaweed extracts have macro and micronutrients, amino acids and plant hormones in their composition; it may have auxins, cytokinins, gibberellins and abscisic acid, and compounds capable of stimulating the production of these hormones by plants (Sharma et al., 2012). The application of products based on algae extract provided positive results, for example, in increasing tomato productivity, production and quality of grapes under alkaline soil conditions, induction of resistance to water deficit in soybean and orange, resistance to pathogen attack in cucumber ; an Ascophyllum nodosum extract applied to 'Sangiovese', 'Pinot Noir' and 'Cabernet Franc' grapevines improved anthocyanin accumulation in all of these cultivars (Gutiérrez-Gamboa et al., 2018a). Also, brown algae, plus boron and zinc applications before the beginning of flowering can allow to improve fruit set, which would affect positively 'Carménère' productivity at harvest (Gutiérrez-Gamboa et al., 2018b) The use of seaweeds in viticulture has allowed to improve grapevine productivity and to enhance grape and wine quality, mostly in terms of their phenolic composition. Their effectiveness seems to be mainly associated to seasonal climatic conditions, to the species of seaweed applied, its structural properties, leaf surface wetting properties of the grapevine variety, the stress and resistance level of the grapevine prior to the application, dosage of the formulation and their implications on relative expression genes in leaves and berries (Gutiérrez-Gamboa and Moreno-Simunovic, 2021).
However, research using algae extract-based biostimulants in vegetative propagation of vine rootstocks is scarce. In this sense, the aim of this study was to evaluate the use of a biostimulant based on algae extract, indolebutyric acid (

MATERIALS AND METHODS
The work was carried out at Santa Catarina Federal University (UFSC), at the Center for Agricultural Sciences (CCA), located in Florianópolis/SC, Brazil, from August to November 2019 and 2020. The plant material had approximately one year and was collected in the period of dormancy (August) of mother plants kept in the germplasm collection of Embrapa Clima Temperado -Canoinhas Experimental Station.
The evaluated rootstocks and their respective genealogies were: '3309 C' and '101- The plant material was segmented into cuttings approximately 30 cm long, selecting those with 8 to 10 mm in diameter. At the base of each cutting, a transverse cut was made, 0.5 cm below the last bud, and at the cutting apex, a cut was made in a bevel, 3 cm above the last bud. Thereafter, the cuttings underwent stratification in a cooling chamber, with an average temperature of 2 ºC ±1 ºC, for 10 days. Afterwards, two lesions were made in the longitudinal direction, approximately 3 cm long, at the base of each cutting (Bettoni et al., 2015).
The cuttings were placed in 8 L pots containing a substrate consisting of a mixture of sand and commercial substrate (Plantmax®). The cuttings were kept in a greenhouse, without temperature control and irrigated two times a week until the experiment was evaluated. In this experiment it was tested four treatments to stimulate rooting (IBA, algae extract, IBA + algae extract and control) and they are described below.
Immersion 2 cm of cutting basal end in a 3000 mg/L solution of indolebutyric acid (IBA) for 20 sec. The IBA was dissolved in 70% alcohol and distilled water was added until the desired concentration was reached. Application of 100 mL of algae extractbased biostimulant SprintAlga TS® (Biolchim) at a concentration of 0.4 mL/L of solution, 25 and 40 days after planting the cuttings. The solution was applied at cuttings base, directly on substrate. The combination of 3000 mg/L of indolebutyric acid (IBA) applied in the form of immersion at cutting base on the day of experiment installation and 0.4 mL/L of SprintAlga TS® (Biolchim) solution, 25 and 40 days after planting the cuttings applied directly on substrate. The control consisted in immersion two centimeters of cutting basal end in distilled water.
The evaluations were carried out approximately 50 days after the last application of biostimulant, and the percentage of survival and rooting of cuttings was determined, through a visual analysis, verifying if the tissues were intact and alive.
