Studies on the Short-Term Effects of the Cease of Pesticides Use on Vineyard Microbiome

*Simona Ghiță, Mihaela Hnatiuc, Aurora Ranca, Victoria Artem and Mădălina-Andreea Ciocan*

## **Abstract**

In this chapter, an overview of the impact of phytosanitary treatments on the vineyard microbiome is provided, together with the results of the research we conducted. The studied plant material consisted of grapevine from the cultivars Sauvignon blanc and Cabernet Sauvignon, cultivated within the plantation of the Research Station for Viticulture and Enology from Murfatlar, Romania. For each cultivar, a treated plot and an untreated plot were established. For each of those, the phyllosphere microbiota was quantified using the epifluorescence microscopy method, followed by automated image analysis using CellC software. At the same time, the soil fungal diversity was evaluated in three stages during the year 2021, using microscopic morphological criteria. The results give useful information regarding the phytosanitary state of the studied plant, as well as the short-term effects produced by the ceasing of pesticide application on the grapevine microbiota.

**Keywords:** grapevine, phyllosphere microbiota, soil fungi, biodiversity, vineyard microbiome

## **1. Introduction**

It is widely known that plants, like all the other living organisms, are colonized by a multitude of microorganisms [1]. Microbial communities are generally described from the composition (abundance and diversity of the populations that establish the community) and function (behavior and metabolic activity) points of view [2]. The extremely complex functions of microbial communities are not yet significantly understood, even though numerous studies were conducted on the composition of microbial communities [3]. Being modulated by abiotic and biotic factors, predation, competition, and cooperation interactions take place between the members of microbial communities. The environmental effects generated by the microbial activity have the ability of altering the aforementioned interactions furthermore [4].

Microorganisms that have a close relationship with the host plant, regardless of the environmental variables, form the core microbiome [5]. This core microbiome comprises keystone microorganisms that possess genes that can improve the fitness of the holobiont, which were selectively chosen from an evolutionary point of view [6]. Bacterial strains belonging to the families *Pseudomonadaceae, Hyphomicrobiaceae,* and *Micrococcaceae* have been found in relation with the grapevine in any environmental condition, being part of its core microbiome [7].

On the other side of the spectrum, microorganisms found in a lower abundance, which are deeply influenced by the geographical location and habitat characteristics, represent the satellite taxa [8]. Despite their low abundance, satellite microorganisms have critical roles, such as protecting the plant against pathogens through the emission of volatile compounds with antifungal properties [9].

Although not long ago, the focus of plant-microorganisms interactions has been pathogenicity [10]; recently, the ability of some plant-associated microorganisms to directly or indirectly improve plant fitness and performance has been described, as well as the potential of using microbes as a replacement for some synthetic phytosanitary products [1]. A better grasp of the concept of the holobiont can lead to a better future of viticulture, as biocontrol, biofertilization, and biostimulation are realistic and achievable options to reduce the impact of biotic and abiotic stressors, as well as the use of chemical pesticides and fertilizers [11].

The aim of this study is to illustrate the impact of ceasing pesticide use for one year in vineyards on phyllosphere microbiota and soil fungi.

## **2. Vineyard microbiota**

The term "microbiota" refers to the ensemble of microorganisms that exist in a defined environment [12]. In the vineyard ecosystem, endogenous factors, such as the plant age and cultivar, or exogenous factors, such as the cultivation system, geographic location of the plantation, farming techniques, seasonality, human intervention, soil characteristics, and surrounding plants, among others, can influence the presence of grapevine associated microorganisms to a certain extent [13]. It has been pointed out that the effects produced by the composition of the vineyard microbial communities may affect the phytosanitary status of the grapevine, and therefore wine quality, in a significant way [14]. The unique combination of bacteria, fungi, and other microscopic organisms that are found in association with the grapevine and the vineyard soil has even been termed "microbial terroir," as it was found in the recent years to imprint distinctive traits on wine [15]. Zoochory, hydrochory, anemochory, and anthropochory are the known microorganisms dispersal methods that transfer microbiota from the surrounding environment to the grapevine or from one grapevine to another [16]. Perennial plant structures, such as canes, spurs, and bark harbor a greater, more stable microbial diversity, being at the same time one of the sources for the microbiota of the ephemeral structures − leaves, flowers, and berries [17].

