**Preface to "Study of the Influence of Abiotic and Biotic Stress Factors on Horticultural Plants"**

In the face of rapidly changing environment and anthropogenic pressure, plants must develop mechanisms allowing them to survive in the adverse conditions. Understanding the alterations under stress and relationships among different organisms or environmental elements can help create more favorable conditions and holistic approach towards not only the survival of plants but also for their optimal growth and development. This reprint book is dedicated to the scientists especially interested in environmental sciences, involved in plant physiology and biochemistry, as well as horticultural and microbiological issues.

Global climate change is expected to be critical over the century, leading to influences on different parameters of the environment. First, biochemical, and physiological changes appear and affect plant biomass and consequently limit the yield of crops. Instead of the up-to-date knowledge, deeper approach about alleviating the stress effects is still vital in understanding the complexity of the problem. Such direction of research may be the basics to obtain environmentally friendly methods and tools to inhibit negative influence of stress agents on plants.

We have a great pleasure to present the scientists a set of studies entitled "Study of the Influence of Abiotic and Biotic Stress Factors on Horticultural Plants". The reprint book contains 12 papers about the influence of the stress factors on the plant growth and soil parameters. The ideas of the papers are gathered around five topics: (1) achieving better quality of plant material for food production by changes made in the growth conditions, metabolic and genetic modifications; (2) increasing the plant resistance to environmental stresses by application of exogenous compounds of different chemical character; (3) reducing plant stress caused by anthropogenic activity applying nonmodified and genetically modified plants (GMP); (4) mitigating drought stress by irrigation; and (5) the positive effect of plant growth-promoting microorganisms on horticulture plants performance during drought stress.

#### **Agnieszka Hanaka, Jolanta Jaroszuk-Sciseł, Małgorzata Majewska ´** *Editors*

### *Editorial* **Study of the Influence of Abiotic and Biotic Stress Factors on Horticultural Plants**

**Agnieszka Hanaka 1,\*, Małgorzata Majewska <sup>2</sup> and Jolanta Jaroszuk-Sciseł ´ <sup>2</sup>**


In changing environmental conditions, horticulture plants are affected by a vast range of abiotic and biotic stresses which directly and indirectly influence plant condition. Moreover, biomass production or some of the plant metabolites are expected to steadily increase. Such expectations lead to research on the influence of different stressors and their potential modifiers. It is extremely important to have a holistic approach to the processes taking place in a plant that is affected by stress factors. At the same time, the plant can be affected by very diverse factors that mutually affect and shape the plant growth environment, such as the state of water supply—extremely low or, conversely, too high—the impact of low or high temperatures, which in turn cause a state of drought, increased osmotic pressure and salinity.

This Special Issue (SI) was planned with a structure to consider a large range of aspects on stress factors affecting horticultural plants. It is a research summary on the influence of the stress factors on plant growth and the soil parameters. The studies were investigated at the cellular, tissue, organ and whole plant level. Authors described the impact of stress caused by both climate change and human activity resulting in disorder of the optimum temperature (low- and high-temperature stresses), water balance (water and drought stress and irrigation) and the subsequent disturbance of soil parameters. The SI gathers eleven research papers [1–11] and one review [12]. Three papers were dedicated to cold stress, two to salt stress, two to inorganic pollutants such as metals and phosphite (Phi), three to climate change (i.e., high temperature, water and drought stress) and two to irrigation. The subject of the studies were different plant species, i.e., watermelon, lettuce, kale, potato, tomato, grapevine, hops, orchid, strawberry and *Buxus megistophylla*.

Among the classical parameters used as indicators of plant condition are morphological, anatomical, physiological, biochemical and genetical ones [12]. Physiological, biochemical and anatomical changes occurring in the plant under the influence of stress factors should be especially strongly noticed and analyzed.

In the presented SI, the most frequently applied morphological features were: seed germination, plant growth, leaf and berry area, leaf number, stem diameter and plant dry weight [1–3,5,7–11]. Anatomical characteristics were based mainly on the evaluation of epidermis and mesophyll quality and number of cells and chloroplasts [8]. Physiological aspects were focused on water content in leaves, mineral elements (nutritional elements and heavy metals, e.g., N, P, K, Mg, Ca, Mn, Fe, Cu, Na, Cl, Co, Cr, Ni, Pb), photosynthetic pigments content (chlorophylls *a* and *b*, carotenoids), polyphenolic compounds content (e.g., polyphenols, phenolic acids and flavonoids), soluble sugars, acids and proline contents and plant growth regulators (e.g., abscisic acid, indole-3-acetic acid, gibberellin A3) [1–11]. Biological traits were mostly lipid peroxidation (i.e., content of malondialdehyde; MDA), level of reactive oxygen species (ROS, e.g., H2O2 and O2 −•), activity of antioxidative enzymes (e.g., superoxide dismutase, peroxidase and catalase) and content of non-enzymatic

**Citation:** Hanaka, A.; Majewska, M.; Jaroszuk-Sciseł, J. Study of the ´ Influence of Abiotic and Biotic Stress Factors on Horticultural Plants. *Horticulturae* **2022**, *8*, 6. https:// doi.org/10.3390/horticulturae 8010006

Received: 3 November 2021 Accepted: 30 November 2021 Published: 22 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

compounds (e.g., vitamin C and lycopene) [2,4–11]. Additionally, expression of ethyleneresponsive factor genes was analyzed [4]. Some of the papers studied soil properties [1,9]. At the same time, attention was drawn to the close links between hormonal balance and induction of plant immunity, in which regulatory functions are performed by signaling substances (such as salicylic acid, jasmonic acid, ethylene) produced in the pathways of phenolic compounds transformation under the influence of marker enzymes of the phenylpropanoid pathway [12]. Stress factors affect both the uptake of mineral compounds and the process of photosynthesis, which can be monitored based on the biometric parameters and the level of photosynthetic pigments (chlorophyll and carotenoids).

All these elements have been taken into consideration in twelve publications presented in this SI, providing meaningful results revealing a series of events occurring as a consequence of the influence of various environmental factors and explaining the operation of plant defense mechanisms. In the largest number of works, the increase in polyphenols and phenolic acids content and the activity of antioxidant enzymes has been shown because of the action of the abiotic stress factors [1,2,4,9]. Accumulation of proline was observed in salinity stress after chitosan (CTS) application [2], in low temperature stress (kale, *Brassica oleracea* var. *acephala*) [3] and in the Phi supply (potato, *Solanum tuberosum*) [4].

The ideas of the research papers gathered under the titled "Study of the Influence of Abiotic and Biotic Stress Factors on Horticultural Plants" were divided into five sections. They were dedicated to the following fields: (1) achieving better quality of plant material for food production by changes made in the growth conditions, metabolic and genetic modifications; (2) increasing the plant resistance to environmental stresses by application of exogenous compounds of different chemical character; (3) reducing plant stress caused by anthropogenic activity applying non-genetically modified and genetically modified plants (GMP) and (4) mitigating drought stress by irrigation, whereas the main goal of the review paper [12] was to discuss (5) the positive effect of plant growth-promoting microorganisms (PGPM) on horticulture plant performance during drought stress.

(1) Better quality of plant material for food production.

To achieve better plant material for food production, changes can be made in the plant growth conditions, its metabolism and genetic material [3,5–7].

Manipulations leading to an increase in the stress tolerance of a plant may not always allow us to obtain new varieties with better parameters. Such an example was the research on *Vitis vinifera* L. varieties [3] in the wine industry, it seems crucial to find the answers to two questions: (1) do the new genotypes of the pathogen-resistant grapevines (new genotypes with low sensitivity to biotic stress) keep good qualities of fruit? and (2) how do the new varieties react to global climate warming? Frioni et al. [3] proved that the production and fruit composition traits during ripening of several new cross-bred pathogen-resistant grapevine varieties (patented and admitted to cultivation) are significantly lower than two *V. vinifera* traditional varieties, Ortrugo and Sauvignon Blanc. In these studies, five white pathogen-resistant varieties (PRV) listed as UD 80-100, Soreli, UD 30-080, Sauvignon Rytos and Sauvignon Kretos were tested. All tested PRV exhibited an earlier onset of veraison and faster sugar accumulation compared to Ortrugo and Sauvignon Blanc. Such effects could suggest an earlier start of the harvest. Therefore, canopy and ripening management strategies must be significantly adjusted compared to the standard practice employed for the parental Sauvignon Blanc. Overall, PRV could perform better in cooler climates, in north-facing hillsides, or at higher altitudes, where their good resistance to mildews could match an adequate grapes' biochemical balance. Moreover, retaining adequate acidity at harvest is crucial to produce high-quality white wines [3].

Not only genetic manipulation leading to obtain the new varieties, using cold-tolerant rootstocks to efficient adaptation plantlets, or regulation of the growth temperature, but also the nearness of other plants can improve growth parameters and fruit quality of the horticultural plants in stress. Such a phenomenon was observed by Karakas et al. [5]. They showed that strawberries, as salt-sensitive plants, reacted strongly to slight or moderate salinity, reducing the crop yield and quality of fruits. Salt stress negatively affected the growth, stomatal conductance, electrolyte leakage, contents of chlorophyll, proline, H2O2, MDA, activity of catalase and peroxidase and content of the health-related compounds such as vitamin C and lycopene. On the other hand, when strawberry seedlings were grown in combination with *Portulaca oleracea* L. under NaCl stress condition, not only an increase in weight of the green parts of the plant and the total fruit yield of strawberry plants, but also an improvement in physiological and biochemical parameters were observed. The cultivation of strawberry plants with *P. oleracea* directly reduced the concentrations of stress metabolites and antioxidant enzyme levels, as well as indirectly contributing to an increase in vitamin C and lycopene contents. Therefore, Karakas et al. [5] suggested the use of *P. oleracea* on the areas with significant salinity as an environmentally friendly method to diminish salt stress.

The plant struggle with stress may be manifested by changes in metabolism and the accumulation of various compounds. For example, kale tolerance to low temperatures is associated with the presence of specialized metabolites such as polyphenols, carotenoids and glucosinolates, which can act not only as protective factors against environmental stress for the plant, but they can also be a source of beneficial compounds for human health [6]. Ljubej et al. [6] observed that a short (24 h) chilling period (8 ◦C) was beneficial for the accumulation of phytochemicals in kale. However, freezing temperatures (−8 ◦C) caused significant stress and decrease in pigments and phytochemical compound levels. The studies suggested that the temperature of kale cultivation should be controlled by producers to achieve production of crops with a high content of health-related compounds [6].

Lu et al. [7] proved that using cold-tolerant rootstocks may be an efficient adaptation strategy for improving stress tolerance in watermelon (*Citrullus lanatus* (Thunb.), cv. ZaoJia 8424). It was demonstrated that the improved cold tolerance was associated with gourdgrafted watermelons compared to non-grafted (control) plants. Grafted plants accumulated lower levels of ROS, consequently representing enhanced antioxidant activity. Under cold stress, higher chlorophyll and proline contents and lower MDA content were also determined [7].

(2) Exogenous compounds in plant resistance to environmental stresses.

To mitigate stress, growth of horticultural plants can be supported with exogenous substances during agrotechnical treatments such as 24-epi-brassinolide (EBR) on tomato [4], 5-aminolevulinic acids (5-ALA) on *Buxus megistophylla* [10] and CTS on lettuce (*Lactuca sativa* L.) [11].

The exogenous EBR used by Heidari et al. [4] as analog of brassinosteroids eliminated the effects of oxidative stress induced by low temperature in cold-sensitive tomato species. 24-epi-brassinolide decreased the ROS content, simultaneously increasing antioxidant enzymes activity, auxin and gibberellin contents, then improved the growth rate of the tomato.

Yang et al. [10] detected that 5-ALA promoted the growth of *B. megistophylla*, improved plant survival, increased leaf color and enhanced the greening effect. The content of several kinds of mineral nutrient elements, such as nitrogen, phosphate, calcium, magnesium, iron, copper and boron in leaves of *B. megistophylla* was strongly increased by 5-ALA treatment. Unfortunately, a negative effect was also observed. Under this treatment, accumulation of cadmium, mercury, chromium and lead in roots increased. Luckily, these toxic elements were intercepted in roots without translocation and accumulation in leaves. The activities of antioxidative enzymes and the stress resistance of plants were enhanced. According to the results, 5-ALA, as a specific activator of biochemical pathways, can lead to both favorable and unfavorable alterations in metabolism. Therefore, Yang et al. [10] recommend application of this non-protein amino acid in urban landscapes to improve stress tolerance of ornamental plants.

It has been proven by Zhang et al. [11] that CTS, the classic, widely commercially used elicitor of plant immunity protecting plants against phytopathogens, can be effective in protecting the plant (*Lactuca sativa*) against the effects of the abiotic factor—excessive salt concentration. Most likely, its direct protective effect under salinity condition was associated with the regulation of intracellular ion concentration, controlling osmotic adjustment and increasing antioxidant enzymatic activity (e.g., peroxidase and catalase) in lettuce leaves. Moreover, results of Zhang et al. [11] showed that exogenous CTS could improve plant growth and biomass under salt stress. There is significant evidence that CTS curbed the accumulation of sodium but enhanced the accumulation of potassium in the leaves of NaCl-treated plants. This fact may be important for obtaining better-quality lettuce and supplementing the deficiencies of K in the human diet [11]. Chitosan, as natural polysaccharide, is a safe and cheap substance promoting plant growth and increasing the biotic and abiotic stress tolerance of plants.

(3) Reduction of plant stress under anthropogenic activity by application of non-genetically modified plants and GMP.

Human beings create habitats unfavorable for the development of plants. This is the result of industrial and agricultural activity. Fortunately, the plants have also learned to deal with this kind of stress. For example, Maleva et al. [8] found the *Neottia ovata* growing in the young forest community formed during the natural revegetation of the fly ash deposits (fly ash dump of Verkhnetagil'skaya Thermal Power Station). In Russia, this orchid species is included in several regional Red Data Books, and it is especially interesting to gather knowledge of the adaptive characteristics of orchids. The study of orchid adaptive responses to unfavorable factors by Maleva et al. [8] will help to run the process of the introduction of the *N. ovata* into new environments. The adaptive changes in the leaf mesostructure organization, such as an increase in epidermis thickness, the number of chloroplasts in the cell and the internal assimilating surface were found for the first time by Maleva et al. [8]. The orchid population colonizing the fly ash deposits was characterized by a relatively favorable water balance and stable assimilation indexes further contributing to its high viability.

Crop production is expensive in the areas with low phosphorus (P) availability, and for this reason Domatey et al. [2] were looking for other compounds that may serve as a useful source of assimilable P for *Solanum tuberosum* L. According to this paper, it is possible to apply Phi as fertilizer, only when plants stay resistant to this phosphorus form. Like herbicides, Phi has an inhibitory effect on plant growth. The authors try to combine these phenomena as a hypothetical advantage. Only if plant genotypes are resistant to Phi could it be used both as herbicide to weed control and the source of bioavailable P. Such a solution would be environmentally friendly. Furthermore, Domatey et al. [2] showed significant genotypic variation in tolerance indices among the five tested genotypes (Atlantic, Longshu3, Qingshu9, Longshu6 and Gannong2). Firstly, antioxidant enzyme activities and proline content increased significantly under Phi treatments compared to control without Phi. Secondly, potato genotypes with larger root systems such as Atlantic and Longshu3 were more tolerant to Phi stress than genotypes with smaller root systems (Qingshu9, Longshu6 and Gannong2) [2].

(4) Mitigation of drought stress by irrigation.

Plants require an adequate amount of water during the growing season. Nowadays, this is a rising problem because there is not enough water in many parts of the world, even in areas where such shortages did not occur in the past. Luckily, water limitations can be partially eliminated using various irrigation methods, e.g., by flooding the inter-row [1] or more precisely drip irrigation that supplies water directly to the place where the plant grows out of the soil [9].

Flooding the inter-row is still the most frequently used irrigation method for hop (*Humulus lupulus* L.) in northern Portugal. Afonso et al. [1] showed that using this type of irrigation to prevent drought stress can worsen the condition of the soil in inter-rows. The irrigation of the hop fields by flooding the inter-row for more than 20 years caused decreased porosity and increased soil bulk density in the 0–10 cm soil layer in comparison to the 10–20 cm layer. Fortunately, it did not damage the soil structure of ridges, which are the place of nutrient accumulation's gradual uptake by hop plants. Although irrigation and soil

tillage have damaged the soil structures, they did not create the negative nutrient gradient along the row. Moreover, they refilled water deficiencies during plant development, and the quality of the hop cone yield was sufficient, but on the other hand, the water consumption was too high [1].

Limited water resources force more economical use of water by precise irrigation techniques. It is important to accurately follow the plants' water needs, and correctly predict the moments of deficiency supplementation. New irrigation techniques based on biological parameters and very precise calculations can serve as an adequate solution. It is important to follow exactly the first reactions of the plants to water restrictions to finally be able to use less water [9].

