The operations developed in the vineyard before the harvest are important to obtain sound and quality grapes. Some of these agronomical operations are focused on reaching the correct nitrogen content in grapes.

Nitrogen (N) is an important element for adequate plant growth and for alcoholic fermentation (AF) completion ( Bell & Henschke, 2005 ). The N fertilizers have been traditionally added to the soil for being absorbed by plants' roots but, taking into account an increase in the pollution of soils derived from their use ( Galloway et al. , 2003 ), new ways of fertilization as foliar application of N sources have been developed ( Haytova, 2013 ). Those N sources are thought to increase the amino acid content of must ( Bell & Henschke, 2005 ). Some of these amino acids are precursors of the aroma synthesis routes so that they can improve the aromatic profiles of wines. Some studies have been conducted on the foliar application of different N sources such as urea and phenylalanine which caused an increase in the amino acid composi­tion of musts ( Ancín-Azpilicueta et al. , 2013 ; Portu et al. 2015a , b ). Other substances can be sprayed over leaves. Thus, some companies as Lallemand S.L. have commercialized products composed by specific fractions of the inactive, dry and non-genetically mo­dified organism (non-GMO) oenological yeast Sac­charomyces cerevisiae (LalVigne ^® Aroma) that are applied over the plants' leaves like a N fertilizer in order to enhance wine aromatic characteristics. Moreover, the N concerned in oenology joins to the objective of performing an eco-friendlier agriculture ( He et al. , 2017 ). The mushroom industry is a quite relevant industry in La Rioja (Spain), for this reason, two types of mushroom wastewaters were considered in the current study. Those wastewaters are thought to be rich in nitrogenous compounds (confidential information pending of patent) so that an improvement of the aromatic features of wines could be achieved with its application.

Additionally, increasing the phenolic compounds in wine is other important aim of the viticulture and oeno­logy. These compounds are known for collaborating in the color, mouthfeel properties and even increasing the stability during ageing what could directly improve wine organoleptic properties ( Villangó et al. , 2015 ). Submitting the plants to an external biotic or abiotic threat is referred to as "elicitation". The elicitation could be an interesting tool to make the phenolic content of grapevines be enhanced because it increa­ses the natural biosynthesis of phenolic compounds - by the phenylpropanoid pathway - as a biological response of surviving ( Suzuki et al. , 2004 ; Farouk et al. , 2017 ). The chitosan is a biopolymer synthesized by the alkaline deacetylation of chitin that has been employed in agriculture for elicitation. The methyl jasmonate is a plant metabolite to manage the defense responses against stress situations; therefore, it can be also considered an elicitor. The main application in agriculture has been aimed to induce resistance against fungal infection to reduce the postharvest diseases of fruits. As other authors have recognized, the products proceeding from yeast extracts and cell walls such as mannoproteins, lipids, glucans and even chitin can act as elicitors for the plant ( Walker, 2009 ). The company Lallemand S.L. also commercialized an elicitor based on specific fractions of the inactive, dry and non-GMO oenological yeast S. cerevisiae (LalVigne ^® Mature) that improves the phenolic content and consequently the phenolic maturity of grapes ( Villangó et al. , 2015 ).

The application of nitrogenous compounds and elicitors may produce a variation in the must amino acid content and in phenolic compounds concentration. Little research about the impact of those physical and chemical shifts on microbial communities - yeasts, molds, lactic acid bacteria (LAB), acetic acid bacteria (AAB) and environmental bacteria (EB) - has been performed until now. In effect, only one previous study performed with two nitrogenous compounds applied to leaves and based on culture-independent methods has been recently published with interesting results, specifically for urea foliar application effect on must microbiota ( González-Arenzana et al. , 2017 ). The present research was intended to analyze for the first time the microbial diversity of must samples from grapevines where nitrogenous compounds and elicitors had been foliar applied.

The microbial diversity of must samples of Tempranillo grapes was analyzed from different approaches to determine the possible effects of pre-harvest applications of three nitrogenous compounds and three elicitors to grapevine leaves. The study was based on culture-dependent methods that allow the detection of viable, cultivable and metabolically active microorganisms.

