Effectiveness of Azospirillum Brasilense Sp245 on Young Plants of Vitis Vinifera L

Open Life Sci. 2017; 12: 365–372 Research Article Susanna Bartolini*, Gian Pietro Carrozza, Giancarlo Scalabrelli, Annita Toffanin Effectiveness of Azospirillum brasilense Sp245 on young plants of Vitis vinifera L. https://doi.org/10.1515/biol-2017-0042 Received April 15, 2017; accepted June 20, 2017 1 Introduction Abstract: Information about the influence of the plant Azospirillum spp. represents one of the best-characterized growth promoting rhizobacteria Azospirillum brasilense free-living diazotrophs among plant growth-promoting Sp245 on the development of grapevine plants could be rhizobacteria (PGPR), able to colonize hundreds of promoted to enhance sustainable agricultural practices plant species [1]. Azospirillum are proteobacteria mainly for globally important fruit crops such as grapes. Thus, associated with the rhizosphere of important cereals this study was initiated to evaluate the potential influence and other grasses; they stimulate the plant growth of A. brasilense Sp245 on two separate experimental trials, through several mechanisms including nitrogen fixation, A) potted young grape plants: cv. ‘Colorino’ grafted onto production of phytohormones and molecules with two rootstocks 420A and 157/11 which received a fixed antimicrobial activity [2]. Azospirillum brasilense was volume of inoculum at different times of vegetative cycle; additionally shown to induce remarkable changes in root B) hardwood cuttings from rootstock mother-plants of morphology [3]. A. brasilense Sp245 was initially isolated 420A and 775P inoculated during the phase of hydration, from the rhizosphere of wheat (Triticum aestivum L.) in before bench-grafting in a specialized nursery. Overall, our Brazil [4] and has been considered a promising strain for results revealed that A. brasilense Sp245 has considerable cereal inoculation during the 1980s. In recent decades, potential to enhance the root apparatus and vegetative due to its strong effect as a plant growth promoter and development in grapevines. Potted plants inoculated with influence on root architecture, A. brasilense Sp245 has A. brasilense Sp245 showed an improved development. been tested in several studies aimed at improving growth Repeated supply was revealed to be more effective, of woody plants, including the propagation of fruit inducing a greater accumulation of dry mass. Interesting rootstocks [5,6] and grapevines [7,8]. Grapevine vegetative findings were obtained in nursery experiments resulting multiplication is based on rooting of cuttings, and usually from a considerable enhancement of the final quality performed in nurseries. The rhizogenetic process leads to of roots. The stimulating effect of A. brasilense Sp245 new adventitious lateral roots, which can regenerate the on vegetative development of rootstocks and vines may plant. Since the late 19th century, modern Vitis vinifera indeed improve sustainability in viticulture, through the cultivars have been grafted onto selected rootstocks, which promotion of healthy growth and a reduced dependence are resistant to insect pathogen, grape phylloxera (Viteus on fertilizers and other chemicals. vitifoliae). Since then, a wide variety of rootstocks have been selected, obtaining hybrids of American species (V. Keywords: grapevine, Colorino, rootstock, 420A, 157/11, berlandieri, V. riparia and V. rupestris), to tolerate diverse 775P, PGPR, bacterial inoculums soil and climate conditions. They are now produced by nurseries worldwide. Some rootstocks, though widely used for their positive characteristics, do not root easily, thus causing significant loss in the production of vines. Additionally, there are numerous other viral, fungal, and insect diseases of grapevine propagation material, which *Corresponding author: Susanna Bartolini, Institute of Life threaten nursery production. Hence, wide application of Sciences, Scuola Superiore Sant’Anna, Piazza Martiri della Libertà chemical compounds, such as fertilizers, pesticides and 33, 56127 Pisa, Italy, E-mail: [email protected] synthetic plant hormones, are being used in conventional Gian Pietro Carrozza, Giancarlo Scalabrelli, Annita Toffanin, Diparti- nurseries to overcome these events. Azospirillum may mento di Scienze Agrarie, Alimentari e Agro-ambientali (DiSAAA-a), Università di Pisa, Via del Borghetto 80, 56124 Pisa, Italy have a role in this context as producers of plant hormones, Open Access. © 2017 Susanna Bartolini et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution- NonCommercial-NoDerivs 4.0 License. 