Ru et al. [9] devoted the article to the above topic. The authors searched a reliable method to easily quantify and monitor the grapevine water status to enable effective manipulation of the water stress of the plants. It was shown that the study on a daily stem diameter variation of grapevine planted in a greenhouse could be helpful to precise irrigation management of plants. The relative daily variation of the grapevine stem diameter from the vegetative stage to the fruit stage was related to different irrigation levels. Both signal intensity calculation of maximum daily shrinkage (SIMDS) and daily increase (SIDI) can be applied as indicators of the moisture status of grapevine and soil. Ru et al. [9] concluded that SIDI was suitable as an indicator of water status of grapevine and soil during the vegetative and flowering stages, whereas SIMDS was suitable as an indicator of the moisture status of plant and soil during the fruit expansion and mature stages. In general, SIMDS and SIDI were very good predictors of the plant water status during the growth stage and their continuous recording can offer the promising possibility of their use in programming automatic drip irrigation of the grapevine [9].

(5) The positive effect of PGPM on plant performance during drought stress.

Trend towards temperature rise contributes to water evaporation and global warming, eventually leading to drought stress in plants. The review paper of Hanaka et al. [12] discussed the positive influence of PGPM on horticulture plant performance during drought stress. Among mechanisms of plant protection by rhizospheric or plant surface-colonizing and endophytic bacteria and fungi are the production of phytohormones, antioxidants and xeroprotectants, and the induction of plant resistance. On one hand, application of various biopreparations containing PGPM seems to be a relatively cheap, easy to apply and efficient method of alleviating drought stress in plants, with implications in productivity and food condition. On the other hand, the vital problems of using biopreparations containing PGPM include limitations in introducing the microbial inoculum to the appropriate conditions and the low repeatability of their activities. Microorganisms that promote plant growth and at the same time induce physical and chemical alterations that result in enhanced tolerance to abiotic stresses, constitute an important separate group called "induced systemic tolerance" (IST). It should be strongly emphasized that a significant enhancement of the protective effect in drought conditions is achieved by using a mixture of bacterial strains (e.g., from genus Bacillus and Serratia), which indicates the synergistic effect of IST strains. It is also very important to emphasize the special role of fungi in protecting plants against drought. Fungi are more tolerant to drought than bacteria and their abundance in soil increases in water-limiting conditions. This is due to the specific fungal growth and traits which allow an intensive soil and plant tissue exploration and colonization and taking water from resources unavailable to other microorganisms.

Studies on the protection of horticultural plants influencing drought stress indicated that the application of well-selected microorganisms can be efficient [12]. Biopreparations should be multicomponent to achieve an appropriate level of microorganism cooperation and the final desired effect. Moreover, the combination of bacterial and fungal strains into one preparation gives even better effectiveness and reliability. Crop specificity should also be taken into consideration.

**Author Contributions:** Conceptualization, A.H. and M.M.; writing—original draft preparation, A.H., M.M. and J.J.-S.; and writing—review and editing, A.H., M.M. and J.J.- ´ S. All authors have read and ´ agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We gratefully acknowledge all the authors that participated in this Special Issue.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Plant Tolerance to Drought Stress in the Presence of Supporting Bacteria and Fungi: An Efficient Strategy in Horticulture**

**Agnieszka Hanaka 1,\*, Ewa Ozimek 2, Emilia Reszczy ´nska 1, Jolanta Jaroszuk-Sciseł ´ <sup>2</sup> and Maria Stolarz <sup>1</sup>**


**Abstract:** Increasing temperature leads to intensive water evaporation, contributing to global warming and consequently leading to drought stress. These events are likely to trigger modifications in plant physiology and microbial functioning due to the altered availability of nutrients. Plants exposed to drought have developed different strategies to cope with stress by morphological, physiological, anatomical, and biochemical responses. First, visible changes influence plant biomass and consequently limit the yield of crops. The presented review was undertaken to discuss the impact of climate change with respect to drought stress and its impact on the performance of plants inoculated with plant growth-promoting microorganisms (PGPM). The main challenge for optimal performance of horticultural plants is the application of selected, beneficial microorganisms which actively support plants during drought stress. The most frequently described biochemical mechanisms for plant protection against drought by microorganisms are the production of phytohormones, antioxidants and xeroprotectants, and the induction of plant resistance. Rhizospheric or plant surface-colonizing (rhizoplane) and interior (endophytic) bacteria and fungi appear to be a suitable alternative for drought-stress management. Application of various biopreparations containing PGPM seems to provide hope for a relatively cheap, easy to apply and efficient way of alleviating drought stress in plants, with implications in productivity and food condition.

**Keywords:** climate change; drought stress; biopreparations; plant stimulation; plant growth-promoting microorganisms

#### **1. Introduction**

The horticulture system is affected by various abiotic and biotic stresses which directly and indirectly influence soil fertility, plant health and crop yield [1–3]. These stresses result in the loss of soil microbial diversity, soil fertility and availability of nutrients [4]. The condition of the soil under drought strictly corresponds to plant performance, showing consequences in plant morphology, anatomy, physiology, and biochemistry. With reduction in seed germination and seedling growth, plant height, nutrition and biomass are weakened resulting in yield limitation. The huge variety of changes taking place in horticultural plants and the mechanisms of counteracting stress they produce result from a very wide range of horticultural plant species, including types of crops such as those distinguished by the International Society for Horticultural Science (ISHS): (1) tree, bush and perennial fruits, (2) perennial bush and tree nuts, (3) vegetables (roots, tubers, shoots, stems, leaves, fruits and flowers of edible and mainly annual plants), (4) medicinal and aromatic plants, (5) ornamental plants, (6) trees, shrubs, turf and ornamental grasses propagated and produced in nurseries for use in landscaping or for establishing fruit orchards or other crop production units [5]. Facing the current, rapid climate changes, the cultivation of

**Citation:** Hanaka, A.; Ozimek, E.; Reszczy ´nska, E.; Jaroszuk-Sciseł, J.; ´ Stolarz, M. Plant Tolerance to Drought Stress in the Presence of Supporting Bacteria and Fungi: An Efficient Strategy in Horticulture. *Horticulturae* **2021**, *7*, 390. https:// doi.org/10.3390/horticulturae7100390

Academic Editor: Alessandra Francini

Received: 1 September 2021 Accepted: 5 October 2021 Published: 11 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

plants is strongly affected by abiotic stresses, which additionally intensify the influence of biotic factors such as pests causing serious plant infections [4]. In this dramatic situation, plant associations with rhizospheric [6,7] and endophytic [8,9] microorganisms colonizing the rhizoplane, rhizosphere and plant tissues should be considered as the main stress relievers [10–14]. Three types of effects of microorganisms associated with plants are distinguished: beneficial, deleterious and neutral ones. Based on the positive effects of microbes, two main groups are listed, plant growth-promoting rhizobacteria (PGPR) or more generally, plant growth-promoting bacteria (PGPB) and plant growth-promoting fungi (PGPF) [14–19]. All mentioned groups of microorganisms can serve as biocontrol agents, biofertilizers, phytostimulators and phytoremediators [2,12,20–22].

The most frequently described biochemical mechanisms of plant protection against drought by microorganisms are the production of phytohormones, antioxidants and xeroprotectants [23]. Trehalose can act as xeroprotectant triggering the plant-defense system to counteract the damage caused by drought. It has been shown that microorganisms with tolerance to desiccation have the ability to protect some plants from drought. It seems to be dependent on the microorganism's ability to regulate the concentration of trehalose in the plant as a signal of drying damage.

In horticultural production, plant–microbe interactions should be considered the main factor of plant growth, protection against abiotic stresses and resistance against adverse conditions [24,25] (e.g., in arid and semiarid areas), and these interactions could also be beneficial in alleviating drought stress in plants [26]. Profound knowledge about the mechanisms of plant–microbe interactions can offer several strategies to increase plant productivity in an environmentally friendly manner [27]. Therefore, in the increasing market for plant growth-promoting products, it is important to develop a successful strategy for microorganism screening [28]. Furthermore, the European Green Deal (EGD), provided by the European Commission in December 2019, is currently focused on the application of natural products in agriculture and horticulture instead of chemical plantprotection products. To cope with this idea, new efficient biological ingredients in the face of changing climate are desired. Nowadays, the most significant consequence of climate change is drought stress [29].

To deal with severe drought stress in the near future, it is strictly necessary to determine the interactions, mechanisms and signaling pathways responsible for increased drought tolerance in terrestrial organisms. The concept of drought and water deficit is difficult to define, but the literature data [30–32] indicate that drought can be defined as a state of the total water capacity being within the range of 12–20% for a period of 16 days. Moreover, the drought state can achieve at least two degrees—mild and severe [33]—while the water deficit [34] refers to the state of water capacity falling below 30%. To handle the drought effect, plants can be supported by both microorganisms inhabiting the rhizoplane (i.e., those adhering to the surface of the roots) and rhizosphere (i.e., living at a further distance within the root secretions) [34,35], as well as endophytic microorganisms inhabiting the inside of the root [36]. The application of plant growth-promoting microorganisms seems to provide hope for a cheap, easy to apply and efficient way of alleviating drought stress in plants with implications in productivity and food condition. The presented review was undertaken to discuss the impact of climate change with respect to drought stress, and to emphasize that modifications in microorganisms composition and their traits should indicate new solutions in the search for efficient compounds of biopreparations supporting plant growth.

#### **2. Climate Change**

Global climate change is expected to be considerably critical over the century, leading to influences on various parameters of the environment [17]. Not only atmospheric CO2 concentrations derived from natural and anthropogenic sources, but also surface temperatures will be increasing gradually, likely from 1.0 to 5.7 ◦C by the end of this century [37]. Moreover, some regions, such as the Eastern Mediterranean and Middle East (EMME), have been classified as a climate "global hot-spot". In the EMME, the temperature is predicted to increase from 3.5 to 7 ◦C by the end of the century [38]. Additionally, it is anticipated that rising air temperatures will increase the frequency of extreme weather disasters such as heat waves, drought and heavy precipitation occurrence to a level that has never been monitored before [37]. These strongly temperature-dependent climate changes, combined with water scarcity, will lead to enhanced drought throughout the globe, hurting whole ecosystems and different organisms, including the distribution of plants and microorganisms [17].

In climate studies, calculations concerning crop evapotranspiration are also important [17]. For instance, in South East Europe, the mean annual crop evapotranspiration in the period 1991–2020 reached from 56 mm to 1297 mm, while averages for the future 30 years (between 2021 and 2050), are expected to vary from 59 mm to 1410 mm [17]. These predictions consider the impact of future climate warming. Global warming increases water evaporation and consequently leads to drought stress [39]. High temperature is the crucial factor in melting glaciers and increasing the sea level [8]. The changes in polar and subpolar climate zones also correspond with climate warming [40–42].

Climate change results in altered environmental conditions and negative effects on natural ecosystems, which are likely to trigger modifications in plant physiology [43] and microbial functioning [44] based on the availability of nutrients [4] or signal compounds [2]. It is certain that not only plants, but also plant-associated microorganisms might be remarkably changed in abundance, diversity and activity [44,45]. Both increased temperature and drought may activate correspondent adjustments in plants and microorganisms and their mutual interactions [17]. The adaptational challenges of horticultural plants are not only associated with long-term average climate change, but also with the short-term changes driven by weather extremes and interannual fluctuations [46]. Drought-related cereal production losses are increasing by more than 3% yr−<sup>1</sup> [46]. In the face of the continuous raise of the world population to an estimated nine billion by 2050 [47], withstanding drought stress according to sustainable agriculture/horticulture is a challenge for the 21st century [48].

#### **3. Plants under Drought Stress**

Drought is an uncontrolled stress which affects almost all stages of plant growth and development directly or indirectly [43]. Most of the drought effects on plants are associated with high temperature. Physiological processes occur mostly in temperatures ranging from 0 ◦C to 40 ◦C. However, the optimal temperatures for the different stages of growth and development are narrower and strongly depend on the species and ecological origin [1,49].

Plants exposed to drought stress develop numerous responses in different areas, from morphological and physiological mechanisms to anatomical and biochemical or molecular ones [1,39,50] (Figure 1).

Four types of morphological and physiological response strategies to drought stress are highlighted, i.e., tolerance, avoidance, escape and recovery [51] (Figure 2). Tolerance is defined as the plant's ability to resist dehydration using osmoprotectants [52]. Avoidance is based on the undisturbed occurrence of physiological processes (such as stomata regulation, root system development). Escape is the adjustment of the plant's life cycle by shortening of the life cycle to avoid drought stress. Recovery is the ability of a plant to restart growth after the exposure to the extreme drought stress [53].

The morphological features of drought stress include limited seed germination and seedling growth, reduced size, area and number of leaves, restricted number of stomata, reduced number of flowers, disturbed stem and root elongation, impaired plant height, growth, development and yield, and reduced fresh and dry biomass [7,39,50].

**Figure 2.** Response strategies to drought stress (modified on the basis of [1,39,50,51]).

In order to adapt to the adverse environment, avoid drought and improve water availability, plants increase the root length and their number [54]. Drought significantly affects the plant's cell elongation and division, its growth and its development, which is mainly caused by the reduction in cellular differentiation, plant growth and yield [50]. The negative effect on the leaf area under the drought condition could be dependent on the reduction in the leaf number, size and longevity, combined with temperature, leaf turgor pressure and assimilation rate [55]. The reduction in plant height and shoot dry weight results in a lower quality of yield [54].

The morphological responses are most frequently combined with anatomical changes in plants exposed to drought, e.g., thickening of cell walls, increased cuticle layer on the leaf surface and improved development of vascular tissues [8,56]. Drought stress results in anatomical changes in the lower and upper epidermis, mesophyll tissue and vascular bundle diameter of leaves [57]. The negative anatomical effects on the leaves are based on a shortage of water supply from the soil, limitations in nutrients uptake, and reduction in photosynthetic rate. Plant hydraulic conductivity is modulated during drought stress leading to the disruption of water flow in the xylem vessels (embolism) or modifications in the vessel size and function [58]. Consequently, the reduced water flow from the root to the shoot causes stomatal closure and transpiration disruption [50].

Drought affects the physiological traits such as the leaf relative water content and water potential, stomatal conductance, transpiration and photosynthetic rates [59,60]. Reduced water content and water conductivity are responsible for the loss of turgidity and limited stomatal conductance resulting in restricted gaseous exchange (the rate of carbon assimilation) [8,61]. Furthermore, climatic conditions, e.g., higher temperature, drought and soil aeration reduce the movement of nutrients in the soil, their uptake by roots and transport in plant tissues [62].

Photosynthesis can be disrupted through the modulation of the electron transport chain and can increase the rate of biochemical reactions catalyzed by different enzymes. Above a certain temperature threshold, enzymes lose their function, influencing the plant tissue tolerance to drought [1,63,64]. Drought stress also affects the translocation of nutrients and the composition of minerals, antioxidants and proteins [39,52]. Under stress conditions, reactive oxygen species (ROS) are highly generated [65,66] causing cell damage and plant necrosis [67]. Additionally, plant hormones and primary and secondary metabolites are modified [1]. Drought is the elicitor that can increase the content of secondary metabolites in plant tissues such as flavonoids, phenolics or more specific molecules, e.g., glycosides and alkaloids [68,69].

Crosstalk between drought and salinity stresses results in secondary stresses such as oxidative and osmotic ones [66]. Drought stress is a major agricultural problem worldwide and almost all of the main agricultural lands are affected by drought stress. The potential mechanisms of drought tolerance include: (1) production of phytohormones (such as indole-3-acetic acid (IAA), cytokinins and abscisic acid (ABA)) (2) synthesis of exopolysaccharides (3) activity of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (4) induced systemic tolerance [66,70].

#### **4. Mechanisms of Resistance in Plants**

Plants are capable of defending themselves against numerous stress factors, both biotic and abiotic ones, by activating very effective pathways of immunity (Figure 3).

**Figure 3.** Immune response of plants against stress factors (modified on the basis of [71,72]); PAL—phenylalanine ammonialyase; TAL—tyrosine ammonia-lyase; ISR—induced systemic resistance, SAR—systemic acquired resistance; JA—jasmonic acid; ET—ethylene; SA—salicylic acid; PR proteins—pathogenesis related proteins.