The application of these different substances was focused on leaves, although these products have probably reach the grape surface by dripping. In general, biofilms in their mature stage might be disrupted by external or internal factors and microbial cells can then reach other locations or surfaces ( Taraszkiewicz et al. , 2013 ). Indeed, foliar application could indirectly cau­se changes in the grape microbial biofilm integrity. Furthermore, these substances are thought to cause variations in the amino acidic and polyphenolic composition of must, therefore both aspects could have influenced the size, diversity and structure of the microbial communities of must samples. These changes could have an impact on the development and completion of AF and malolactic fermentation (MLF).

In the must samples, the microbial groups traditionally described are yeasts, AAB and LAB ( Ribéreau-Gayon et al. , 2007 ), but ubiquitous EB were also identified in must samples of the current study. Therefore, the inherent microbial diversity of samples was considerable. The counts of GYP plates were in concordance with the described by other authors ( Santamaría et al. , 2005 ; Garijo et al. , 2011 ) and, apparently, the foliar applications did not cause significant modification of the size of the yeast population and of the genera detected on GYP plates. A slight difference was detected with the DBDM cul­ture media, which consisted of significantly highest population after the foliar application of the two types of wastewater from the mushroom industry in comparison to the population after the YE2 application. Despite DBDM was aimed to detect Brettanomyces/Dekkera genus ( Rodrigues et al. , 2001 ), this species was not found probably because it was not present in the must samples. In contrast, the DBDM allowed the identification of spoilage yeast genera such as Candida, Schizosaccharomyces and Trigonopsis . The high po­pu­lation of microorganisms isolated in this DBDM medium after the foliar application of wastewater could be detrimental for must quality. A case in point was the presence of Schizosaccharomyces on DBDM plates. This species produce negative metabolites for wine quality and also degrades malic acid ( Mylona et al. , 2016 ), consequently its occurrence may not be desirable.

Regarding bacterial community size, the LAB detected during this study in every sample with both culture media (MRS and M30) was quite small and significant differences did not exist between treatments. Small LAB populations are quite common from grapes to the completion of AF ( Bae et al. , 2006 ). None of the employed culture media was specific, so that other bacteria genera, different to LAB, were encountered. Therefore, in the MRS medium, besides Pediococcus, Staphylococcus , which is considered a novel histamine producer ( Benavent-Gil et al. , 2016 ), was detected. On M30, employed for enhancing Oenococcus genus ( Gon­zález-Arenzana et al. , 2012 ), this genus, Pediococcus and the genera of AAB Gluconobacter were also found.

The size of the population found on Mann and ZPP culture media were between yeasts and LAB. On Mann plates, the wastewater foliar application provided higher bacteria population in comparison to the fo­liar elicitation of YE2. Nevertheless, on ZPP plates - culture media used for promoting EB - differences in EB sizes were not registered. Some of the genera detected on Mann plates were AAB, but other bacteria such as the LAB Pediococcus or several EB were also found. Certainly, ZPP provided similar information about the genera than Mann. The majority of the EB genera detected in both media (Mann and ZPP) has been sometimes described as opportunistic pathogen ( Osdaghi et al. , 2015 ; Cao et al. , 2017 ) but their role in oenological industries is not well-known although, for instance, some interesting results have been reported about Enterococcus from wines by Pérez-Martín et al. (2014) . The higher population of the microorganisms isolated in Mann media after the foliar application of wastewater might not be beneficial for must quality. Considering the raw wastewater from mushroom in­dustry an important source of EB, the detection of those genera in the must should not be surprising if a probable transference of those microorganisms from leaves to grapes in the vineyard were supposed to occur. Nevertheless, detecting such an enormous diversity of viable EB in must samples is a result that should be taken into consideration for future research.

Analyzing the species detected in control samples regarding the species found after the foliar application of nitrogenous compounds and the elicitation provided very interesting results. An obvious example was that the control sample and the methyl jasmonate elicitation samples had the lowest number of yeast species while the foliar elicitation of YE2 and chitosan were featured by the presence of a higher number of yeasts species. Among yeasts, the genera Candida and Hypopichia were the only two represented by two species. Most of the species detected matched with non- Saccharomyces species that are precisely more frequently found in the early stages of the vinification. Some of those yeasts species have been traditionally thought to be negative for wine quality ( Hutzler et al. , 2012 ); in contrast, they are being currently studied owing to their ability of enhancing some color and aromatic characteristics of wines ( González-Arenzana et al. , 2016 ). Curiously the mold Phaeocremonium sp. that is considered a pathogen for some plants ( Gramaje et al. , 2015 ), only was found after the elicitation of chitosan in spite of being used in food and beverages industries as a fungicide ( Rinaudo, 2006 ). Furthermore, the foliar application of chitosan to Tempranillo grapevines did not cause a significant improvement on the grape and wine phenolic compounds ( Portu et al. , 2016 ) and its application did not seem positive due to the yeast species composition of the sample.