#### **2.1 Grapevine as a holobiont: The phyllosphere**

Like many other eukaryotes, the grapevine is colonized by a multitude of microorganisms that play a certain role in its growth and survival. First used in 1991 [18], the term "holobiont" evolved to describe a host and the microbial community associated with it [19]. The holobiont concept states that, as it is the case for the animal kingdom, the plant's health state is deeply influenced by the composition of its microbiota [16]. Depending on their role in relation to the plant, microbial species may be beneficial, pathogenic, or neutral [4].

#### *DOI: http://dx.doi.org/10.5772/intechopen.105706 Studies on the Short-Term Effects of the Cease of Pesticides Use on Vineyard Microbiome*

Microorganisms may be found on the surface of grapevine organs, composing the epiphytic microbiota [20], or they may reside inside the plant tissues, making up the endophytic microbiota [21]. Some microbial species can be found both outside the plant structures and inside their tissues [16], meaning that microorganisms find gateways in piercing wounds caused by insects, stomata, or intercellular junctions, among others [22].

The sum of aerial plant organ surfaces represents the phyllosphere [23]. Depending on the plant organ they populate, phyllosphere microorganisms can be a part of the microbiota of the following plant compartments: leaves − phylloplane; flowers − anthosphere; fruits − carposphere; and stems − caulosphere. The phyllosphere is an open system colonized by complex microbial communities, even though the habitat can be considered hostile, as it is exposed to temperature oscillations, UV radiation, and plant-secreted antimicrobial compounds, as well as low water and nutrient accessibility [24]. The phyllosphere is dominated by the phylloplane, represented by the photosynthetically active foliar surface [23]. Considering the fact that the grapevine is a woody perennial plant that sheds leaves each autumn, the phylloplane is an ephemeral environment [24].

From a nutrient perspective, the foliar ecosystem is oligotrophic, due mainly to the presence of the hydrophobic cuticle that prevents plant metabolites from leaching and reduces water evaporation [25]. However, the presence of stomata, hydathodes, veins, and trichomes can assure a better nutrient supply for microorganisms [24]. Due to the distribution of such structures at the foliar level, the abaxial and adaxial sides of the leaf are colonized by different microorganisms [26]. In most cases, bacterial and fungal cells are found forming aggregates, held together by extracellular polymeric substances that can prevent desiccation [24]. Some phyllosphere inhabitants have also been found to protect their host plants through the substances they produce, that act like pesticides, stimulators, or fertilizers [27].

The other more intensely studied ephemeral grapevine organ is the fruit, the interest shifting often in this case from the health state of the plant to the impact on wine-making [16].

### **2.2 Vineyard soil microbiota**

The soil is an everchanging complex environment, dominated by microbial activity [28]. As they have an important role in the cycle of nutrients and the decomposition of organic matter, microorganisms are a decisive factor in determining soil quality. A wide range of organisms coexists in the soil, including bacteria, fungi, archaea, viruses, oomycetes, protists, and arthropods, which are involved in complex trophic networks [29]. Bacteria and fungi are the dominant taxonomic groups, accounting for approximately 90% of the microbiota found in soil samples [30]. Bacteria are the most abundant soil microorganisms and are the first to react and reproduce when their optimal conditions are met [31]. However, in spite of having longer generation times, fungi are more efficient at decomposing organic substrates and have more stable populations [32, 33]. Although some species are phytopathogenic, there are numerous fungal species capable of antagonizing plant pathogens, stimulating vegetative growth, and decomposing plant residues [28].