> The plant might acquire resistance against phytopathogenic infection due to the induction of plant defense responses driven by the very wide range of interactions with above- and below-ground microorganisms [6,73,74]. Several microbial species have displayed plant-priming phenomena. The priming process of plants is typically known in induced systemic resistance (ISR) and systemic acquired resistance (SAR), and microbeassociated molecular pattern (MAMP)-triggered immunity. ISR is mediated through the involvement of phytohormones, e.g., ethylene (ET) and jasmonic acid (JA), and the defense responses against phytopathogenic microorganisms are activated very quickly. SAR and MAMP-triggered immunity are induced as a first line, and unlike ISR, they utilize salicylic acid (SA) as signal substance of the plant resistance pathways [6,73,74]. The nonpathogenic microorganisms and various organic elicitors, mainly derived from microorganisms, act by inducing systemic acquired resistance (SAR) [75]. To elicit defense responses in plants, microorganisms secrete several molecules such as antibiotics (i.e., 2,4-diacetylphloroglucinol, phenazines synthesized by *Pseudomonas* species, and cyclic lipopeptides such as surfactin, synthesized by *Bacillus* strains), volatiles, quorum-sensing signals (N-acyl homoserine lactone of Gram-negative bacteria), proteins and small lowmolecular weight compounds [6]. Natural bioactive compounds of microbial, Protist or plant origins with the ability to protect plants against phytopathogens can have fungicidal effects (can kill pathogens) or fungistatic effects (can limit development of phytopathogens), as well as being able to induce plant defense reactions as elicitors [71,76]. Every factor (physical, chemical, biotic, abiotic and their mixture) that induces plant immunity or stimulates the defense mechanisms in a plant is called an elicitor and is defined depending on its origin and molecular structure [68]. MAMP-type molecules are exoelicitors of microbial origin. Pathogen-associated molecular pattern (PAMP)-type molecules are exoelicitors of pathogenic organism origin. Damage/danger-associated molecular pattern (DAMP)-type

molecules are endoelicitors of plant origin released during phytopathogen infection or produced under various stresses, [77–79]. Receptor proteins in the plasma membrane-pattern recognition receptors (PRRs) recognize particular molecular patterns of MAMP/PAMP and DAMP molecules [80,81]. The priming or PAMP-triggered, (PTI)-type local immunity, which arises in the absence of virulent pathogens, is due to the rapid onset of intracellularsignaling-pathway activation leading to a very fast and effective defense responses in the plant [82].

In plants, a range of abiotic and biotic elicitors can strengthen tolerance to drought stress, including alginate-derived oligosaccharides, ketoconazole, 2-aminoethanol, ABA, brassinosteroids, and beneficial microorganisms such as *Rhizobium* strains, endo- and exomycorrhizal rhizospheric and endophytic nonpathogenic fungi. These elicitors reduced the content of monodehydroascorbate, prevented the accumulation of ROS, increased activities of antioxidant enzymes, and maintained fresh and dry weights, grain yield, and relative water content in a variety of plants in response to drought stress [83]. The term "induced systemic tolerance" (IST) has been suggested for PGPB-induced physical and chemical alterations that result in enhanced tolerance to abiotic stresses [70,84,85].

#### **5. Bacteria Supporting Horticultural Crops**

The most promising solution for the future of modern horticulture seems to be the skillful use of biopreparations in the conventional crops and not limiting their use to a narrow range of ecological or organic farming crops. Biopreparations include at least three types of products: (1) biocontrol, or biological plant protection inhibiting directly (antagonism, competition) or indirectly (defense responses) the growth of phytopathogenic fungi or bacteria and other pests such as insects and nematodes, (2) biostimulation, positively affecting the plant development, increasing the plant biomass and yield, and (3) biofertilization, which provides nutrients and enhances plant nutrient uptake [6,29,71], (Table 1). The components of these biopreparations are very diverse, ranging from various inoculum types of microorganisms (either single or consortia of endophytic bacteria, fungi and Protista strains belonging to the plant growth-promoting group), through to metabolites, including phytohormonal and hormonelike substances or parts of microorganism cells, to various metabolites and structural compounds derived from microorganisms, Protista and plants often acting as the plant resistance elicitors [6,86]. Interestingly, the components of biopreparations are composed in such a way that, while performing biocontrol, biofertilizer and biostimulant functions [87–91], they reduce the impact of stresses caused by the numerous and dynamically changing environmental factors. Among these factors are rapid shifts in the temperature and humidity leading to the formation of drought, which reduces the availability of nutrients.

A very common approach is the isolation and application of active microorganisms to similar or the same plant and conditions, e.g., a *Pseudomonas* IACRBru1 strain isolated from *Eruca versicaria* (rucola) tissues improved *Lactuca sativa* (lettuce) biomass (up to 30%) [92]. One of the critical steps for the successful application of microorganisms is their survival and development in the new environment. In drought-stressed soils, the highest efficiency of this inoculation could be achieved using drought-tolerant bacteria isolated from arid soils or drought-resistant plants [93–95]. Bacteria classified as *Bacillus subtilis*, *Bacillus altitudinis*, *Brevibacillus laterosporus* and *Bacillus mojavensis* were isolated from *Cistanthe longiscapa*, a plant native to Atacama Desert in Chile [94]. A consortium of these microorganisms, with various complementary properties such as phosphate solubilization, the ability to grow on N-free culture, IAA, ACC-deaminase, and exopolysaccharide (EPS) synthesis, were applied onto tomato seeds, improving seedlings growth under drought stress.

**Table 1.** Selected activities of beneficial bacteria under drought-stress conditions in horticultural plant species; H—higher level/content; L—lower level/content; GPX—glutathione peroxidase; CAT—catalase; Chl—chlorophyll; RWC—relative water content; Fv/Fm—quantum efficiency of photosystem II; SOD—superoxide dismutase; MDA—malondialdehyde.


It is worth emphasizing that among the most frequently mentioned PGPR strains of different genera, e.g., *Pseudomonas, Bacillus, Klebsiella, Azotobacter,* many *Pseudomonas* species show a very high diversity of traits stimulating plant growth. For this reason, many scientific laboratories are looking for such valuable isolates adapted to drought conditions [101–103] (Table 1). *Pseudomonas putida*, isolated by Kumar et al. [101], synthetized IAA, siderophore, ACC-deaminase, formed biofilm and solubilized phosphate. *Pseudomonas aeruginosa* strain, isolated from North East India, additionally showed HCN synthesis and endogenous osmolyte accumulation under the drought condition [103]. Sandhya et al. [102] also selected drought-tolerant *Pseudomonas* spp.: *P. monteilli*, *P. putida, P. stutzeri, P. syringae* from the rhizosphere of crop plants.

Niu et al. [93] isolated drought-tolerant plant growth-promoting bacteria from *Setaria italica* (foxtail millet) cultivated in arid soils. The bacterial strains identified in the roots and bulk soil (e.g., *Pseudomonas fluorescens* DR7 and DR11, *Pseudomonas migulae* DR35 and *Enterobacter hormaechei* DR16) synthetized ACC-deaminase under drought conditions. All the isolates produced EPS, but IAA activity was confirmed only in DR35 culture. Similarly, *Pseudomonas* sp. isolated from Californian soil exposed to frequent drought also showed significant production of EPS in response to desiccation [104].

Belonging to the PGPR family, *Azospirillum* spp. (Table 1) are a group of free-living soil bacteria mainly known for their ability to fix atmospheric nitrogen but also for releasing phytohormones, enhancing root growth, water and mineral uptake and plant resistance to drought stress [105,106] (Table 1). As a microbial inoculant, *Azospirillum* spp. could be crucial to improve fruit-tree acclimatization when transferred to the *post-vitro* environment [106].

Mariotti et al. [105] revealed that *Azospirillum baldaniorum* cells and their metabolites promote *Ocimum basilicum* cv. Red Rubin (purple basil) growth under the water stress condition. This action was attributed to the synthesis and transport of phytohormones that promoted plant growth and conferred tolerance to the abiotic stress. The plant leaves treated with a relevantly high dose of the filtered culture supernatants of *A. baldaniorum* contained significantly higher concentration of chlorophyll *a* and *b*, total chlorophyll, carotenoids, and anthocyanins. In the presence of these bacteria, in the tissues of purple basil, the concentration of stress-related phytohormones, ABA, JA and SA were higher. *Azospirillum brasilence* accompanied by *Pseudomonas* sp. and *Bacillus lentus* also caused a higher level of chlorophyll content in *Ocimum basilicum* grown under drought stress [107].

Moreover, at the end of the growing season, certain soil species, including soil-borne endophytic microorganisms promoting plant growth (e.g., including *Bacillus, Clostridium* and *Sporolactobacillus* genera), form endospores capable of remaining dormant in the soil. It is extremely important that in adverse environmental conditions (e.g., drought, very high or low temperature or higher amounts of incoming solar radiation) [1,108,109], when spores encounter the appropriate conditions (for example in the next growing season), they survive, germinate and the vegetative cells develop in the soil and are able to inhabit plants [109].

A mixture of three PGPR strains (*Bacillus cereus* AR156, *Bacillus subtilis* SM21, and *Serratia* sp. XY21) (Table 1) stimulated IST in drought stress in cucumber plants by maintaining the root recovery intensity, reducing plasmalemma peroxidation, stabilizing the osmotic potential, increasing photosynthesis efficiency and activities of SOD and cytoplasmic ascorbate peroxidase (APX) in the leaves, without involving the action of ACC deaminase to the lower plant ethylene levels [83].

#### *5.1. Bacillus Species in Drought Stress*

Among the features of the soil-aerobic, rod-shaped cells of *Bacillus* species (Table 1) contributing to the biocontrol mechanism is the synthesis and secretion of various antimicrobial peptides and very diverse antibiotics, enzymes, other proteins and organic compounds [110,111]. Inoculation of *Cucumis sativus* (cucumber) with *Bacillus cereus* and *Bacillus subtilis* strains along with *Serratia* sp. induced systemic tolerance to drought stress in plants by maintaining photosynthetic efficiency, root vigor, increasing proline content and enhanced SOD and CAT activities in the leaves [83]. In another experiment, to enhance *Lycopersicon esculentum* (tomato) drought tolerance, *Bacillus cereus* AR156 supernatant was applied. In the treated plants, chlorophyll *a* and *b* contents, as well as the activities of SOD, POD and CAT were increased markedly after culture supernatant application [112].

Plant small heat shock proteins (sHSPs) act as molecular chaperones that prevent irreversible aggregation of denatured proteins [85]. During drought stress, pepper plants inoculated with *Bacillus licheniformis* K11 exhibited enhanced transcription of Cadhn, VA, sHSP, and CaPR-10 genes [113,114]. In the study of Lim and Kim [113], the *Capsicum annuum* (pepper) seedlings were treated with a *Bacillus licheniformis* strain originated from Korean soil. Plants inoculated with drought-tolerant bacteria achieved higher shoot length and dry weight, and the analysis of gene expression in pepper roots indicated higher levels of expression of four genes related to drought and cold stresses. *Bacillus* sp. selected for high levels of cytokine synthesis was introduced into 12-day old *L. sativa* grown in dry soil. After 3 weeks of seedlings inoculation, the increased amount of cytokinin and higher fresh and dry weights of shoots were confirmed [115].

At the beginning of vegetative season, higher temperature induces microbial metabolism (including releasing of inorganic available P to the soil solution by phosphate solubilizing microorganisms (PSM)) [116]. Gradually, the lack of adequate precipitation, insufficient soil moisture and high temperature decreased the soil microbial activity and the movement of nutrients in the soil [62]. *Bacillus* strains are commonly known to be great phosphate solubilizers [110]. Ying et al. [18] revealed high phosphatase activity of *Bacillus megatherium* and inorganic phosphate solubilization of *Bacillus saryghattati* strains under drought stress. *Bacillus* spp. (*B. cultidtuctinus*, *B. subtilis*, *B. polymyxa* and *B. mojavensis*) isolated from the *Cistanthe longiscapa* rhizosphere grown in the Atacama Desert (Chile) also exhibited phosphate-solubilizing activity [94].

It is worth noting that the activity of phosphate-solubilizing *Bacillus* strains support very energy-consuming processes of nitrogen fixation. Available P is a crucial ingredient of the energy source ATP. It can also replace conventional fertilization. An effective action on N and P uptake by the *Vicia faba* (faba bean) seeds and straws was confirmed after inoculation with the well-known phosphate-solubilizing bacterium *Bacillus megatherium* [117]. After inoculation of the apple trees cv. 'Topaz' with 'Mycostat' (containing *Bacillus subtilis* among strains promoting plant growth) the P root content was the same as in the tissues treated with chemical NPK fertilizer [89]. In addition, when soil moisture declined, the limited diffusion rate of nutrients, particularly P, from the soil matrix into the absorbing surface negatively affected nodulation and biological nitrogen fixation [118].

Plants with symptoms of potassium deficiency show accelerated wilting and lower yield, causing the loss of control of turgor-driven leaf movements [119]. *Bacillus* strains can secrete acidic metabolites (e.g., oxalic, fumaric, lemon, tartaric acids) that dissolve various minerals. Avakyan [120] demonstrated the ability to produce a thick EPS envelope by the strain *Bacillus mucilaginosus*. Secretion of the acidic metabolites by *B. mucilaginosus* cells creates a zone of strong acidification at the soil minerals' surface and allows the dissolution of mineral compounds. The polysaccharides secreted by these microorganisms additionally strongly adsorb SiO3 <sup>−</sup><sup>2</sup> leaving bioavailable K cations for plants in the soil solution [121].

The plant root is involved in the perception and transduction of stress signals via phytoregulators such as ET [122]. The increased level of ET causes premature aging of fruits and vegetables; wilting of flowers and leafy vegetables and defoliation of the mature leaves. Additionally, higher concentrations of ET in the rhizosphere inhibits arbuscular mycorrhizal fungi colonization and the root nodulation of legumes. A *Bacillus subtilis* (LDR2) strain isolated from the rhizosphere of drought-stressed plants, synthetizes ACC deaminase-regulating ET concentration. In the experiment, a LDR2 strain revealed protective mechanisms against the low water availability in soil, and improved *Trigonella* plants' weights (by 56%). Barnawall et al. [122] also demonstrated the enhanced nodulation and arbuscular mycorrhizal fungi colonization in the plants, which caused better nutrient uptake after inoculation of plants with *B. subtilis*.

In the face of climate change, certain future adaptations can be predicted by observing the functioning of organisms in extreme environments. In addition, in natural adverse ecosystems, except for the ability to form spores, microorganisms support plant growth and simultaneously provide an optimal environment for the development of plants tissues [111,117,123], e.g., *Bacillus mojavensis* was isolated from the very extreme environment of the Mojave Desert in California [111]. All the strains belonging to this group are described as endophytic and antagonistic to fungi [124]. The endophytic microorganisms (including both obligate and facultative species) are microbial symbionts residing within plants, mostly influencing host physiology [36,111].

The *B. mojavensis* strain isolated from the soybean plant rhizosphere was a very antagonistic strain, effectively controlling *Rhizoctonia solani*, a pathogenic fungus causing huge harvest losses of horticultural crops [125,126]. The presence of endophytic microorganisms with the biocontrol actions of soil-borne pathogens and the ability to stimulate the growth

of cultivated plants from the early stages of its development seems to be a crucial solution for plants under unfavorable climate conditions. The inoculation of soybean seeds with the *Bacillus mojavensis* PB 35(R11) strain enhanced the growth of plant inoculated with *R. solani* (about 30% higher plant fresh weight and over 100% higher plant dry weight) [111]. Moreover, quantitative assays of the PB-35(R11) strain showed HCN, ammonia and siderophore production, as well as phosphate solubilization and chitinase activity. The treatment of seeds gives several advantages for the control of pathogenic fungi as a promising alternative to the use of synthetic pesticides. The endophytic *Bacillus* inoculants are also known for controlling *Fusarium* species, especially *Fusarium verticillioides* [124,127].

#### *5.2. Actinomycetes Species in Drought Stress*

A more advantageous strategy is the selection of microorganisms adapted to functioning in the conditions of temporary lack of water, drought, or rapid changes in temperature, because the metabolically active forms of microorganisms may support the growth of sensitive horticultural crops. *Actinomycetes* are Gram-positive, mostly aerobic, saprotrophic bacteria of diverse phenotypes (from cocci to highly differentiated mycelia).

Tangles of filaments grow similarly to filamentous fungi. This pseudomycelial growth (surface, plunge or air) provides penetration of a larger soil volume and into pores of soil, easing access to valuable minerals and simultaneously making them available to plants [128].

The main place of *Actinomycetes* occurrence is the soil (warm and humid or dry), but they are also identified in desert sands, on leaves and in plant tissues [129]. Reproduction of these bacteria occurs by fragmentation of pseudomycelium and spore formation. This group of microorganisms are mostly chemoorganotrophs with the ability to break down difficult decomposing substrates, e.g., cellulose, chitin, steroids, higher fatty acids or aromatic compounds. These activities allow them to survive and outcompete the native microflora in various ecological niches [130]. Lawlor et al. [131] revealed a higher number of *Actinomycetes* colony forming units (CFU) (about 10<sup>6</sup> to 10<sup>7</sup> g−<sup>1</sup> of dry weight of soil) than CFU of fungi (10<sup>4</sup> to 10<sup>5</sup> g−<sup>1</sup> of dry weight of soil).

*Actinomycetes* are known to be producers of bioactive compounds (antibacterial, antifungal), exhibiting great potential in promoting plant growth [129]. Sousa et al. [3] investigated that the *Streptomyces* strains produce siderophores, phytohormones (IAA), and solubilizing phosphate compounds, and exhibit chitinase, xylanase, cellulase, amylase and lipase activities. Additionally, the number of plant growth-promoting *Actinobacteria* is 1.3 times higher than that of the other bacteria [132].