Then, the total number of roots emitted from cutting base was counted, as well as the measurement of the largest root length (cm), with the aid of a graduated ruler. The number of leaves emitted, and the shoot length (cm) were also assessed.
The destructive evaluations were carried out in the laboratory, where fresh and dry mass of leaves and roots were determined. The fresh mass was measured on a precision scale (Bel Engineering, Precision balance L, Brazil), and the dry mass was obtained after drying the material in a forced air oven (QUIMIS Q317M, Brazil) at 70 °C for 72 hours.
The experimental design was completely randomized with four replications, arranged in a 17 x 4 factorial scheme, involving 17 rootstocks associated with four treatments to stimulate rooting (IBA, algae extract, IBA + algae extract and control), with three cuttings per plot, totaling 816 cuttings. The data obtained were submitted to the Scott-Knott test (p<0.05). Survival and rooting percentage data were . transformed into arc sine √x/100. The data of root number and shoot number were transformed into log (x + 1) and submitted to statistical analysis.
In fact, most vine rootstocks do not present great difficulties in rooting when propagated by woody cuttings, but when this is done, this characteristic is inherited from their parents, mainly from the species Vitis riparia and Vitis rupestris, which root easily (Willians and Antcliff, 1984). This result confirms the good rooting capacity of Vitis riparia species and its hybrids, such as 'Kober 5BB' and 'SO4', when propagated by woody cuttings (Monteguti et al., 2008). In previous studies, different responses between cultivars regarding the ability to form adventitious roots in cuttings were observed. One of the main explanations for this behavior is the genetic constitution of the cultivars, which would result in a different rooting potential between them (Dutra et al., 1998).
In the present work, the rootstock 'Paulsen 1103' showed low survival and rooting rates; these results differ from those reported by other researchers. Previous works stated that 'Paulsen 1103' is easy to root, being quite adaptable to various types of soils, and also that the use of IBA is unnecessary, as it does not induced significant differences (Bordin et al., 2005). It is believed that the low rooting and survival rates observed for this rootstock are related to specific conditions of the mother plant from which the cuttings were taken. Factors such as cutting diameter, degree of lignification and concentration of endogenous hormones can markedly interfere the rooting (Hartmann et al., 2011).
Hybrid rootstocks, such as 'Richter 99' and 'VR043-43', have potential for cultivation, as they present a genotype that is tolerant or resistant to the etiological agents of vine decline and dieback. The 'Richter 99' ('V. berlandieri' x 'V. rupestris') is recommended for regions with fusariosis occurrence (Garrido et al., 2004). In southern Brazil, the rootstock 'VR 043-43' is widespread due to its tolerance to fusariosis and Brazilian ground pearl (Eurhizococcus brasiliensis) (Broetto et al., 2011;Souza et al., 2015). The hybrid between vinifera x rotundifolia 'VR043-43' is especially difficult to root dormant cuttings. Because 'Vitis rotundifolia' different number of chromosomes from others vitis species, presenting a lower development rate when traditional propagation techniques are used. The causes of low rooting are possibly related to the presence of inhibitors or the absence of promoters (Pires and Biasi, 2003). However, it is possible to find higher rooting rates of the VR043-43 rootstock when herbaceous cuttings are used and growth regulators are applied (Lone et al., 2010).
Woody cuttings from rootstocks '3309 C', '101-14 Mgt', '99 R' and 'Freedom' produced the highest number of roots, between 14 and 16, while cuttings from rootstocks 'Paulsen 1103', '420 A Mgt', 'IAC 313', 'Salt Creek' and 'VR 043-43' produced less roots, between 1.8 and 4.1 (Table I). In a study that evaluated the same rootstocks, the highest number of roots was produced by rootstocks '101-14 Mgt', '3309 C', 'Gravesac' and 'Harmony', and the lowest number of roots was observed in '110R', '420 A Mgt' and 'VR 043-43' (Würz et al., 2022), thus complementing the results of the present work. The longest root length, between 19.5 and 20.7 cm, was obtained in 'IAC 572' and 'Freedom' rootstocks, while the shortest root lengths were presented by the 'Paulsen 1103', '420 A Mgt' and 'VR 043-43' rootstocks, between 2.8 and 6.0 cm (Table I). According to Regina et al. (2012), these results indicate that '420 A Mgt' was not recommended to produce rooted vine cuttings by the table grafting technique. In addition, the authors also indicated low rooting potential of this rootstock. Similar results were also obtained by Würz et al. (2022) when evaluating the rooting potential of different vine rootstocks.