366 S. Bartolini, et al. such as mainly auxins, which are well known to positively but received the same volume of water. For each grafting influence the rooting process. Therefore, it would likely combination, 90 plants were used. Of the first batch, 45 improve the performance of rootstocks, particularly plants were used for the first-year observations, and the those which have difficulty rooting. Our understanding remaining plants were left over the successive dormancy of the influence of A. brasilense Sp245 on development season for spring observations. These latter plants were of grapevine plants is still poor, and should be promoted pruned at the end of the first vegetative year, leaving in order to enhance sustainable agricultural practices on 2 buds per spur. A randomized block design (3, with 5 worldwide important fruit species such as grapevines. replications ) was adopted. Thus, this study was conducted to evaluate the potential Treatments for adequate development of plants (T1, influence of Azospirillum brasilense Sp245 on nursery T2, T3), such as control of weeds, fungicide application propagation of rootstocks and during the vegetative and irrigation were also performed. Since bud burst, the development of young grapevine plants. shoots were tied and maintained in an upright position, thus ensuring an equal distribution of solar radiation. From the beginning of bud burst to plant senescence, 2 Materials and Methods periodic observations (from 15 to 3-4 days intervals one/ twice a week depending on the phenological stage) were Two separate experimental trials on (A) young potted grape performed to track evolution of phenological phases by the plants and (B) hardwood cuttings, were performed using BBCH scale [9], shoot number and length measurements, bacterial cells of A. brasilense Sp245 cultured in liquid shoot rate growth, and leaf growth rate and number. At Nutrient medium (Oxoid), harvested by centrifugation the end of active growth phase of the first year, plants (10 min at 4300 x g, at 4°C), washed and suspended in were harvested to evaluate the produced biomass which equal volumes of 0.9% NaCl solution exactly as described was divided into leaves, shoots and roots for further in Russo et al., 2008 [5]. Cell number was assessed by measurements of fresh and dry weight (held at 65°C until microscopy with Burker counting chambers and verified reaching constant weight). by plate counts on Nutrient agar medium (Oxoid). A final For plants in the second batch, the survival rate cell concentration of 107 CFU/mL was obtained by dilution was evaluated after bud dormancy overcoming, at the in sterile water before use as inoculum. resumption of active growth. A) Young grape plants of Vitis vinifera L. (cultivar B) Harwood cuttings (n = 100/rootstock) from rootstock ‘Colorino’) were observed following grafting onto two mother-plants of 420A (V. berlandieri x V. riparia) and rootstocks 420A and 157/11 (V. berlandieri x V. riparia). 775P (V. berlandieri x V. rupestris) were inoculated during Before bud swelling (first ten days of April), roots of the the phase of hydration before bench-grafting in nursery grapevine rootstocks were immersed overnight at room (‘Vivai La Vite’, Cenaia, Pisa-Italy) (Fig. 1). The following temperature in a suspension of Azospirillum brasilense parameters were considered: callus diameter at the graft Sp245 which was diluted 1:10 in tap water to a final cell level, at the end of the grafted cutting forcing (15 days); concentration of 106. Plants were then transplanted into rooting percentage, number of adventitious roots and 25 L pots (33 x 33 cm) containing a sterile 1:1 mixture biomass of the root system, at the end of winter storage. of peat and inert perlite. Pots were placed outdoor at The root biomass was evaluated by fresh and dry weight the Department of Agriculture, Food and Environment (held at 65°C until reaching constant weight). Moreover, (University of Pisa) located in central Italy (Pisa, Tuscany, samples of roots were processed in order to determine the Lat. 43°43’17” N, Long. 10°51’55” E). To prevent external content of: total phenolic compounds in fresh material infection, pots were isolated from the ground with a layer (0.6 g), according to Ait Barka et al. [10]; content of main of black plastic sheet. During the vegetative season, 1L of mineral macro-elements (N, P, K) and the micro-element the bacterial suspension diluted 1:10 in tap water to a final Fe in ashes (0.5 g of dried material heated to 600°C for cell concentration of 106, was added to the pot mixture 14 hours), according to Sabir et al. [7]. Chemical analyses surface at different intervals. The inoculated plants were were performed at least in triplicate both in control and divided into two groups that were treated differently by treated cuttings. adding a fixed volume of inoculum. The first group (T1) Data, expressed as means, were subjected to received only one treatment (end of May), the second

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