Khamna et al. [133] identified about 30 *Streptomyces* isolates in Thai soil samples collected from the rhizospheres of plants such as *Curcuma magga*, *Zingiber officinale* (ginger), *Ocimum sanctum* (holy basil), and *Cumbopogon citratus* (lemongrass). After 3-day incubation, the *Streptomyces* CMU-H009 strain synthetized the highest concentration of IAA (about <sup>144</sup> <sup>μ</sup><sup>g</sup> · mL<sup>−</sup>1) and its culture filtrates stimulated *Vigna unguiculata* (cowpea) seed germination. El-Tarabily [134] isolated over 60 *Streptomyces* spp. strains from a tomato rhizosphere in the United Arab Emirates and some of them revealed ACC-deaminase and IAA synthesis. The most efficient *Streptomyces filipinensis* 15, *Streptomyces atrovirens* 26 and *Streptomyces albovinaceus* 41 strains increased *Lycopersicon esculentum* (tomato) root and shoot length and dry weight. A higher level of endogenous IAA in the roots and shoots in these plants was also confirmed. *Actinomycetes* exhibited great potential in promoting rice, sorghum [135], tomato [134], maize [136] and soybean seedling growth [137].

It is worth emphasizing that some *Actinomycetes* (e.g., *Frankia* sp.) function in a symbiosis with higher plants, fixing nitrogen, while the plant provides the bacteria with sugars and minerals [136]. Such a favorable relationship has been observed in soybeans, peas, *Elaeagnus umbellata* and *Eleangus angustifolia* (Russian olive) [138].

A variety of activities improving plant development have been indicated in the *Acitnomycetes* species, and their efficiency obtained by adapting to adverse climatic conditions enables them to receive commercial products containing PGPA (plant growth-promoting

actinobacteria), such as *Streptomyces lydicus* strains. *Acitnomycetes* species (*Streptomyces kasugaensis*, *Streptomyces griseus* and *Streptomyces cacoi* var. *asoensis*) producing antibacterial and antifungal bioactive compounds are components of biocontrol products applied against plant pathogens [129]. *Actinomycetes* synthesize enzymes such as lysozyme, glucanases, peptide–peptide hydrolases, mannanase and chitinase, which are involved in the lysis of the cell walls of other microorganisms [128]. El Tarabily et al. [134] investigated the promotion and biological control of seedlings and the mature plants of *C. sativus* using endophytic *Actinomycetes* (*Actinoplanes campanulatus*, *Micromonospora chalcea* and *Streptomyces spiralis*). *Pythium aphanidermatum* (oospore-producing soil-borne pathogen) causes seedling and root diseases of cucumber, causing damage to horticultural crops. This experiment proved that *Actinomycetes* colonize the roots of inoculated plants, promoted their growth and reduced the impact of *P. aphanidermatum*. Furthermore, El Tarabily et al. [134] compared the impact of *Actinomycetes* with chemical fungicide (metalaxyl) and demonstrated the possibility of replacing fungicide with plant inoculation with endophytic *Actinomycetes*.

#### **6. Plant Growth-Promoting Fungi in Horticultural Crops**

Among microorganisms, fungi can be much more drought-tolerant than bacteria [139] due to a number of mechanisms to overcome drought stress, including osmolytes, thick cell walls, and melanin [140]. Yeast cells are encased in a protective cell wall and cells of filamentous fungi can be connected, allowing water and solutes to flow between them. The filamentous fungi produce extremally long hypha, enabling the extraction of water from remote sites in the soil. Fungal abundance in the soil can increase under drought. They can remain active and even grow under extremely dry conditions. Their resistance to drought allows them to conduct the basic processes of decomposition of polymer compounds and the circulation of C and N [139]. Fungi, bacteria, seaweeds and plants are able to accumulate osmoprotectants, for instance amino acids (e.g., proline, glutamate), carbohydrates (trehalose), sugar alcohols (inositol, mannitol), quaternary ammonium compounds (glycine betaine) and tertiary sulphonium compounds (e.g., dimethylsulphoniopropionate) [141].

Plants use various mechanisms to protect against water deficiency, but some of them are associated with the presence of fungi with special activities (Table 2). Eukaryotic plant endophytes belong mainly to the fungi kingdom [142,143] and the most numerous among these endophytes are Glomeromycota (40%), Ascomycota (31%), Basidiomycota (20%), Zygomycota (0.1%) and unidentified phylla (8%). The Glomeromycota phyllum includes only arbuscular mycorrhizal fungi (AMF), whose species protect against phytopathogens, promote plant growth and counteract diverse stresses (mainly drought and salinity) by activating stress responsive/induced genes in plants. AMF are able to create a symbiosis with many horticultural plants belonging to various families, e.g., *Alliaceae*, *Apiaceae*, *Asteraceae*, *Fabaceae*, *Solanaceae*, *Rosaceae*, and *Oleaceae* [144–146]. Arbuscular mycorrhiza (AM) is the endomycorrhizal symbiotic association improving the nutrient uptake and growth of plants which may protect the host plants from pathogens and the harmful effects of drought [144,146,147]. Interactions with AMF, through an extensive network of hyphae, supply the plant with water from distant places. Studies have shown that AMF mainly use plant-derived carbohydrates in symbiosis with plants, and the plant receives access to the bioavailable minerals absorbed by the fungus from the soil (especially phosphorus). Moreover, the hyphae of fungal strains can uptake phosphorus and ammonium ions much more efficiently than the plant roots [148].

Moreover, fungi may influence the hormonal balance of plants by producing phytohormones (auxins, gibberellins) and through tolerance and resistance pathways, which protect the plant against biotic and abiotic factors. Both ectomycorrhizal (EMF, e.g., *Laccaria* spp.) and endomycorrhizal-AMF fungi (e.g., belonging to the *Glomus*, *Rhizophagus*, *Funneliformis* genera) are capable of inducing the ISR resistance pathways involving JA as a signaling substance, or SAR, in which signaling occurs thanks to the SA molecules [149]. The production of auxins by the fungal endophytes increases the growth of plants under stress [150]. After the plant under stress is colonized by endophytes, stress-induced levels of ABA and some

genes' expressions (e.g., zeaxanthin epoxidase, 9-cis-epoxycarotenoid dioxygenase 3 and ABA aldehyde oxidase 3) were decreased. Similar effects were achieved in the promotion of plant growth and yield under stress conditions, after exogenous phytohormone application such as gibberellic acid [151]. Sometimes endophytes do not have positive effects on plant growth during drought, but they improve plant recovery after water shortage [152].

The presence of some fungal endophytes (e.g., in the *Nicotiana benthamiana* seedlings) increased the leaf area, chlorophyll content, photosynthetic rate, antioxidative enzyme activities, accumulation of osmoprotectants (sugar, protein and proline) and enhanced expression of drought-related genes [153,154]. On the other hand, in drought conditions, the relative water content in leaves and soluble protein content in the tissues of *Cinnamomum migao* did not change after 120-day inoculation with *Glomus lamellosum* [155] (Table 2). In drought-suffering plants, after endophyte inoculation, a lower level of biomolecule degradation was observed as a consequence of the reduced level of ROS production, e.g., in tomato [156]. In the face of drought stress, inoculation with *Piriformospora indica* (Table 2) mobilized activities of peroxidase (POX), catalase (CAT) and superoxide dismutase (SOD) in the leaves [157]. The endophytic fungal strains of *Ampelomyces* sp. isolated from soil exposed to drought enhanced drought tolerance in tomato [143,158]. Inoculation of tomato seedlings with *Alternaria* spp. strains under drought conditions resulted in the maintenance of the photosynthetic efficiency and effective reduction of ROS accumulation [156].




**Table 2.** *Cont.*

The presence of beneficial fungal strains in soil and plants might specifically induce resistance by releasing elicitors belonging to fungal-derived compounds, e.g.: chitin, chitosan, ergosterol, β-glucans [151]. Moreover, among the pathogenic microorganisms, the filamentous fungi are responsible for horticultural crop diseases. It should be also noted that fungal endophytes belonging to PGP (e.g., *Colletotrichum* sp., *Alternaria* sp., *Fusarium* sp. and *Aspergillus* sp.) induce plant resistance and increase plant tolerance to drought, but may also produce mycotoxins in plants [162]. To increase specificity and enhancement of the induction, elicitors can be derived from the nonpathogenic fungi belonging to the same genus as the pathogenic strains causing plant diseases, e.g., *Fusarium* or *Trichoderma* [152]. In grapevine with black-foot disease (*Dactylonectria* and *Cylindrocarpon* genera), the relative abundance of the potential biocontrol agent *Trichoderma* in the root endosphere, rhizosphere, and bulk soil under drought stress (25% irrigation regime) was significantly lower than in control conditions (50–100% irrigation regime) [163]. Moreover, enrichment in AMF *Funneliformis* during drought was observed.

Recent and particularly promising studies have focused on the determination of the effectiveness and reliability of a mixture of bacterial and fungal strains (Table 3).


**Table 3.** Selected activities of beneficial consortia (bacteria with fungi) under drought stress conditions in horticultural plant species; H—higher level/content; L—lower level/content; CAT—catalase; APX—ascorbate peroxidase; Chl—chlorophyll.

#### **7. Conclusions**

The EGD emphasizes sustainable food production by the crucial reduction in the use of pesticides, biocides and chemical mineral fertilizers and increase of organic (ecological) production. Consequently, in many European countries, continuous research has been carried out on natural biopreparations (biocontroling, biofertilizers) containing selected microorganisms with different activities, and/or their metabolites.

The key problems of using biopreparations containing various microorganisms include limiting the possibility of introducing the microbial inoculum to the appropriate conditions and the low repeatability of their activities. This might be due to drought stress during the vegetative period in comparison to microorganisms tested in the optimal conditions.

The resistance of plants that interact with microorganisms in drought conditions is enhanced because it is induced by both abiotic (stress factor) and the biotic (microorganism) elicitors. In drought conditions, many cultivated horticultural plants use their own numerous mechanisms (morphological, physiological, anatomical, biochemical or molecular) to counteract the negative effects and are supported by endophytes that constantly inhabit them, and rhizospheric microorganisms existing in the vicinity of roots.

The use of preparations containing fungal strains, which are more tolerant to drought than bacteria, provides many tolerance mechanisms, and their abundance increases in water-limiting conditions. Fungi, through their specific growth and traits, allow intensive soil exploration, water extraction and penetration of plant tissues influencing the plant and might be more effective compared to bacteria with the same activity.

Studies on the influence of drought stress on horticultural plants have indicated that the application of various microorganisms allows efficient protection of plants, despite our restricted knowledge about these mechanisms of action. Due to such a high variability of the environment, biopreparations should be multicomponent in order to achieve appropriate levels of microorganism cooperation and the final desired effect. The combination of fungal and bacterial strains into one preparation gives even better effectiveness and reliability, allowing us to consider higher crop-specificity, and seems to be particularly promising.

**Author Contributions:** Conceptualization, A.H.; E.O. and J.J.-S.; writing—original draft preparation, ´ E.O.; A.H.; E.R. and J.J.-S.; writing—review and editing, A.H.; E.O.; E.R. and M.S.; visualization, A.H.; ´ E.O.; E.R. and M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Investigating Evolution and Balance of Grape Sugars and Organic Acids in Some New Pathogen-Resistant White Grapevine Varieties**

**Tommaso Frioni \*, Cecilia Squeri, Filippo Del Zozzo, Paolo Guadagna, Matteo Gatti, Alberto Vercesi and Stefano Poni**

> Department of Sustainable Crops Production, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy; cecilia.squeri@unicatt.it (C.S.); filippo.delzozzo@unicatt.it (F.D.Z.); paolo.guadagna@unicatt.it (P.G.); matteo.gatti@unicatt.it (M.G.); alberto.vercesi@unicatt.it (A.V.); stefano.poni@unicatt.it (S.P.) **\*** Correspondence: tommaso.frioni@unicatt.it; Tel.: +39-05-2359-9384

**Abstract:** Breeding technologies exploiting marker-assisted selection have accelerated the selection of new cross-bred pathogen-resistant grapevine varieties. Several genotypes have been patented and admitted to cultivation; however, while their tolerance to fungal diseases has been the object of several in vitro and field studies, their productive and fruit composition traits during ripening are still poorly explored, especially in warm sites. In this study, five white pathogen-resistant varieties (PRV) listed as UD 80–100, Soreli, UD 30–080, Sauvignon Rytos, Sauvignon Kretos were tested over two consecutive seasons in a site with a seasonal heat accumulation of about 2000 growing degree days (GDDs), and their performances were compared to two *Vitis vinifera* L. traditional varieties, Ortrugo and Sauvignon Blanc. Berries were weekly sampled from pre-veraison until harvest to determine total soluble solids (TSS) and titratable acidity (TA) dynamics. All tested PRV exhibited an earlier onset of veraison and a faster sugar accumulation, as compared to Ortrugo and Sauvignon Blanc, especially in 2019. At harvest, Sauvignon Blanc was the cultivar showing the highest titratable acidity (8.8 g/L). Ortrugo and PRV showed very low TA (about 4.7 g/L), with the exception of Sauvignon Rytos (6.5 g/L). However, data disclose that Sauvignon Rytos higher acidity at harvest relies on higher tartrate (+1.1 to +2.2 g/L, as compared to other PRV), whereas in Sauvignon Blanc, high TA at harvest is due to either tartaric (+1 g/L, compared to PRV) and malic (+2.5 g/L, compared to PRV) acid retention. Overall, Sauvignon Rytos is the most suited PRV to be grown in a warm climate, where retaining adequate acidity at harvest is crucial to produce high-quality white wines. Nevertheless, canopy and ripening management strategies must be significantly adjusted, as compared to the standard practice employed for the parental Sauvignon Blanc.

**Keywords:** *Vitis* spp.; piwi cultivars; disease-resistant varieties; malic acid; ripening; fruit composition; downy mildew

#### **1. Introduction**

Warming trends are severely endangering viticulture and its sustainability [1,2]. In many wine regions, the incidence of drought and the rise of temperature are affecting vineyard performances by compromising vine physiology, compressing phenology, and boosting grapes' metabolism. In white varieties, especially when intended for sparkling wine, accelerated ripening leads to excessive sugars and inadequate acidity and aromas at harvest, resulting in unbalanced wines [1,3]. Moreover, the advancement of veraison increases the susceptibility of grapes to sunburn and dehydration phenomena by exposing ripening berries to the hottest days of the year, when the evaporative demand is maximum [2]. As a consequence, several wine regions are seeking either new cultural practices viable to decompress sugar accumulation and acidity decrease, or late-ripening cultivars, especially when the target hits white and/or sparkling wines [1,4–6]. In addition, contrary

**Citation:** Frioni, T.; Squeri, C.; Del Zozzo, F.; Guadagna, P.; Gatti, M.; Vercesi, A.; Poni, S. Investigating Evolution and Balance of Grape Sugars and Organic Acids in Some New Pathogen-Resistant White Grapevine Varieties. *Horticulturae* **2021**, *7*, 229. https://doi.org/ 10.3390/horticulturae7080229

Academic Editors: Agnieszka Hanaka, Jolanta Jaroszuk-Sciseł ´ and Małgorzata Majewska

Received: 13 July 2021 Accepted: 2 August 2021 Published: 6 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

to what one might think, fungal diseases are a serious issue also in warm and hot regions. Pathogen spread varies according to the seasonal weather evolution; the increase of average temperatures broadens the time window available for pathogens to complete additional biological cycles within one year [7]. Consequently, numerous pesticides applications are still frequently needed during the season to control downy mildew (DM) and powdery mildew (PM), even in warm climates. For instance, in northern Italy, where average heat accumulation easily exceeds >2000 growing degree days (GDDs), several pesticides residues have been detected in the groundwater [8].

Pathogen-resistant grapevine varieties (PRV) are complex interspecific hybrids obtained by multiple crossings of resistant *Vitis* spp. accessions (mostly *V. amurensis*, *V. rupestris*, and *V. berlandieri*) with selected cultivars of *Vitis vinifera* which, conversely, lacks genetic resistance towards *Plasmopara viticola* (Pv) and *Erisyphae necator* (En). In most Mediterranean grape growing regions, adoption of PRV has been hindered for many decades. Hindrance to PRV was not merely due to their botanical origins, yet to the fact that non-*vinifera* genotypes often carry undesired or atypical flavour, and wines are not appreciated [9–12].

Over the last decades, however, the increasing concerns of consumers about ecological issues and product safety, the rapid surge of organic (or environmentally certified) wines, and the spread of new awareness and values in modern society, have led to a renovated interest in PRV. Moreover, new breeding technologies have opened up new frontiers [9,11]. For instance, in Italy, a few breeding programmes have originated new pathogen-resistant varieties by marker-assisted selection (MAS) sorted out within progenies obtained by crossing *Vitis vinifera* cultivars with complex hybrids having genetic resistance to DM and PM. MAS has considerably shortened the time needed to identify the crossings carrying one or more quantitative trait loci (QTL), inducing higher tolerance to Pv and En. Within this pool of individuals, the genotypes having the most similar must biochemical composition to the *V. vinifera* parentals were identified. Currently, several selected resistant cultivars are available for growers [11,13–15]. At the same time, also national and European regulations changed and, from 2009 to date, many hybrids PRV have been admitted for cultivation.