Considering the LAB species, in general, they were underrepresented and, in some samples, such as elicitation of chitosan and methyl jasmonate, they were not found. Regarding the AAB species observed, they were in many cases quite similar among samples and three representative species of the genera Gluconobacter and Acetobacter were noticed. Apparently, the LAB and AAB present in sam­ples were not really influenced by the foliar appli­cations. In relation with the EB species detected, it was found that only two species were noticed in the control sample and just after the foliar application of the treated wastewater from the mushroom industry and the methyl jasmonate and chitosan elicitations. In contrast, the foliar application of wastewater and the elicitation of the two LalVigne products were characterized by holding a quite high number of EB species. The genera Pseudomonas and Curtobacterium were represented by at least two species, while the other genera were represented by no more than one species what could indicate a very diverse group of bacteria. Curiously, after the foliar application of raw mushroom wastewater two species of EB, Methylobacterium sp. and Pantoea agglomerans , were found; these species were not present in other samples, what could be related to the nature of this mushroom wastewater that could contain higher diversity of EB than the treated version of the wastewater.

Although the species information is important, it is only a qualitative information useful for making an exhaustive description of the species richness of each sample. Precisely with the aim of obtaining some interesting measurable information, a bioinformatic software for biodiversity quantification was used. The software takes into account the number of species and the representative colonies of each species in the different replicates from a sample to asses diversity indices. These indices meant a quantitative description of the alpha diversity of samples. Accor­ding to this fact, significant differences observed between samples were scarce; in effect, only indices regarding the richness of species were different after some treatments while indices linked to structure of community were not described. The ANOVA statistical analysis showed that control samples had similar richness of species (S and Menhinick index) to the methyl jasmonate elicitation. The richness of species of YE1 was significantly higher than the described for the other samples based on the number of species. In the same way, the richness of species after the elicitation of the YE2 was significantly higher than the described for the other samples, in this case according to the Menhinick index.

Aimed to obtain a more general approach of results, a hierarchical cluster was constructed with average and standardized diversity indices. Similar to the ANOVA results, the control sample and the methyl jasmonate elicitation were clustered together, what means that both treatments were quite similar having a minimal impact on microbial community. Albeit, Portu et al. (2015c) described a positive effect of the methyl jasmonate foliar application to grapevines during two different vintages, increasing the grape and wine phenolic compounds; and Garde-Cerdán et al. (2016) observed an increase of some amino acids content in musts after the foliar application of methyl jasmonate. The methyl jasmonate elicitation might improve some oenological characteristic ( Portu et al. , 2015c ) and its application would not greatly modify the microbial community. Curiously, the YE1 and YE2 were clustered together, so that both could have a similar effect on the microbial population being applied for reaching different aims. Overall, considering the diversity alpha after application of LalVigne products did not cause any important impact on the structure of microbial community. In contrast, they were responsible for a significant increase of the richness of species what could be linked to its nutritive composition of fractions of the inactive and dry yeast S. cerevisiae . Generally, threaten environments lack diversity of species ( Flores-Rentería et al. , 2016 ) so that observing the opposite effect might indicate that the application of both products might have influen­ced positively the microbial community of musts. The remaining samples stayed together in the hie­rarchical dendrogram what means that had not an evident im­pact on microbial alpha diversity of musts.

Generally, the treatments did not cause significant changes nor in the population size of yeasts and bacteria neither in the observed genera and species. Furthermore, dramatic or negative changes were not noticed in any case regarding to the diversity alpha of the microbial communities of the samples. The methyl jasmonate elicitation had similar values of microbial diversity than the control sample what means that its application could be advisable aimed to improve wine organoleptic characteristics. The LalVigne ^® Aroma and Mature products caused an increase of the richness of species what might be positive for microbial community depending on the species springing up after treatments. Assuming that only one vintage was tested, this study showed interesting indicative results for future research aimed at establishing some possible patterns of microbial community response to foliar treatments.