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

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.

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].

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].

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].

3. Materials and methods

3.1 Experimental site

Samples were collected from the vineyards of the Research Station for Viticulture and Enology from Murfatlar (RSVEM), Romania. The biological material consisted of grapevines of the Cabernet Sauvignon and Sauvignon blanc cultivars, grafted on Vitis berlandieri × V. riparia Selection Oppenheim 4 rootstock. The plantations are situated at 50 and 27 m altitude, respectively, and both have a North−−South row orientation. The GPS coordinates are 44 10′48.84″ N 28 25′29.18″ E for the Cabernet Sauvignon plantation and 44 10′30.94″ N 28 25′16.70″ for the Sauvignon blanc plantation. Vines were planted in 2011 and 2008, respectively, at 2,2/1,1 m distances and are trained on a double-Guyot training system. The management of the soil, under and between the rows, is done by keeping the soil bare. The soil is of the calcic Chernozem type, with a medium texture and a humus percentage of 2.3%.

For each cultivar, a treated and an untreated plots were established. For the untreated plots, no treatments were applied in the year 2021 in order to observe the short-term effects of ceasing pesticide use on grapevine microbiota. For the conventionally treated plots, the usual treatment scheme has been applied, which involved 8 treatments during the studied year: the first treatment was applied during the dormancy period, consisting of calcium polysulfide; the second during BBCH 53, with products that have cymoxanil, mancozeb, copper oxychloride, and sulfur as active ingredients; the third during BBCH 60, with oxathiapiprolin, folpet, fenhexamid, proquinazid, and alpha cypermethrin; the fourth during BBCH 69, with oxathiopiprolin, folpet, proquinazid, fludioxonil, and cyprodinil; the fifth during BBCH 73, with fosetyl Aluminum, folpet, myclobutanil, and emamectin benzoate; the sixth during BBCH 77, with dimethomorph, mancozeb, metrafenone, boscalid, and hexythiazox; the seventh during BBCH 81, with dithianon, dimethomorph, sulfur, and emamectin benzoate; and the eighth, during BBCH 85, with copper hydroxide, sulfur, and fenhexamid.

IoT sensors were installed in the experimental plots, which were used to monitor, among others, leaf moisture. The PHYTOS 31 leaf wetness sensor measures the dielectric constant on the upper surface of the device, the value being proportional to the present water amount.

At harvest, the average production per vine for each of the 4 studied variants was calculated, in order to assess the impact of pesticide use cease on grape production.

3.2 Phyllosphere microbiota visualization and quantification

For the study of phyllosphere microbiota, sampling was carried out in 2021 during the phenophase BBCH 79 (when most of the bunches were compacted). The samples consisted of 6 leaves taken from one grapevine per variant, on which the sensors were placed, from the base, middle, and top of the canopy. The samples were processed immediately in the microbiology laboratory of Constanta Maritime University.

Considering the fact that the phyllosphere is an oligotrophic system, in which the distribution of nutrients is heterogeneous, squares of approximately 1 cm² were randomly chosen for each part of the leaf, which were cut with a sterile scalpel. In order to observe the cut sections under the epifluorescence microscope (N-400FL type with blue filter), they were subjected to an adhesive tape gluing process. The adhesive tape was stained with specific fluorochromes and then placed on a microscopic slide. By applying this technique, it is possible to recover the cuticle from leaves, trapping the microorganisms between the tape and the cuticle. Thus, a very high recovery of cells is permitted, while preserving spatial information.

Using this method, a total number of cells/analyzed surface is obtained, at the same time observing the physiological state of the microorganisms, using specific fluorochromes: SYBR Green (SYBR Green I nucleic acid gel stain. 10,000× in DMSO, Sigma Aldrich 5 ml) and Propidium Iodide (≥94% HPLC, Sigma Aldrich 10 mg). The staining solution is prepared in a ratio of 1:1, and applied for 8−10 minutes, according to [47, 48, 49].

The efficiency of fluorescent compounds for evaluating the integrity of cell membranes is determined by selectivity, brightness, excitation, and maximum emission. The final SYBR Green concentration is 10 μl/ml and 10 μg/ml for Propidium Iodide. For each sample, 20 microscope fields were quantified. To visualize the bacteria and fungi on the phyllosphere, the blue filter with a wavelength of 450−480 nm was used, specifically for the chosen fluorochromes. Images were taken with a digital camera and further used for automatic processing, using the “CellC” cell counting software, according to [50].

