**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 [47–49].

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**).

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.

#### **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).*

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.

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].

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

**Figure 3.** *Leaf wetness in the studied plots.*

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

#### **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.*

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.

None of the isolated fungal strains presented sexual structures, only the anamorphic stage being observed. As a taxonomic classification, all genera belong to

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

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.

## **Acknowledgements**

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.

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