The composition of soil microbial communities differs significantly both from a quantitative and qualitative point of view, being deeply influenced by the presence of nutrients, water, applied substances, and farming techniques, to name a few [34]. Most of the times, the exogenous factors play a significant role, but it has been

pointed out that plant genotypes also possess the tools to intervene in the selection of root-associated microorganisms [35]. Soil is often regarded as a reservoir for the microbiota of the leaves, flowers, and grapes, as more similarities have been described between each of those aerial plant compartments and the soil than between each other [16]. Understanding the interaction between the plant and soil microbiota is essential in order to have a better grasp of the way farming practices affect the soil habitat [36].

Depending on the soil's relation to the plant, several compartments are distinguished: the endorhizosphere, the rhizosphere, and the bulk soil [37]. The rhizosphere is the most intensely studied soil region in relation to the plant, represented by the soil located in the immediate proximity to the plant root system. Microorganisms found in bulk soil are mostly inactive in comparison with those found in the rhizosphere [36] because the latter is characterized by a high nutrient content due to the release of rhizodeposits. These secretions contain sugars, amino acids, organic acids, flavonoids, and terpenoids [11, 38], which trigger a chemotactic response for some microorganisms [39]. The composition of rhizosphere microbial communities fluctuates in accordance with the root exudate patterns specific to the plant's vegetative cycle and health status [40, 41]. However, it has been pointed out that the high nutrient content makes the microbial diversity poorer in the rhizosphere in comparison with the bulk soil [36], as carbon inhibits the growth of some microorganisms when it is found in such quantities [6].

#### **2.3 The impact of pesticide use on vineyard microbiota**

Like any other crop, grapevine is susceptible to diseases, which are most often controlled by using chemical pesticides. Fungal diseases pose the biggest threat, making it necessary to use fungicides constantly [16]. Due to the fact that grapevine is one of the crops that require very frequent applications of phytosanitary products, the number of pesticide treatments and the maximum allowed quantity per year has been regulated by the European Commission [42]. In conventionally treated vineyards, chemical pesticides are used, raising the incidence of problems regarding pesticide resistance and the presence of residual pesticides [43]. In organically treated vineyards, copper-based fungicides are viewed as the most important treatments for the most commonly occurring diseases, although recently copper was added to the list for substitution candidates [42].

It is a known fact that phytosanitary treatments present the unwanted potential of affecting the structure and function of the microbiota, as their spectrum is too broad to include only the target microorganisms [27]. The composition of the soil microbiota is sensitive to the action of the chemical treatments. As it was pointed out in a study comparing conventional, organic, and biodynamic cultivation systems, the greatest microbial diversity and richness were found in the soil where grapevine was grown organically [16]. As soil is one of the main reservoirs for the phyllosphere microbiota, the composition of its microbial communities has a critical role in determining the microbial communities found in relation with other plant compartments [44]. Environmental factors, pathogens, and the plant itself are elements that have a well-established role in the manifestation of diseases. The other factor that intervenes in disease development is thought to be the composition of the microbiota [45], although the mechanisms that can successfully manipulate the microbial communities in order to inhibit the occurrence of diseases is not particularly well understood [43].

*DOI: http://dx.doi.org/10.5772/intechopen.105706 Studies on the Short-Term Effects of the Cease of Pesticides Use on Vineyard Microbiome*

An essential aim of organic viticulture is reducing the use of pesticides without affecting the yield and production of grapes. Biological fungicides based on microorganisms have been recently developed and present an advantage, as they may be applied at any given time without worrying about the residual presence of pesticides on grapes [46]. Reducing the input of synthetically obtained pesticides can also be achieved by certain farming practices, such as altering the plant microclimate in order to avoid the optimal conditions for the development of pathogens, reducing the overwintering inoculum, or applying treatments only when alerted by devices that use mathematical models and monitor environmental conditions [43].