3.3 Soil fungi identification

For the identification of soil fungi, sampling was carried out in three stages during the year 2021, according to the BBCH phenophases of the grapevine: the first stage was BBCH phenophase 11 (appearance of the first leaf), the second was BBCH phenophase 79 (when several bunches were compacted), and the third was BBCH phenophase 97 (end of leaf fall). From each plot, the soil was collected from the horizon 0−10 cm, from the base of the grapevine trunks, analyzing a total of 12 samples.

Soil samples were processed in the RSVEM microbiology laboratory. The applied technique involves the cultivation of fungi on solid culture media, using the method of serial decimal dilutions. A volume of 0.1 ml of each dilution was spread on the surface of the Rose Bengal CAF Agar (RBCA) medium in triplicate. The plates were incubated at 25° Celsius, being checked initially after 72 h, then daily to observe the growth of the colonies. To avoid redundant isolation of the strains, for each morphotype with specific traits, the colonies present on the 3 plates were counted for the optimal dilution, after which they were isolated on potato dextrose agar (PDA). The modified slide culture method [51] was applied in order to allow a more efficient observation of the fungal structures under the microscope. The fungal strains were identified based on morphological criteria to the genus level, according to [52, 53]. Although this method offers information on the main fungal taxa present in soil, it is important to mention that a more detailed research would have been possible with the aid of molecular identification techniques.

3.4 Data analyses

For the identified soil fungi, the frequency for each genus was calculated according to the formula Di = (Ni/N) ×100, where Di = the frequency of genus i; Ni = UFC number for gender i; and N = total number of CFUs. According to this formula, the genus frequency can be grouped into several classes: <0.5% = rare, ≥0.5 < 1.5% = occasional, ≥1.5 < 3.0% = common, and ≥3.0% = abundant [54]. The ANOVA test was applied to determine whether there were statistically significant differences between the number of CFUs for the sampling phenophases, and the t test was used for treatment types and grapevine cultivars, taking into account a significance level of 5%. For the fungal populations, Sørensen’s similarity index and Shannon diversity index were calculated.

4. Results and discussion

4.1 Phyllosphere microbiota

Microscopy analyses revealed that bacteria are prevalent in epidermal cell grooves, around trichomes and the stomatal opening, and less prevalent on the elevated surface of epidermal cells. Bacteria are the most abundant microbial group in the phyllosphere, followed by fungi. The measured density of bacteria is from 10³ to 10⁷ cells per square centimeter of leaf tissue (Figure 1), while the density of fungal structures is ranging from 10² to 10⁴ cells per square centimeter of leaf tissue (Figure 2).

Figure 1.

Average number of bacterial cells ×10⁵ on leaves from the base (a), middle (b), and top (c) of the canopy, from the adaxial (AD) and abaxial (AB) sides, for sauvignon blanc (SB) and cabernet sauvignon (CS).

Figure 2.

Average number of fungal structures ×10⁴ on leaves from the base (a); ×10³ on leaves from the middle (b), and top (c) of the canopy, from the adaxial (AD) and abaxial (AB) sides, for sauvignon blanc (SB) and cabernet sauvignon (CS).

As it can be seen, the untreated plots show a significantly higher number of microorganisms per square centimeter, at least an order of magnitude greater in comparison with the treated ones, for both cultivars and for all canopy compartments. Microorganisms are placed in higher density on the abaxial side of the leaf, respectively, on the leaves from the base of the canopy. The difference in microbial density between the two vine varieties can also be attributed to the mesoclimate (hill vs. valley), as the plots where Cabernet Sauvignon is cultivated are located at a higher altitude and micro currents can form, that can reduce the humidity conditions favorable to the microbiota development.

The presence of the analyzed microbiota on the abaxial surface of the leaf is probably due to stomata, which represent a natural entry pathway for endophytic microorganisms. The laminar layer may also play a significant role, as moisture emitted by stomata can be retained at this level, reducing the water stress of epiphytic microorganisms. Leaf wetness was analyzed in both plots using IoT sensors (Figure 3). In the studied period, the highest leaf wetness values were observed for the leaves from the untreated Sauvignon blanc plots, which also harbored the greatest number of microorganisms.

Figure 3.

Leaf wetness in the studied plots.