A fairly high number of papers have described the tolerance to DM and PM of these new PRV under various environmental conditions. Depending upon the number and types of QTLs, these PRV exhibit a different degree of tolerance to Pv and En; nonetheless, these varieties have been confirmed to be more tolerant than traditional *V. vinifera* [9,14–19].

Conversely, the literature shows a paucity of scientific papers evaluating the productive performances and fruit composition of such new PRV. Poni et al. [20] have reported that potted vines of the red resistant accession UD 72–096 (Sangiovese x Bianca) had comparable yield with a standard susceptible Sangiovese clone but, when harvested on the same date, UD 72–096 showed significantly higher TSS and TA than Sangiovese. Moreover, UD 72–096 grapes had higher concentrations of skin acylated and coumarated anthocyanins, lower mono-glucosidic, and higher di-glucosidic anthocyanin forms, as well as lower quercetin 3-O-glucoside concentration, as compared to the susceptible Sangiovese. In north-eastern Spain, the resistant genotype Sauvignon Kretos (Sauvignon Blanc x Kozma 20-3) exhibited a similar yield and must composition to the parental Sauvignon Blanc. In this case, the two varieties were picked at different dates, but the date of harvest of Sauvignon Blanc was not provided [21].

To the best of our knowledge, no other scientific papers have tested fruit ripening kinetics or enological parameters of new PRV. Several technical reports suggest that these varietals are poor in di-glucoside anthocyanin forms and free from furaneol and other undesired volatile compounds typical of non-*vinifera* cultivars [22,23]. Analytical and sensorial traits of musts and wines are overall promising and confirm that these varietals, if adequately managed in the field and the winery, can produce wines comparable to those obtained from the *V. vinifera* parentals [22–25].

A shared result of these technical reports is the earlier annual cycle of PRV [22]. The high heritability of earliness traits, when crossing grapevine cultivars, is something

well known, and some PRV originating from pioneer breeding programmes, such as Bronner, Solaris, or Souvignier Gris, show accelerated ripening and fast sugar accumulation [9,22,26–31]. This can be linked to the locations and the times of the selection of these genotypes, obtained by breeding programmes set in cool climates, and well before warming trends affected Mediterranean wine regions. PRV introduction in warm regions, where global warming has already caused a significant compression of the annual growth cycle and a consistent advancement of phenological stages, seems to be an additional matter of concern.

The aim of this work was to evaluate vine performances and fruit-ripening dynamics of five new white PRV, in a region where local viticulture is suffering the negative effects of warming trends on grape biochemical balance. The resistant genotypes were compared to Sauvignon Blanc and Ortrugo, two of the most cultivated white *Vitis vinifera* genotypes in the area. Our hypothesis was that the tested PRV might have a different degree of suitability to warm and hot sites according to three specific traits, namely, (i) onset of veraison time, (ii) malic acid degradation rates, and(iii) maintenance of a minimum acid pool at harvest.

#### **2. Materials and Methods**

#### *2.1. Experimental Site and Treatment Layout*

The study was carried out for two years (2019–2020) in a varietal collection located at Vicobarone (Ziano Piacentino, Italy, 44◦59 31.7" N 9◦21 27.8" E, 268 m a.s.l.). In the vineyard, 5 recently obtained white PRV were planted in 2016. The PRV present in the collection were obtained by crossing a *V. vinifera* parental (namely Sauvignon Blanc or Friulano) with an interspecific *Vitis* hybrid (namely, Kozma 20-3 or Bianca) able to confer resistance to PM and DM [32,33]. The five PRV were UD 80–100 (Friulano x Bianca), Soreli (Friulano x Kozma 20-3), UD 30–080 (Sauvignon Blanc x Kozma 20-3), Sauvignon Kretos (Sauvignon Blanc x Kozma 20-3), and Sauvignon Rytos (Sauvignon Blanc x Bianca) (Figure S1). To date, Sauvignon Rytos, Sauvignon Kretos, and Soreli are already admitted for cultivation in Italy, whereas UD 80–100 and UD 30–080 are still under evaluation. Ortrugo (VCR245) and Sauvignon Blanc (R3) vines, planted in the same year nearby these 5 PRV, were selected as *V. vinifera* references. Ortrugo was chosen since it is the most common white variety in the region and also is susceptible to summer temperatures in terms of fast acidity loss [6]. Sauvignon Blanc was instead included in the study since it was one of the parentals of three PRV among the five planted in the vineyard. All the cultivars, grafted on SO4 rootstock, were planted at 2.4 m × 0.8 m spacing (between row and within row distance, respectively) for a resulting density of 5125 plants/hectare. The vineyard has a soft slope (about 6◦) and an east-facing aspect, with rows following E–W orientation. Vines were trained to a unilateral Guyot with about 10 nodes on the primary horizontal cane and two more on a spur left for annual cane renewal.

Each cultivar was present in one row of 80 m, encompassing 100 vines. The vineyard was divided into three uniform blocks along the rows. Nine test vines per varietal (three vines per cultivar per block) were randomly chosen in 2019, tagged, and then maintained also for the following season. These selected vines were used for detailed assessment of vegetative growth, yield components, and grape composition at harvest. The vineyard is typically non-irrigated, whereas fertilisation was uniform across all the vineyard surfaces and conducted based on local sustainable practices. Vines were trimmed once shoots outgrew 20 cm above the top wire. In order to prevent the spread of pathogens on both PRV and reference varieties, control of diseases was differentiated based on the degree of tolerance of the different genotypes. Details of pest management layout are provided in Table S1. In both seasons, none of the experimental vines showed symptoms of DM and PM at harvest. The minimum, mean, and maximum daily air temperature (◦C) and daily rainfall (mm) from 1 January (DOY 1) to 31 December (DOY 365) were recorded in each season by a nearby weather station. Cumulative GDDs were then calculated according to Winkler [34].

#### *2.2. Phenological Stages, Vegetative Growth, and Yield Components*

In both years, bud break (BBCH09) and the onset of veraison (BBCH81) were assessed on each tagged vine according to Lorenz et al. [35]. Each season, in late spring (end of May–beginning of June), the number of inflorescences bore on each shoot was recorded according to the position of the shoot onto the horizontal cane. Total vine fruitfulness was then calculated as total inflorescences/total shoots ratio for the entire vine and for basal nodes (base node + count nodes 1 and 2).

All the experimental vines were picked on the same day, when a berry total soluble solids concentration of about 20 ◦Brix was achieved for Ortrugo, according to the optimal ripening threshold for this cultivar identified by Gatti et al. [36]. At harvest, tagged vines were individually picked, the mass of grapes was weighed, and the total bunch number per vine was counted. The average bunch weight was then calculated. Concurrently, three representative bunches per vine, usually inserted on basal, median, and apical cane portions, were taken to the laboratory. On each bunch, the number of berries was counted, and the mass of berries was weighed. Rachis length was measured, and bunch compactness was expressed as the ratio of total berry mass to rachis length. Berries were crushed, and the obtained must was then used for technological maturity and organic acids determination (see next paragraphs).

At harvest, the leaves inserted at nodes 3, 6, 9, 12, 15, 18, and 21 of the distal shoot of each tagged vine were collected with all the leaves from two lateral shoots developing below the trimming cut. The area of each leaf was measured with an LI-3000A leaf area meter (LI-COR Biosciences, Lincoln, NE, USA). Immediately after leaf fall, the number of nodes per cane and the number of nodes of each lateral cane were counted. The final leaf area was then estimated from the main and lateral shoots per vine on the basis of node counts and leaf-blade areas. Total vine leaf area was calculated as a sum of the two components. Leaf area to yield ratio (LA/Y) was finally calculated by dividing the total leaf area and yield of each tagged vine.

#### *2.3. Grape Composition*

Each year, from veraison (TSS ~4.5 to 5 ◦Brix) until harvest, three 100-berry samples were taken weekly from untagged vines of each varietal. These samples were not taken from the tagged vines so that the natural dynamic of grape ripening would not be altered due to the progressive reduction of the pending yield. During sampling, it was assured that the removed berries were taken from bunches located on both sides of the row and, within each bunch, the top, median, and bottom portions were also represented. In 2020, untagged Ortrugo and Sauvignon Blanc were not picked, and two additional post-harvest samplings were conducted. Sampled berries were brought to the laboratory, weighed, and crushed to obtain a must. Musts were analysed immediately for TSS using a temperature-compensated desk refractometer, whereas pH and TA were measured by titration with 0.1 N NaOH to a pH 8.2 end point and expressed as g/L of tartaric acid equivalents. TSS/TA ratio at harvest was then calculated.

TSS accumulation rates (◦Brix day−1) were calculated from pre-veraison to harvest dividing the difference in TSS between two subsequent samplings by the number of elapsed days. The same procedure was carried out based on malic acid concentration in order to calculate degradation rates (g/L day<sup>−</sup>1).

For better readability of data, when appropriate, seasonal trends and correlations of specific parameters were graphed separately for PRV having Sauvignon Blanc as *V. vinifera parental* (UD 30–080, Sauvignon Kretos, and Sauvignon Rytos, compared with Sauvignon Blanc) and for PRV obtained crossing Friulano (UD 80–100 and Soreli, compared with Ortrugo).

#### *2.4. HPLC Analysis*

To assess tartaric and malic acid concentrations in all samples taken seasonally and at harvest, an aliquot of the must was diluted four times, then filtered through a 0.22 μm

polypropylene syringe for high-performance liquid chromatography (HPLC) analysis and transferred to auto-sampler vials. All solvents were of HPLC grade. Water Milli-Q quality, acetonitrile, and methanol were obtained from VWR. L-(+)-tartaric acid and L-(-)-malic acid standards were purchased from Sigma-Aldrich. The chromatographic method was developed using an Agilent 1260 Infinity Quaternary LC (Agilent Technology) consisting of a G1311B/C quaternary pump with an inline degassing unit, G1329B autosampler, G1330B thermostat, G1316B thermostated column compartment, and a G4212B diode array detector (DAD) fitted with a 10 mm path, 1 μL volume Max-Light cartridge flow cell. The instrument was controlled using the Agilent Chemstation software version A.01.05. The organic acids analysis used an Allure Organic Acid Column, 300 × 4.6 mm, 5 μm (Restek). Separation was performed in isocratic conditions using water, pH-adjusted to 2.5 using ortho-phosphoric acid, at a flow rate of 0.8 mL/min. The column temperature was maintained at 30 ± 0.1 ◦C, and 15 μL of the sample was injected. The elution was monitored at 200 to 700 nm and detected by UV–Vis absorption with DAD at 210 nm. Organic acids were identified using authentic standards, and quantification was based on peak areas and performed by external calibration with standards. The ratio between tartaric acid and malic acid (HT/HM) was then calculated.

#### *2.5. Statistical Analysis*

Vine performance data were subjected to a two-way analysis of variance (ANOVA) using IBM SPSS 25 (IBM, Chicago, IL, USA). Treatment comparison was performed using the Student–Neuman–Keuls test at *p* ≤ 0.05. Year × treatment interaction was partitioned only when the F test was significant.

Repeated measures of the same parameters (TSS, pH, TA, tartaric acid, malic acid) taken at different dates along the season were analysed with the repeated-measure analysis of variance (ANOVA) routine embedded in IBM SPSS Statistics 25. Equality of variances of the differences between all possible pairs of within-subject conditions was assessed via Mauchly's sphericity test. The least squared (LS) mean method at *p* ≤ 0.05 was used for multiple comparisons within dates.

Data about grapes TSS, TA, malic acid, and tartaric acid progression were also reelaborated to predict fruit composition at the thresholds of TSS = 20 ◦Brix and, separately, TA = 7.5 g/L, for all the tested varieties. The balance among malate, tartrate, and TA or TSS for grapes harvested at those thresholds was compared by a one-way analysis of variance (ANOVA) using IBM SPSS 25 (IBM, Chicago, IL, USA).

The correlations existing between TSS and TA values of grapes sampled during the season were subjected to regression analysis, using SigmaPlot 11 (Systat Software Inc., San Jose, CA, USA).

#### **3. Results**

#### *3.1. Weather Conditions and Phenology*

At the experimental site, 2019 was marked by a wet and cold spring (Figure 1A). From 1 April to the end of May, 274 mm of rain and only 260 GDDs were recorded, vs. 62 mm and 369 GDDs recorded in the same period of 2020 (Figure 1B). Then, summer 2019 was conversely hotter than summer 2020, so that at the end of August, a comparable amount of GDDs were accumulated from 1 April in the two years (1568 GDDs in 2019, 1571 GDDs in 2020). From 1 April to 31 October, 2007 GDDs were accumulated in 2019 vs. 1917 GDDs in 2020.

All the genotypes tested in the trial had a similar bud-break date (BBCH09) in both seasons, with cv. Ortrugo showing a delay of a few days, as compared to other varieties (Table 1). Sauvignon Kretos was the cultivar showing the earliest onset of veraison (BBCH81) on both seasons (between DOYs 195–197). The onset of veraison was anticipated in 2020 in Sauvignon Rytos, Sauvignon Blanc, and Ortrugo by 8 days, compared to the previous year, whereas other genotypes did not exhibit a similar variation between seasons.

Considering only resistant varieties, UD 30–080 was the cultivar exhibiting, in both years, the later onset of veraison.

**Figure 1.** Seasonal daily trends of minimum temperature (Tmin), mean temperature (Tmean), maximum temperature (Tmax), rainfall (blue bars), and heat accumulation (cumulative GDDs) calculated following Winkler [34] in 2019 (**A**) and 2020 (**B**). DOY = day of the year.

**Table 1.** Date of achievement of main phenological stages and key ripening thresholds for five pathogen-resistant grapevine genotypes and two non-resistant *Vitis vinifera* cultivars in 2019 and 2020.


<sup>1</sup> DOY = day of the year. <sup>2</sup> Phenological stages were identified according to Lorenz et al. [35]. <sup>3</sup> TSStr = achievement of grapes total soluble solids threshold of 20 ± 1 ◦Brix; MAtr = achievement of grapes malic acid threshold of 2.5 ± 0.5 g/L.

#### *3.2. Vegetative Growth and Shoot Fruitfulness*

UD 30–080 had a significantly higher main shoot leaf area/vine at the end of the season, as compared to Sauvignon Rytos and UD 80–100, with other genotypes scoring intermediate values (Table 2). Lateral shoot leaf area/vine was unaffected by the genotype, so that differences in total vine leaf area mostly tracked those in the main shoot leaf area.

**Table 2.** Vegetative growth and shoot fruitfulness of five pathogen-resistant grapevine genotypes and two non-resistant *Vitis vinifera* cultivars in 2019 and 2020.


<sup>1</sup> V = variety; Y = year. <sup>2</sup> Means within columns noted by different letters are different by SNK test. <sup>3</sup> \*, \*\* and \*\*\* indicate significant difference per *p* ≤ 0.05, *p* ≤ 0.01 and *p* ≤ 0.001, respectively. ns: not significant.

> Soreli and Sauvignon Rytos exhibited the highest shoot fruitfulness (1.76 and 1.73 inflorescences per shoot, respectively). UD 80–100 and UD 80–030 showed a slightly lower shoot fruitfulness (Table 2), with Sauvignon Kretos setting at intermediate levels. Sauvignon Blanc and Ortrugo were the two cultivars having the lowest number of inflorescences per shoot (1.23 and 0.93, respectively). This trend was substantially confirmed when considering only basal nodes' fruitfulness (i.e., base bud until count node 2).

#### *3.3. Yield, Bunch Morphology, and Vine Balance*

Harvest was performed on DOY 245 in 2019 and on DOY 238 in 2020. Soreli was the variety having the highest yield per vine (3.53 kg). All the remaining cultivars exhibited a significantly lower yield (from 2.08 to 2.43 kg/vine), with the sole exception of Sauvignon Blanc (Table 3). The high productivity of Soreli was associated with the high number of bunches per vine (30). However, the number of bunches per vine in Sauvignon Rytos and Sauvignon Kretos was not significantly lower than Soreli; rather, these two genotypes had a lower bunch weight and number of berries per bunch (−29% and −23%, respectively). Ortrugo was the variety with the lower number of bunches per vine (13), paralleled by the highest bunch weight (181 g) and number of berries per bunch (170). Sauvignon Rytos had small bunches (85 g) with a low number of berries per bunch (83). All PRV had medium bunch compactness, significantly lower than Ortrugo.

UD 30–080 showed the highest LA/Y ratio (1.64 m2/kg), whereas all the other cultivars had a lower LA/Y ratio (from 0.92 to 1.33 m2/kg), except for Ortrugo, which set at intermediate levels (1.42 m2/kg).


**Table 3.** Yield, vine balance, and bunch morphology of five pathogen-resistant grapevine genotypes and two non-resistant *Vitis vinifera* cultivars in 2019 and 2020.