Regarding the effect of treatments, results of Table  III show that cuttings submitted to the control treatment presented significantly lower results than the other treatments, which in turn did not differ from each other. These outcomes reveal a positive effect of phytohormones and biostimulants used in the vegetative propagation of vine rootstocks.
Auxins play an important role in controlling the growth and development of the plants; besides, they also influence the production of primary, secondary, and adventitious roots (Šebánek, 2008). Some of the processes regulated by IBA include formation of embryo in development, induction of cell division, stem and coleoptile elongation, apical dominance, induction of rooting, vascular tissue differentiation, fruit development, and tropic movements such as bending toward light (Kumar et al., 2019). The historical use of IBA phytohormone has been widely used in vegetative propagation, both for rootstocks and for grapevines. Wide bases of studies corroborate what was observed in the present work, in which the use of this hormone proved to be efficient in the rooting and vegetative development of woody and semi-woody grapevine rootstock cuttings (Doğan et al., 2019;Uddin et al., 2020).
In some studies, the use of the synthetic hormone IBA did not show a significant effect in increasing the rooting index of 'IAC 766' rootstock (Driusso and Trevisan, 2020). However, the use of this technique showed a positive influence on the development of shoots and roots produced from rootstock cuttings (Bettoni et al., 2014;Uzunoğlu and Gökbayrak, 2018). It is also worth mentioning that the 3000 mg/L concentration used in this study is considered efficient for a range of materials; the use of less diluted solutions tends to reduce the rooting efficiency of woody rootstock cuttings (Salibe et al., 2010).
Regarding the effect of algae extracts in vegetative propagation protocols, studies have shown that such products promote the development of quality planting material in rose and dragon fruit (Parađiković et al., 2019;Freitas et al., 2021). Significant results using biostimulants in the 6 production of planting material have also been observed in cuttings multiplication of species that are difficult to propagate. The use of these products showed an encouraging performance compared to the use of rooting inducers already consolidated (Trofimuk et al., 2020). In a previous work, the use of algae extract-based biostimulant promoted a significant increase in shoot length, root density, root area, root length, root dry matter and shoot dry matter of cv. 'Crimson Seedless' (Ribeiro et al., 2017). The combination of IBA and biostimulants also proved to be efficient in rooting other fruit species (Freitas et al., 2021).  According to the results of the analysis of variance (p value < 0.05), it can be stated that there was a statistical difference for the variables analyzed as a function of the different rootstocks. Thus, it is possible to state that this parameter influences all analyzed variables. For the treatment parameter, there was a statistical difference in all analyzed variables, except root dry mass. Concerning the rootstock x treatment interaction, no significant effect was observed for the analyzed variables (Table  IV).

CONCLUSIONS
The use of indolebutyric acid (IBA) and algae extract-based biostimulant proved to be efficient in rooting woody cuttings from different vine rootstocks.
The use of growth regulator, biostimulant and IBA combined with algae extract-based biostimulant resulted in higher rates of rooting and vegetative development than the control.
The rootstocks showing the highest rooting rates and the highest vegetative development were 'IAC 572' and 'Freedom'. 'Gravesac', '101-14 Mgt' and '99 R' exhibited higher rates of rooting and root development, but less shoot development.
The rootstocks presenting the lowest rooting rates and the lowest vegetative development were 'Salt Creek', 'VR 043-43' and '110 R'.