Epiphytic microbiota has a first contact with the leaf cuticle, which may contain a higher or lower amount of wax that may prevent bacterial colonization [55]. Bacterial aggregates can lead to the formation of biofilms on the leaf surface, which represent a form of adaptation that offers protection against desiccation. Phyllosphere-colonizing bacteria can alter the environment in order to modify the plant’s immune system, reflected in differential host responses. Clearly, these bacteria are very dense (10⁷/cm²) and contribute to many processes in the behavior of the individual plants. The results of the analysis done directly on the leaf surface show that biofilms may be tens of micrometers thick and could form extensive networks that cannot be quantified (Figure 4). Biofilms contain multiple microbial species and could create physical barriers on the leaf surface and establish chemical gradients, promoting metabolic exchange. The biofilm could protect the microbial community under adverse conditions and confer them a survival and colonization selective advantage. Extracellular polymeric substances are usually produced, having the role of maintaining the foliar surface hydrated and concentrating detoxifying enzymes at the same time [24].

Figure 4.

Fluorescence micrograph of the microorganisms colonizing a grapevine leaf. Yellow arrow − Bacteria present on plant veins; black arrow − fungal hypha; and white arrow − fungal structures.

Even though the results illustrate the fact that pesticide use influences phylloplane microbiota in a negative way, it is important to mention the impact of ceasing pesticide use on grape production, as the untreated grapevines were affected by diseases; for the Sauvignon blanc cultivar, there was a 50.6% decrease in grape production for the untreated variant when compared with the treated one, while for the Cabernet Sauvignon cultivar, the untreated variant had on average 33.6% lower production in comparison with the treated variant. Thus, the variant that harbored the highest number of microorganisms per square centimeter also showed the lowest grape production.

4.2 Soil fungi

A total of 123 strains were isolated, 44 for the BBCH 11 phenophase, 29 for the BBCH 79 phenophase, and 50 for the BBCH 97 phenophase. In terms of frequency (Figure 5), out of the 12 genera identified, the following were classified as abundant: Penicillium (37.87%), Aspergillus (26.08%), Fusarium (10.77%), Paecilomyces (5, 00%), Cladosporium (4.26%), and Botrytis (3.00%). Due to the absence of sporulation, for 8,51% of the strains, an indentification based on morphological criteria was not possible.

Figure 5.

Frequency of the isolated fungal strains. AB – Abundant; OC – Occasional; R – rare.

None of the isolated fungal strains presented sexual structures, only the anamorphic stage being observed. As a taxonomic classification, all genera belong to the phylum Ascomycota, except the genus Rhizoctonia, which belongs to the phylum Basidiomycota. The most predominant class is Eurotiomycetes, represented by 3 genera (68.96%), followed by class Sordariomycetes, represented by 4 genera (12.77%).

A very recent study pointed out that most of the isolated genera, such as Acremonium, Alternaria, Aspergillus, Fusarium, and Penicillium are very common in vineyard soil [56]. The genera Penicillium, Aspergillus, Cladosporium, Acremonium, Alternaria, Botrytis, and Scopulariopsis have also been reported as endophytes of the grapevine [57].

From a statistical point of view, the differences between the treated and the untreated experimental plots in terms of the diversity of isolated genera are not significant (P = 0.55, F < F crit).

No statistically significant differences were reported with respect to the studied phenophases or the grapevine cultivars compared to the types of identified fungi. The calculated Shannon index had a higher value for the untreated plots (2.253), in comparison with the treated plots (2.139), whereas the calculated value for Sørensen’s similarity index was 73.68%.

Fungal communities found in agricultural soils are influenced by factors, such as soil type, available nutrients, edaphic properties, plant communities, and agrotechnical practices, as well as climatic conditions [58]. The importance of the latter has been highlighted in a study that showed that climatic factors were probably the leading element that caused a variation in fungal communities from 1 year to another [59]. Water stress is a factor known to impact the composition of soil fungal communities [60].

A great number of soil micromycetes are active where readily assimilable elements are found, thus making the soil a “world of asexual microfungi” [61]. Fungi are generally involved in the decomposition of organic matter, the cycling of nutrients, soil aggregates formation, and the mobilization of minerals, among others [62]. Moreover, fungi are extremely adaptable, as they are able to react to detrimental conditions by modifying their form [63].

5. Conclusions

Concerning phylloplane microbiota, the differences between the treated and untreated plots were obvious, with the untreated leaves showing considerably greater numbers of microorganisms for both of the studied cultivars. Thus, the effects of ceasing pesticide use can be readily seen on ephemeral plant structures, such as the leaves.

However, when comparing soil fungi from a quantitative point of view, no significant differences can be seen after only 1 year between the treated and the untreated plots, statistically speaking. This can be due to the fact that pesticides can still persist in the soil residually, affecting microbial populations.

Acknowledgments

This work was funded by the Romanian National Authority for Scientific Research and Innovation, CCCDI - UEFISCDI, for the COFUND-ICT-AGRI-FOOD-MERIAVINO-1, project number 203, within PNCDI III.