<sup>1</sup> V = variety; Y = year. <sup>2</sup> Means within columns noted by different letters are different by SNK test. <sup>3</sup> \*, \*\* and \*\*\* indicate significant difference per *p* ≤ 0.05, *p* ≤ 0.01 and *p* ≤ 0.001, respectively. ns: not significant.

#### *3.4. Grapes' TSS, pH, and TA during Ripening*

All PRV showed earlier berry sugar accumulation than Ortrugo and Sauvignon Blanc (Figure 2). In both years, Sauvignon Kretos had a significantly higher TSS than any of the other genotypes at any sampling date. UD 30–080 and Sauvignon Rytos had lower sugars than the other PRV right after veraison. However, later on, both genotypes showed a faster TSS accumulation rate, reducing the gap with other PRV. The threshold of 20 ◦Brix was achieved much earlier by the PRV in both years (Table 1). Overall, Sauvignon Blanc and Ortrugo lagged behind PRV by 15 days in 2019, and by approximately 10 days in 2020.

Similarly, must pH in PRV was constantly higher in both years (Figure 2C,D). In Sauvignon Rytos, only the dynamic of pH was more similar to the one of Ortrugo and Sauvignon Blanc, especially in 2020.

Figure 2E,F shows the early loss of TA by UD 80–100, Soreli and Sauvignon Kretos. In both years, Sauvignon Rytos maintained higher TA than other PRV until the end of the season. Ortrugo and Sauvignon Blanc showed a delayed acidity loss than Sauvignon Rytos, especially in 2019; however, Ortrugo crossed values lower than 5 g/L (on DOY 245 in 2019, on DOY 230 in 2020), whilst Sauvignon Blanc maintained a TA higher than 8 g/L until the end of the season. Over the last sampling dates, in 2019, Sauvignon Rytos tracked Ortrugo in terms of TA, whereas, in 2020, it set at intermediate levels between Ortrugo (and PRV) and Sauvignon Blanc.

All correlations between TSS and TA fit a quadratic model for any of the tested cultivars (Figure 3). The model shows that in 2019 Sauvignon Blanc, Sauvignon Kretos and UD 30–080 had similar TA for any TSS level, up to the threshold of 15 ◦Brix (Figure 3A). At higher TSS, Sauvignon Kretos and UD 30–080 had a TA lower than Sauvignon Rytos and Sauvignon Blanc. In 2020 (Figure 3B), conversely, Sauvignon Blanc had lower TA for any TSS below 15 ◦Brix, whereas, above this threshold, Sauvignon Blanc, Sauvignon Rytos, and Sauvignon Kretos grouped together and only UD 30–080 had lower TA. In both years, Ortrugo and Soreli had quite low TA for any TSS level, whereas, conversely, UD 80–100 exhibited the opposite behaviour (Figure 3C,D).

**Figure 2.** Seasonal evolution of grapes total soluble solids (TSS, panels **A**,**B**), pH (**C**,**D**) and titratable acidity (**E**,**F**) in 2019 (**A**,**C**,**E**) and 2020 (**B**,**D**,**F**) for 5 pathogens-resistant varieties (white symbols) and 2 reference *V. vinifera* cultivars (black symbols). Each point represents the average of three replicates ± SE. DOY = day of year.

#### *3.5. Trends for Grapes Organic Acids Concentration*

In both years, UD 80–100 was the variety showing the highest malic acid concentration pre-veraison (about 35 g/L). Sauvignon Kretos had the earliest decrease of malic acid, achieving the threshold of 2.5 g/L on DOY 217 in 2019 and on DOY 213 in 2020 (Figure 4A,B, Table 1). In 2019 all the PRV had an earlier peak of malic acid and onset of its degradation, as compared to Ortrugo and Sauvignon Blanc. In 2020, this was confirmed as related to Sauvignon Blanc, even if the gap was ostensibly narrower, whereas in Ortrugo the trend of malic acid degradation was comparable to the one exhibited by Soreli and Sauvignon Rytos. In both years, at the end of the season, Ortrugo reached a minimum malic acid concentration comparable to those of PRV (below 1 g/L), whereas Sauvignon Blanc maintained significantly higher malic acid concentrations (above 2.5 g/L).

**Figure 3.** Seasonal variation of titratable acidity expressed as a function of total soluble solids (TSS) in 2019 (**A**,**C**), and 2020 (**B**,**D**), for 5 pathogens-resistant varieties (white symbols) and 2 reference *V. vinifera* cultivars (black symbols). Data were fit to the following equations: UD 30–080 2019 y = 70.07 <sup>−</sup> 5.62x + 0.12x2, R2 = 0.994; UD 30–080 <sup>2020</sup> y = 60.68 <sup>−</sup> 4.10x + 0.06x2, R2 = 0.996; Sauvignon Kretos 2019 y = 67.86 <sup>−</sup> 5.21x + 0.11x2, R2 = 0.976; Sauvignon Kretos 2020 y = 63.80 <sup>−</sup> 4.54x + 0.08x2, <sup>R</sup><sup>2</sup> = 0.991; Sauvignon Rytos <sup>2019</sup> y = 58.94 <sup>−</sup> 4.03x + 0.08x2, R2 = 0.991; Sauvignon Rytos 2020 y = 59.89 <sup>−</sup> 4.39x + 0.09x2, R2 = 0.993; Sauvignon Blanc 2019 y = 49.32 <sup>−</sup> 2.53x + 0.02x2, <sup>R</sup><sup>2</sup> = 0.971; Sauvignon Blanc 2020 y = 53.47 <sup>−</sup> 4.20x + 0.09x2, R2 = 0.980; UD 80–100 2019 y = 72.20 <sup>−</sup> 5.60x + 0.12x2, R2 = 0.989; UD 80– 100 2020 y = 73.80 <sup>−</sup> 5.53x + 0.11x2, R2 = 0.992; Soreli 2019 y = 57.47.86 <sup>−</sup> 4.91x + 0.11x2, <sup>R</sup><sup>2</sup> = 0.966; Soreli 2020 y = 56.61 <sup>−</sup> 4.66x + 0.10x2, R2 = 0.989; Ortrugo 2019 y = 49.13 <sup>−</sup> 4.00x + 0.09x2, R2 = 0.983; Ortrugo 2020 y = 50.06 <sup>−</sup> 4.23x + 0.10x2, R2 = 0.991. All the correlations listed were significant per *p* < 0.05.

In both years, Sauvignon Rytos had the highest grapes tartaric acid concentration during ripening, if considering only PRV (Figure 4C,D). In 2019, Ortrugo and Sauvignon Blanc had lower tartaric acid than Sauvignon Rytos after veraison, even if later in the season Sauvignon Blanc maintained significantly higher tartaric acid than Ortrugo and Sauvignon Rytos (approximately +3 g/L). In 2020, conversely, Sauvignon Blanc had a similar decrease of tartaric acid to the one exhibited by Ortrugo, whereas in Sauvignon, Rytos tartaric acid concentration was consistently higher (+3.3 g/L on DOY 237).

#### *3.6. Sugar Accumulation and Malic Acid Degradation Rates*

In 2019, UD-30–080, Sauvignon Kretos and Sauvignon Rytos exhibited relatively high TSS accumulation rates (Figure 5A), ranging between 0.40 and 0.65 ◦Brix day−<sup>1</sup> until DOY 231. Conversely, Sauvignon Blanc jumped from 0.1 to 0.4 TSS day−<sup>1</sup> on DOY 217 and, after peaking at 0.65 TSS day−<sup>1</sup> on DOY 224, it decreased, together with the PRV. On the other hand, Ortrugo did not exceed 0.6 ◦Brix day−1, although it maintained higher sugar accumulation rates late in the season (Figure 5C). In 2020 (Figure 5B,D), PRV had a higher peak of TSS accumulation rates (0.8–1.0 ◦Brix day<sup>−</sup>1), as compared to the previous year, yet occurring approximately at the same DOYs (209–223). Conversely, in Sauvignon

Blanc and Ortrugo, maximum TSS accumulation rates occurred much earlier than in 2019 (approximately 15–20 days). Moreover, in Ortrugo, the maximum TSS accumulation rate, recorded on DOY 214, reached the peak of 1.4 ◦Brix day−1, before declining to values comprised between 0.2 and 0.4 ◦Brix day−<sup>1</sup> for the rest of the season.

**Figure 4.** Seasonal evolution of grapes malic acid (panels **A**,**B**) and tartaric acid (**C**,**D**) in 2019 (**A**,**C**) and 2020 (**B**,**D**) for 5 pathogens-resistant varieties (white symbols) and 2 reference *V. vinifera* cultivars (black symbols). Each point represents the average of three replicates ± SE. DOY = day of year.

Sauvignon Kretos was also the cultivar showing the earliest peak of malic acid degradation rate, in both years (Figure 5G,H). In 2019, Sauvignon Blanc was the variety showing the most delayed onset of malic acid degradation, with a sudden peak of 2.1 g/L day−<sup>1</sup> occurring only on DOY 224. UD 30–080 and Sauvignon Rytos set at intermediate levels between Sauvignon Blanc and Sauvignon Kretos, in terms of timing and magnitude of malic acid degradation rates increase.

In 2020, UD 30–080 and Sauvignon Rytos had a dynamic of the degradation of malic acid comparable to the one exhibited by Sauvignon Blanc, even if this latter peaked at 2.4 g/L day−<sup>1</sup> vs. 1.6–2.0 g/L day−<sup>1</sup> exhibited by UD 30–080 and Sauvignon Rytos. UD 80–100 and Soreli had a malic-acid-degradation-rate dynamic comparable to other PRV (Figure 5I,J). In 2020, UD 80–100 scored, on DOY 209, the highest malic acid degradation rate recorded during the experiment (3.1 g/L day<sup>−</sup>1).

**Figure 5.** Seasonal evolution of grapes total soluble solids accumulation rates (panels **A**–**D**), malic acid degradation rate (panels **G**–**J**) and minimum temperature (blue line), mean temperature (grey line), and maximum temperatures (red lines) of the period (panels **E**,**F**) in 2019 (**A**,**C**,**E**,**G**,**I**) and 2020 (**B**,**D**,**F**,**H**,**J**) for 5 pathogens-resistant varieties (white symbols) and 2 reference *V. vinifera* cultivars (black symbols). Each point represents the average of three replicates ± SE. DOY = day of year. T = temperature.

#### *3.7. Fruit Composition at Harvest*

At harvest, all the PRV had higher TSS than Ortrugo and Sauvignon Blanc (from +2.5 ◦Brix in Soreli to +5.5 ◦Brix in Sauvignon Kretos), with the sole exception of UD 30–080 (Table 4). Sauvignon Blanc was the variety showing the higher TA at harvest (8.81 g/L). Ortrugo, UD 30–080, Soreli, UD 80–100 had quite low TA, comprised between 4.08 and 5.07 g/L. By contrast, Sauvignon Rytos maintained a TA of 6.50 g/L, halfway between this group of cultivars and Sauvignon Blanc. The high TA of Sauvignon Blanc is linked to a high malic acid concentration (3.45 g/L). Conversely, in Sauvignon Rytos, the malic acid concentration at harvest was similar to those of cultivars having low TA. Sauvignon Rytos had, instead, the highest tartaric acid concentration (7.44 g/L), together with Sauvignon Blanc.

**Table 4.** Fruit composition at harvest of five pathogen-resistant grapevine genotypes and two non-resistant *Vitis vinifera* cultivars in 2019 and 2020.


<sup>1</sup> V = variety; Y = year. <sup>2</sup> Means within columns noted by different letters are different by SNK test. <sup>3</sup> \*, \*\* and \*\*\* indicate significant difference per *p* ≤ 0.05, *p* ≤ 0.01 and *p* ≤ 0.001, respectively. ns: not significant.

> Sauvignon Blanc was the variety scoring the lowest TSS/TA and HT/HM ratio (2.16 and 2.21, respectively). Sauvignon Rytos had a TSS/TA ratio of 3.60, comparable to Ortrugo, and an HT/HM of 8.36, similar to Sauvignon Kretos (8.48). Ortrugo had a lower HT/HM than Sauvignon Rytos and Sauvignon Kretos (6.93), yet higher than all the other PRV.

> Figure 6A shows that, if the tested varieties had been harvested at TSS = 20 ◦Brix, the acid pool of Sauvignon Rytos would be mostly contributed by tartaric acid, whereas the acidity of Sauvignon Blanc would be also driven by higher malic acid. Sauvignon Kretos and UD 30–080 would exhibit comparable tartaric acid to Sauvignon Blanc but lower malic acid, resulting in a lower TA. Figure 6B shows the low TA of Ortrugo and Soreli at 20 ◦Brix, linked to a very low malic acid concentration and relatively low tartaric acid level. Conversely, UD 80–100 would have retained a good balance between organic acids and total acids.

> Simulated harvest at TA of 7 g/L reveals that both Sauvignon Kretos and Sauvignon Rytos would have shown higher TSS than Sauvignon Blanc, but the TA would be again linked to high tartaric acid and low malic acid, compared to Sauvignon Blanc (Figure 6C). Ortrugo and Soreli would exhibit instead very low TSS, whereas UD 80–100 shows again a good balance among TSS, malic acid, and tartaric acid (Figure 6D).

**Figure 6.** Balance among titratable acidity and organic acids in grapes of 5 pathogens-resistant varieties and 2 reference *V. vinifera* cultivars, at the total soluble solids threshold of about 20 ◦Brix (panels **A**,**B**) and balance among total soluble solids and organic acids in grapes of the same varieties, at the titratable acidity threshold of about 7 g/L (panels **C**,**D**). Scaling for titratable acidity and tartaric acid is 0–12 g/L, for malic acid is 0–6 g/L, and for total soluble solids is 12–24 ◦Brix. Data pooled over two seasons.

#### **4. Discussion**

Compared to Ortrugo and Sauvignon Blanc (Table 2), all tested PRV had a mediumto-high yield per vine linked to a higher shoot fruitfulness. Nowadays, remunerative productivity is a necessary requirement for new genotypes and it should be associated with desired grape quality and low to moderate susceptibility to biotic and abiotic stress [2]. Moreover, high basal bud fruitfulness allows the implementation of spur-pruning systems prone to the mechanisation of winter operations [2,37,38].

Although bud break of all tested genotypes occurred within a quite narrow time window (i.e., 4–5 days), the unseasonal low temperatures recorded in 2019 between DOYs 100–160 likely pushed back veraison in Ortrugo and Sauvignon Blanc, whereas PRV were substantially unaffected by this weather trend. This suggests that PRV have lower requirements in terms of heat accumulation to shift from vegetative activity to a prioritised reproductive activity. This trait could indicate, at the same time, a weak point or an advantage. In fact, if the advancement of phenological stages might indeed increase grapes susceptibility to sunburn and biochemical unbalances in several traditional wine districts [1,39], the PRV early veraison observed in 2019 renders these varieties more suitable to exploit cool climates or areas located at higher altitudes [40–42].

Overall, TSS accumulation had an earlier onset and faster pace in PRV varieties, as compared to Ortrugo and Sauvignon Blanc. The two reference varieties lagged behind PRV by 10 to 20 days in terms of TSS accumulation (Figure 2A,B), and sugar accumulation never caught up to the final TSS of any of the PRV in both seasons. Moreover, the time interval when TSS accumulation rates were higher than 0.3 ◦Brix day−<sup>1</sup> was much longer in PRV than in Ortrugo and Sauvignon Blanc, especially in 2020 (Figure 5A–D). While Ortrugo and Sauvignon Blanc are well known for their slow sugar accumulation and low maximum sugar thresholds [42,43], such fast sugar accumulation dynamic exhibited especially by Sauvignon Kretos and UD 80–100 could easily lead to excessive wine alcohol content and unbalanced aroma, especially in white wines [1]. Our results are overall similar to the findings of Poni et al. [20], who compared Sangiovese and UD 72–096 (a Sangiovese x Bianca PRV).

Interestingly, the fostered sugar accumulation found in Sauvignon Kretos and some others PRV was not associated with a higher LA/Y ratio when compared to Sauvignon Blanc and Ortrugo. The only variety showing a higher LA/Y ratio was UD 30–080 (1.64 m2/kg) that, concurrently, was the PRV having the most delayed TSS pattern in both years and the lowest TSS at harvest (Figure 2A,B and Table 3). Therefore, the fast sugar accumulation observed in PRV was likely due to the actual genotype efficiency in accumulating sugars and not to differential source/sink relations among cultivars. Moreover, our data suggest that canopy management of PRV should substantially differ from that of *V. vinifera* cultivars such as Ortrugo and Sauvignon Blanc. For instance, Gatti et al. [44] demonstrated the effectiveness of bunch thinning in promoting faster sugar accumulation making it possible to target the enological standards required for producing Ortrugo sparkling wines (TSS of 20–21 ◦Brix and TA of 6.5 g/L). Accordingly, if, in the two reference cultivars, a good choice could be increasing the LA/Y ratio in order to promote TSS concentration corresponding to optimal TA, then in the PRV tested in this trial, the best strategy should be, on the contrary, to reduce LA/Y ratio trying to slow down sugar accumulation rates [37].

In ripening fruits, acidity is driven by the relative changes in the abundance of organic acids [4]. Malic acid is one of the main substrates for respiration in grapevine berries, and it is maximum pre-veraison and minimum at the end of the season [45,46]. Malic acid respiration rates are mainly driven by temperatures and the abundance of the substrate [47]. However, each genotype has a specific pattern in malic acid degradation rates and, perhaps more importantly, a different maximum and minimum organic acid concentration [48,49]. For instance, our study shows that UD 80–100 boasts the highest pre-veraison malic acid concentration among the tested varieties and that all PRV retain very low malic acid at high TSS concentrations (Figure 4A,B). The dynamic of malate degradation rates (Figure 5G–J) reveals that daily loss of malic acid is a complex function of the onset of veraison time, the pool of pre-veraison malic acid, and air temperature. In fact, in 2019, Sauvignon Blanc and Ortrugo grapes, due to their later onset of veraison, somewhat mitigated the loss of malic acid under the high temperatures recorded between DOYs 200 and 210. Contrariwise, on the same dates, all PRV had already crossed veraison and suffered a drastic malic acid loss. However, in 2019, maximum malic acid loss per day was exhibited by Sauvignon Blanc immediately after veraison, confirming that air temperature is not the only factor driving degradation rates. In 2020, when the onset of veraison time was similar for all tested cultivars, a stronger role was likely played by the availability of malate for respiration. In fact, genotypes showing the highest malic acid degradation rates corresponded with those varieties showing the highest pre-veraison malic acid pool (Figures 4B and 5H,J). Moreover, our data demonstrate that malic acid degradation rates are not the main factor determining final malic acid at the end of the season. Sauvignon Blanc was indeed the variety that exhibited in both years the more intense malic acid degradation rates and the highest minimum malic acid concentration rates at the end of the season (Figure 4A,B and Figure 5G,H).

If the primary goal is screening for varieties capable to maintain adequate acidity in a hot region, two cultivars seem to be of some interest among the five tested PRV: UD 80–100 and Sauvignon Rytos. UD 80–100 shows adequate acidity at high TSS levels and optimal balance between malic and tartaric acid (Figure 3C,D and Figure 6C,D, and Table 4); however, its sugar accumulation is excessively fast, and grapes should be harvested very early (Figure 2A,B and Figure 5C,D). Sauvignon Rytos was the only PRV showing higher TA than Ortrugo in 2020, whereas, in 2019, these two varieties showed very similar malate concentration from DOY 208 to the end of the season (Figure 2A,B). Ortrugo is a variety

that suffers a fast acidity loss after veraison to very low values, often not compatible with white and sparkling wine-making standards [6,44]. This is also confirmed by our data, showing poor balance among sugars, acidity, and malic vs. tartaric acid in Ortrugo, either if harvested at optimal TSS or at optimal TA thresholds (Figure 6B,D). However, Table 1 and Figure 2 show that in 2019, Ortrugo had a delayed veraison and TA loss, together with higher minimum acidity, compared to data recorded in the subsequent season. Notably, in Sauvignon Rytos, as compared to Ortrugo, optimal acidity matched higher TSS concentration, meaning that harvest might be anticipated with no detrimental effects on sugars (Table 4 and Figure 6C,D). However, data also support that Sauvignon Rytos acidity cannot be even close to that of Sauvignon Blanc, a variety that is known for the high acidity retained late in the season [50], a trait confirmed by our results (Figure 2E,F). If our data suggest that, in our conditions, Sauvignon Rytos was the PRV more capable to retain some acidity, Figure 4 reveals a relevant difference between Sauvignon Rytos and its parental Sauvignon Blanc: the former holds high acidity mostly due to the high concentration of tartaric acid, with a very limited contribution of malic acid; Sauvignon Blanc acidity at harvest relies instead on both malate and tartrate high concentration late at the end of the season (Figure 4A,B and Table 4). Different from malic acid, tartaric acid is not subjected to respiration, and changes in its abundance are mainly due to dilution and salts formation with K+ [4,45,47]. Interestingly, QTL, responding to grapes' cation mobility and organic acid metabolism, and catabolism have been identified, and today, several breeding programmes based on MAS or new breeding technologies are in progress to obtain new varieties less responsive to organic acid depletion under high temperatures [51–55].

Comparing balance between organic acids at optimal TSS or TA confirms that the difference in the ratio between organic acid concentration in Sauvignon Rytos and Sauvignon Blanc grapes is consistent, either choosing optimal TSS or optimal TA as the key parameter for placing harvest time (Figure 6A,C). This is quite essential information because it suggests that acidity preservation in these two varieties should be pursued based upon contrasting strategies: in Sauvignon Blanc, in order to preserve malic acid from respiration, grapes should be protected from excessive radiation and temperature, choosing trellis system shielding bunches from direct radiation and selecting appropriate aspects and locations [42,56–58]. Contrariwise, in Sauvignon Rytos actions should aim at limiting berry dilution, for instance, by calibrating adequate competition for water and nutrients, and by carefully managing K+ availability and nutrition in order to contrast potassium salts formation [47].

#### **5. Conclusions**

To the best of our knowledge, ripening patterns and organic acid depletion rates in new PRV were never studied in detail. Even if many technical reports suggest that wines obtained from these varieties could have similar aromatic traits to their *Vitis vinifera* parentals, our data demonstrate that, in regions with significant heat summation during the summer (~2000 GDDs), PRV exhibit anticipated veraison, as compared to early ripening *V. vinifera* cultivars, and that their organic acids balance and sugar accumulation rates largely differ from those observed in *vinifera* genotypes. Sauvignon Kretos, Sauvignon Rytos, Soreli, and UD 80–100 exhibited an early and fast grape sugar accumulation. Among the tested PRV genotypes, Sauvignon Rytos was the only one capable of maintaining higher titratable acidity at harvest, due to a significant tartaric acid pool. Conversely, in the Sauvignon Blanc parental, the high acidity at harvest was linked to high final malic acid concentration, resulting in a different acidic balance.

Our work also suggests that all new PRV should be subjected to different canopy and ripening management strategies, as compared to the two reference *V. vinifera* cultivars, given the faster sugar accumulation rates at comparable levels of LA/Y ratio.

Overall, our data indicate that PRV could perform better in north-facing hillsides, in cooler climates, or at higher altitudes, where their good resistance to mildews could match an adequate grapes' biochemical balance.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/horticulturae7080229/s1, Table S1: Integrated pest management protocol applied for pathogenresistant varieties (PRV) and non-resistant cultivars, Figure S1: Ripe clusters of the five resistant cultivars evaluated in the study and their parentals with respective pedigree.

**Author Contributions:** Conceptualisation, T.F. and S.P.; methodology, S.P. and M.G.; investigation, T.F., C.S., F.D.Z. and P.G.; resources, S.P.; data curation, T.F.; writing—original draft preparation, T.F.; writing—review and editing, M.G., S.P., A.V., C.S, F.D.Z. and P.G.; funding acquisition, S.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** Research partially supported by the ValorInVitis project, funded by the Emilia Romagna Region under the RDP program (PSR Emilia Romagna 2014–2020 Mis. 16.1.01 FA 2A), Grant No. 5004320, and by Catholic University of Sacred Heart D1 funds.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors would like to thank Cantina Sociale di Vicobarone, Vivai Cooperativi Rauscedo, and Roberto Miravalle, for establishing the experimental vineyard; Stefano Carrà and Luca Ampeli, for the technical support in the vineyard; Maria Giulia Parisi, Lily Ronney, and Alessandra Garavani, for the technical help; Alice Richards, for revising the written English manuscript; Roberto Miravalle, Marco Profumo, Mauro Mazzocchi, Claudio Gazzola, Andrea Pradelli, and Andrea Illari, for topic discussion.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **The Use of Halophytic Companion Plant (***Portulaca oleracea* **L.) on Some Growth, Fruit, and Biochemical Parameters of Strawberry Plants under Salt Stress**

**Sema Karakas 1,\*, Ibrahim Bolat <sup>2</sup> and Murat Dikilitas <sup>3</sup>**


**Abstract:** Strawberry is a salt-sensitive plant adversely affected by slightly or moderately saline conditions. The growth, fruit, and biochemical parameters of strawberry plants grown under NaCl (0, 30, 60, and 90 mmol L<sup>−</sup>1) conditions with or without a halophytic companion plant (*Portulaca oleracea* L.) were elucidated in a pot experiment. Salt stress negatively affected the growth, physiological (stomatal conductance and electrolyte leakage), and biochemical parameters such as chlorophyll contents (chl-*a* and chl-*b*); proline, hydrogen peroxide, malondialdehyde, catalase, and peroxidase enzyme activities; total soluble solids; and lycopene and vitamin C contents, as well as the mineral uptake, of strawberry plants. The companionship of *P. oleracea* increased fresh weight, dry weight, and fruit average weight, as well as the total fruit yield of strawberry plants along with improvements of physiological and biochemical parameters. This study showed that the cultivation of *P. oleracea* with strawberry plants under salt stress conditions effectively increased strawberry fruit yield and quality. Therefore, we suggest that approaches towards the use of *P. oleracea* could be an environmentally friendly method that should be commonly practiced where salinity is of great concern.

**Keywords:** abiotic stress; strawberry; companion plants; phytoremediation

#### **1. Introduction**

Salinity is one of the most devastating environmental problems limiting crop productivity and quality in many regions of the world. This problem is more prevalent in arid and semi-arid climatic regions. It affects approximately 20% of the cultivated and 50% of irrigated agricultural lands [1,2]. It has now been estimated that 1.5 million hectares of lands have been lost every year due to salinity problems. If salinization goes with this trend, nearly 50% of cultivable lands will be lost by the mid-point of this century [3,4].

Salinity negatively affects plant growth in terms of osmotic, ionic, and nutrient imbalance [5]. These disorders cause oxidative stress on plants. If plants cannot get enough water under high salt stress, turgor pressure significantly decreases, and thus the closure of the stomata of plants becomes inevitable to conserve water [6]. This significantly affects the photosynthetic capacity of plants. Ionic toxicity, on the other hand, inhibits cellular metabolism and biochemical pathways. For example, Na<sup>+</sup> ions at the root cell disturb enzymatic activities and inhibit the uptake of other minerals such as K<sup>+</sup> and Ca++ [7]. The high accumulation of Na<sup>+</sup> and Cl<sup>−</sup> ions result in many morphological, physiological, molecular, and biochemical pathways in plants. Due to disturbed mechanisms in plants, NaCl stress leads to the development of leaf chlorosis and necrosis, as well as the loss of quality in crops. As a consequence, the assimilation of carbohydrates and sugars allocated

**Citation:** Karakas, S.; Bolat, I.; Dikilitas, M. The Use of Halophytic Companion Plant (*Portulaca oleracea* L.) on Some Growth, Fruit, and Biochemical Parameters of Strawberry Plants under Salt Stress. *Horticulturae* **2021**, *7*, 63. https://doi.org/10.3390/ horticulturae7040063

Academic Editors: Agnieszka Hanaka, Jolanta Jaroszuk-Sciseł and ´ Małgorzata Majewska

Received: 10 February 2021 Accepted: 23 March 2021 Published: 26 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

for fruit development is significantly reduced due to stress development and defense mechanisms [8,9].

Plants can be divided into halophytes and glycophytes as responses to salinity stress. Most glycophytes are salt-sensitive, even at low concentrations, while halophytes are highly salt-tolerant plants, which enables them to survive and thrive in extremely saline environments [10,11]. Salt ions have to be taken up by halophytes and deposited in the vacuoles of leaf or root tissues or in separate organelles. In general, salt secretion takes place through the shedding of salty leaves and salt glands or specialized leaf cells [12]. Most halophytes are able to survive by maintaining negative water potential under extreme salt concentrations. Therefore, a true halophyte is considered to maintain its viability and complete the life cycle at NaCl levels between 200 and 1000 mmol L<sup>−</sup>1. These concentrations are very close to the concentrations of seawater level. Some halophytes, on the other hand, tolerate much higher concentrations of NaCl [10,13]. Halophytes such as *Atriplex* spp., *Chenopodium* spp., *Portulaca* spp., *Suaeda* spp., and *Salsola* spp. uptake salt ions through their roots and store them in their leaves. It is quite possible that these plants could be used as companion plants with crop plants, especially salt-sensitive glycophytes, to reduce the negative effects of NaCl through the uptake of toxic ions [14,15]. For example, *Portulaca oleracea* L. (purslane) (which is a member of Portulacaceae)*,* is a drought- and salt-tolerant annual plant. The plant is a promising crop species in saline–alkali soils [16,17]. Moreover, *P. oleracea* could effectively absorb salts from soil media to remediate saline–alkali soils [18]. Previous studies have investigated the effects of salinity on *P. oleracea* growth. For example, Grieve and Suarez [19] evaluated *P. oleracea* responses with saline irrigation, and they showed that the plant could survive at a salinity of 28.5 dS m−1. The authors further elucidated the performances of salt-tolerant halophyte species of *P. oleracea* and *Salsola soda* against increasing NaCl levels. They reported that *P. oleracea* and *S. soda* seeds were effectively germinated between 250 and 350 mmol L−<sup>1</sup> NaCl levels [20].

Strawberry is an economically important fruit crop that is globally cultivated. It belongs to the *Fragaria* genus in Rosaceae family, which contains 23 species [21,22]. The popularity of strawberry fruit crops is increasing in the world due to increasing consumption. Its popularity is also increasing along with the generation of new varieties. Strawberry cultivation has therefore become an important greenhouse and open field crop in the Mediterranean area, although drought and salinity have played significant roles in limiting crop production [23,24].

Strawberry is considered to be sensitive to NaCl salinity due to increased osmotic pressure and Na<sup>+</sup> or Cl<sup>−</sup> ion toxicity. NaCl salinity not only reduces the crop yield but also deteriorates the quality parameters in many crops, including strawberries [25,26].

In the present study, we elucidated the effects of different NaCl concentrations on strawberry plants grown with or without the halophytic companion plant *P. oleracea* L. to remediate the physiological and biochemical parameters of strawberry plants.

#### **2. Materials and Methods**

#### *2.1. Experimental Design and the Growth of Plants*

The experiment was performed between September 2018 and January 2019 in a semicontrolled greenhouse at the University of Harran, Sanliurfa, Turkey. Fresh strawberry (Rubygem variety) plants were grown alone or in combination with *P. oleracea* seedlings in 8-L pots containing peat (Klasmann TS 1) under natural light conditions. Peat is a very porous substrate with an excellent water capacity. Its slow degradation rate, high porosity, and high-water holding capacity makes it one of the most commonly used growth medium, especially for saline-related studies in vegetables and ornamental plants [27,28]. It has low nutrient values, so it is highly unlikely to affect the mineral uptake of macro and micronutrient elements.

The average temperatures for day and night were 35 ± 2/28 ± 2 ◦C during the course of the experiment. The trial was carried out in a randomized block design. The first group of strawberry plants grown under differing NaCl conditions (0, 30, 60, and

90 mmol L−1) was designated as S0, S30, S60, and S90; the second group of plants grown with *P. oleracea* under the same NaCl conditions was designated as SP0, SP30, SP60, and SP90. Treatments in each group were replicated five times. Seedlings were individually transplanted to the pots. Strawberry seedlings following one week of establishment growth in pots were accompanied with *P. oleracea* seeds that were sown and germinated at a rate of 25 companion plants per pot. After germinations (five weeks), the pots were irrigated with or without salt to the full pot capacity throughout the treatment period (twelve weeks). The plants were fertigated with Hoagland's nutrient solution once a week. The experimental trial from the very beginning of obtaining strawberry seedlings to the end of harvest took five months. The plants were harvested at the optimum stage of physiological maturity for the evaluation of salinity responses (Figure 1).

**Figure 1.** Strawberry plants were grown with or without *Portulaca oleracea* under different NaCl conditions.

#### *2.2. Plant Growth and Fruit Properties*

Strawberry fruits were harvested when 90% of the fruit surface had reached a fully red color. At the end of the experimental period, total fruit weights were determined and the average fruit yield was calculated.

Plant crown and root fresh weight (Fwt) were analyzed immediately after the harvest. The dry weight (Dwt) of plant organs was determined following the drying of plant samples at 70 ◦C until a constant weight.

Total soluble solids (TSS) were assessed from the fruit juice with a hand refractometer. The results are expressed in percent (%) Catania et al. [29].

The lycopene content of strawberry fruits was assessed according to the method of Barrett and Anthon [30] with minor modifications [31]. One gram of strawberry fruit was homogenized with 10 mL of an ethanol:hexane solution (4:3). The mixture was then centrifuged at 10,000× *g* for 10 min at room temperature. The supernatant (100 μL) was added to 7 mL of the ethanol:hexane solution (4:3) mixture and vortexed. After 1 h of incubation at room temperature, 1 mL of H2O was added to the tubes and vortexed. The tubes were incubated in the dark to form two different phases. The top phase was taken and read at 503 nm against a hexane blank with a UV microplate spectrophotometer (Epoch, SN: 1611187, Winooski, VT, USA). The lycopene content was calculated according to the following formula (Equation (1)).

$$\text{mg Lycopene g}^{-1} \text{Fwt} = \frac{A\_{503} \times 2.7}{172 \times 0.1} \times 537 \tag{1}$$

where 537 g/mole is the molecular weight of lycopene, 2.7 mL is the volume of the hexane layer, 172 mmol−<sup>1</sup> is the extinction coefficient for lycopene in hexane, and 0.1 g is the weight of the strawberry.

The vitamin C content of strawberry fruits was assessed according to the method of Oz [32] with small modifications [31]. Strawberry fruits (5 g) were extracted with 25 mL of oxalic acid. The mixture was centrifuged at 10,000× *g* for 10 min. Then, 1 mL of this mixture was added to 7 mL of a 1% oxalic acid solution and 8 mL of a dye reagent. The dye reagent was prepared according to the recipe of [31]. The mixture was filtered through Whatman No.2 filter paper and diluted to 100 mL with deionized H2O. Then, 25 mL of this solution were taken and diluted to 500 mL with deionized H2O, vortexed, and kept at 4 ◦C until use. The mixture was once more vortexed before measurement at 518 nm against the oxalic acid and dye mixture with a UV microplate spectrophotometer (Epoch, SN: 1611187, Winooski, VT, USA).

Electrolyte leakage (EL) was assessed following the method of Lutts et al. [33] using leaf discs for each treatment. Fully expanded young leaves were cleaned three times with deionized H2O to remove dust and surface-adhered electrolytes. Leaf discs were placed in closed vials containing 10 mL of H2O and incubated at 25 ◦C on a rotary shaker for 24 h; subsequently, the electrical conductivity of the solution (EC1) was measured. The final electrical conductivity (EC2) was determined following the autoclaving of the leaf samples at 120 ◦C for 20 min. Leaf samples were then equilibrated at 25 ◦C, and the EL was calculated as follows (Equation (2)).

$$EL\ \left(\%\right) = \frac{EC1}{EC2} \times 100\tag{2}$$

Stomatal conductivity (SC) was determined on the youngest fully expanded leaves on the upper branches of the strawberry plants with a leaf promoter (SC−1) at midday. Measurements were performed by clamping the leaves in the leaf chamber. The actual vapor flux from the leaf through the stomata is expressed as mmol m−<sup>2</sup> s<sup>−</sup>1, following the work of Karlidag et al. [34].

#### *2.3. Biochemical Parameters*

Strawberry plant leaf chlorophyll content (Chl-*a* and Chl-*b*) was extracted following the method of Arnon [35] with minor modifications [31]. A sample of the fresh leaf (0.5 g) was homogenized with 10 mL of an acetone:water (80/20, *v*/*v*) mixture and filtered through Whatman No.2 filter paper and then put into the dark tubes. The Chl-*a* and Chl-*b* contents of the filtrate was measured with a UV microplate spectrophotometer (Epoch, SN: 1611187, Winooski, VT, USA) at 663 and 645 nm, respectively, against an 80% acetone blank. The findings were expressed as mg L−<sup>1</sup> and calculated as mg g−<sup>1</sup> Fwt.

The proline concentration was determined following the method of Bates et al. [36] with minor modifications [31]. Leaf samples (0.5 g) were extracted with 10 mL of 3% *w*/*v* sulphosalicylic acid using a mortar and a pestle. The extract was filtered through Whatman No.2 filter paper. Then, the 2 mL filtrate was mixed with 2 mL of acid–ninhydrin in a test tube and boiled at 100 ◦C for 1 h. The reaction was terminated in an ice bath. Then, the mixture was extracted using 5 mL of toluene. The tubes were vortexed for 20 s and then

left for 20 min at room temperature to achieve two layers of separation. The organic phase was collected, and the absorbance of the extracts was read at 515 nm using a toluene blank. Proline concentration was made from a standard curve using L-proline (Sigma-Aldrich, Taufkirchen-Germany). The results are expressed as μmol g−<sup>1</sup> Fwt.

Hydrogen peroxide (H2O2) levels were assessed following the method of Velikova et al. [37] with small modifications [38]. Leaf samples (0.5 g) were extracted with 5 mL of 0.1% (*w*:*v*) trichloroacetic acid (TCA). The extract was centrifuged at 12,000× *g* at 4 ◦C for 15 min, and the supernatant (0.5 mL) was added to 0.5 mL of a 10 mmol L−<sup>1</sup> potassium phosphate buffer (pH 7.0) and 1 mL of a 1 mol L−<sup>1</sup> potassium iodide. The absorbance was read at 390 nm in a UV microplate spectrophotometer (Epoch, SN: 1611187, Winooski, VT, USA). The H2O2 content was expressed as μmol g−<sup>1</sup> Fwt.

The malondialdehyde (MDA) content was assessed following the method of Sairam and Sexena [39] with minor modifications [38]. The leaf samples (0.5 g) were extracted with 10 mL of a 0.1% (*w*/*v*) TCA solution. The extract was centrifuged at 10,000× *g* for 5 min. Four milliliters of 20% *v*/*v* TCA containing 0.5% *v*/*v* thiobarbituric acid (TBA) was added to 1 mL of the supernatant. The mixture was kept in boiling water for 30 min, and then the reaction was stopped in an ice bath. The mixture was once more centrifuged at 10,000× *g* for 5 min and then read in a UV microplate spectrophotometer (Epoch, SN: 1611187, Winooski, VT, USA) at 532 and 600 nm. The MDA content was calculated and expressed as nmol g−<sup>1</sup> Fwt (Equation (3)).

$$\text{MDA} \left( \text{mmol g}^{-1} \right) = \frac{\text{Extract volume (ml)} \times \left[ (\text{A}\_{52} - \text{A}\_{60}) / \left( 155 \,\text{mM}^{-1} \,\text{cm}^{-1} \right) \right]}{\text{Sample amount (g)}} \times 10^3 \tag{3}$$

Catalase enzyme activity (CAT; Enzyme Code. 1.11.1.6) was determined following the method of Milosevic and Slusarenko [40] with minor modifications [38]. Leaf samples (0.5 g) were extracted with 10 mL of a 50 mmol L−<sup>1</sup> Na-phosphate buffer solution, and then 50 mL of the extract were added to a 2.95 mL reaction mixture (50 mmol L−<sup>1</sup> Na-phosphate buffer, 10 mmol L−<sup>1</sup> H2O2, and 4 mmol L−<sup>1</sup> Na2EDTA) and read with a UV microplate spectrophotometer (Epoch, SN: 1611187, Winooski, VT, USA) at 240 nm for 30 s. One CAT unit (U) was defined as an increase in absorbance of 0.1 at 240 nm. The activity is expressed as enzyme unite mg−<sup>1</sup> Fwt.

Peroxidase enzyme activity (POX; Enzyme Code. 1.11.1.7) was assayed following the method of Cvikrova et al. [41] with minor modifications [38]. For the analysis, 100 mL of the homogenate (obtained as above) was added to 3 mL of the reaction mixture (50 mmol L−<sup>1</sup> Na-phosphate, 5 mmol L−<sup>1</sup> H2O2, 13 mmol L−<sup>1</sup> guaiacols, and pH 6.5). Activity was defined by the range of change in absorbance at 470 nm with a UV microplate spectrophotometer (Epoch, SN: 1611187, Winooski, VT, USA). One unit of POX was defined as a change of 0.1 absorbance unit per minute at 470 nm. Activity is expressed as enzyme unit mg−<sup>1</sup> Fwt.

Leaf mineral (K+, Na+, Ca2+, Mg2+, and Cl−) contents were determined according to the procedure made by Chapman and Pratt [42] with minor modifications [31]. Dry plant samples (0.5 g) were ground in porcelain crucibles. The porcelain crucibles were placed into a muffle furnace, and the temperature was gradually increased up to 500 ◦C. Following cooling, the ash was dissolved in 5 mL of 2 N hydrochloric acid. After 30 min, the volume was made up to 50 mL with distilled H2O, and the supernatant was filtered through Whatman No.42 filter paper. The resulting supernatant containing Na+, K+, Ca+2, and Mg+2 ions were assessed by Inductively Coupled Plasma (ICP, Perkin Elmer). Chloride was determined using ion chromatography after the filtration through Whatman No.42 filter paper.

Duncan's multiple range test (DMRT) was used to evaluate the data using SPSS 22 (ANOVA test) at a significance level of *p* ≤ 0.05 using. Data are presented as a mean value ± with standard error.

#### **3. Results**

Strawberry plant growth, fruit properties, biochemical parameters, and mineral contents were significantly affected by all salinity levels. The crown fresh and dry weights of strawberry plants in saline conditions were significantly lower in plants grown alone in saline conditions when compared to those of plants grown in combination with *P. oleracea* under the same conditions. For example, the crown fresh weights of the plants were 55.16, 37.62, and 35.16 g plant−<sup>1</sup> grown alone in S30, S60, and S90 mmol L−<sup>1</sup> NaCl conditions, respectively. When plants were grown in combinations with *P. oleracea,* their conditions were significantly improved at all NaCl conditions. The fresh weights of plants increased to 64.38, 44.76, and 44.49 plant−<sup>1</sup> at the SP30, SP60, and SP90 mmol L−<sup>1</sup> NaCl conditions, respectively (Table 1). Similar improvements were recorded for the dry weights of plants (Table 1). In general, the combination of companion plants (*P. oleracea*) was found to be effective in increasing the Fwt and Dwt under each NaCl condition.

**Table 1.** Growth and physiological parameters of strawberry plants grown alone or in combination with *P. oleracea* at differing NaCl levels.


Significance level at *p* ≤ 0.05 was determined for the salt treatment using Duncan's multiple range test. Different letters in each column indicate statistical differences. S: a strawberry grown alone; SP: the strawberry and *P. oleracea* companionship; EL: electrolyte leakage; SC: stomatal conductivity.

> EL is considered an important criterion for salt stress parameters. EL was increased with increasing levels of salt. For example, leaf EL was found to be 11.90 and 11.07% at S0 and SP0, respectively. Increases of EL were trended from 15.61 to 25.34% with respect to conditions from S30 to S90, respectively. When *P. oleracea* was accompanied with strawberry plants in the NaCl conditions, the increase of EC was so minimal that only 11.10 and 15.32% were recorded at SP30 and SP90, respectively; see Table 1.

> Stomatal conductivity in saline conditions was gradually decreased as the concentration of NaCl increased in plants grown alone in saline conditions (Table 1). However, the cultivation of *P. oleracea* improved the SC of strawberry plants under all NaCl conditions when compared to those grown alone in saline conditions. The improvement of SC was evident in that the increases were from 183.26 to 230.80% from S30 to SP30 cultivation conditions, respectively. At the higher NaCl concentrations of S90 and SP90, the SC was still improved with a lesser efficiency from 94.10 to 131.08%, respectively.

> The average fruit weight and yield of strawberry plants under NaCl conditions were reduced in plants grown alone, but the co-cultivation of strawberry plants with *P. oleracea* increased the average and total fruit weight (Table 2).

> The employment of *P. oleracea* not only increased the crop yield and physiological parameters but also improved the quality of fruits in terms of lycopene and vitamin C contents. Lycopene and vitamin C contents were gradually decreased as the concentration of NaCl increased. Again, the employment of *P. oleracea* increased the lycopene and vitamin C at all NaCl levels (Table 2). For example, the remarkable effect was more evident at the 90 mmol L−<sup>1</sup> NaCl conditions, as the both lycopene and vitamin C contents were increased when grown with *P. oleracea* as compared to those of plants grown alone in saline conditions.


**Table 2.** Yield and some fruit properties of strawberry plants grown alone or in combination with *P. oleracea* at differing NaCl levels.

Significance level at *p* ≤ 0.05 was determined for the salt treatment using Duncan's multiple range test. Different letters in each column indicate statistical differences. S: a strawberry grown alone; SP: the strawberry and *P. oleracea* companionship; TSS: total soluble solids.

> Unlike other parameters, the TSS contents of the fruits in saline conditions were significantly lowered. The co-cultivation of *P. oleracea* did not significantly improve the conditions of strawberry plants (Table 2).

> Chl-*a* and Chl-*b* were significantly affected by salinity at the S60 and S90 mmol L−<sup>1</sup> NaCl levels (*p* ≤ 0.05). For example, the Chl-*a* and Chl-*b* were determined as 0.70 and 0.36 mg g−<sup>1</sup> Fwt, respectively, at S90 mmol L−<sup>1</sup> NaCl levels in strawberry plants. The positive effects of *P. oleracea* on the Chl-*a* and Chl-*b* contents at SP90 mmol L−<sup>1</sup> NaCl were evident, as the Chl-*a* and Chl-*b* contents were 1.01 and 0.51 mg g−<sup>1</sup> Fwt, respectively, in strawberry plants (Figure 2A,B).

**Figure 2.** Leaf Chl-*a* (**A**) and Chl-*b* (**B**) contents of strawberry plants grown alone or in combination with *P. oleracea* at differing NaCl levels (0, 30, 60, and 90 mmol L<sup>−</sup>1). S: a strawberry grown alone; SP: the strawberry and *P. oleracea* companionship; TSS: total soluble solids.

Leaf proline content significantly increased as the concentration of NaCl levels increased as a response to salinity stress. (*p* ≤ 0.05); see Figure 3A. The highest proline level was determined as 13.77 μmol g−<sup>1</sup> Fwt with the S90 mmol L−<sup>1</sup> NaCl treatment, whereas at the SP90 condition, the proline level decreased to 4.40 μmol g<sup>−</sup>1. Therefore, the combination of *P. oleracea* not only improved the physiological and biochemical conditions of strawberry plants but also reduced the stress metabolite levels.

**Figure 3.** Proline (**A**), H2O2 (**B**), and malondialdehyde (MDA) (**C**) contents; peroxidase enzyme activity (POX) (**D**) and catalase enzyme activity (CAT) (**E**) antioxidant enzyme contents of strawberry plants grown alone or in combination with *P. oleracea* at differing NaCl levels (0, 30, 60, and 90 mmol L−1). S: a strawberry grown alone; SP: the strawberry and *P. oleracea* companionship; TSS: total soluble solids.

The companionship of *P. oleracea* had a remarkable effect to reduce the impact of NaCl stress in strawberry plants. Again, leaf H2O2 and MDA contents increased with the increasing levels of salt stress. The highest H2O2 and MDA levels were determined as 74.72 and 13.58 nmol g−<sup>1</sup> Fwt, respectively, at the S90 mmol L−<sup>1</sup> NaCl level. The co-cultivation of *P. oleracea* with strawberry plants reduced the contents of H2O2 and MDA levels down to 31.56 and 4.15 nmol g−<sup>1</sup> Fwt, respectively (Figure 3B,C).

POX and CAT antioxidant enzymes showed parallel patterns to those of previous parameters. The co-cultivation of *P. oleracea* significantly decreased antioxidant enzyme levels at the 60 and 90 mmol L−<sup>1</sup> NaCl conditions, (Figure 3D,E).

#### *Leaf Mineral Contents*

The concentrations of beneficial ions such as those of K<sup>+</sup> and Ca2+ decreased with the increases in salinity levels in strawberry plants. The lowest K+ and Ca2+ ions were determined at the S90 level. The leaf Mg2+ content was not significantly affected upon NaCl stress. *P. oleracea* co-cultivation with strawberry plants enhanced the Mg2+ ion level at NaCl treatments; see Table 3. Under saline conditions, gradual increases of Na<sup>+</sup> and Cl<sup>−</sup> ions were evident in strawberry plants grown at increasing NaCl salinity, but the employment of *P. oleracea* significantly decreased the Na<sup>+</sup> and Cl<sup>−</sup> ion contents; Table 3.

**Table 3.** Strawberry leaf mineral contents of strawberry plants grown alone or in combination with *P. oleracea* at differing NaCl levels.


Significance level at *p* ≤ 0.05 was determined for the salt treatment using Duncan's multiple range test. Different letters in each column indicate statistical differences. S: a strawberry grown alone; SP: the strawberry and *P. oleracea* companionship.

#### **4. Discussion**

Salinity stress is one of the most devastating issues that damages crop plants in terms of quantity and quality. Increased salinity levels not only damage plants during vegetative stages but also negatively affect reproductive stages. Under salt stress, Na+ is extensively accumulated in the shoots and roots of cultivars and K<sup>+</sup> content is decreased [43]. Quality parameters such as vitamin contents, aromatic substances, and pigments are remarkably reduced. Leaf proline content, as a response to stress, tends to increase. Increasing proline content under salinity conditions indicates the adverse effects of osmotic stress on the plant. Proline and soluble carbohydrates (also known as compatible solutes) are expected to be accumulated under salinity in strawberry [44]. This can be considered to be a criterion for stress tolerance [45]. This study showed that *P. oleracea* in combination with strawberry decreased proline levels under salinity along with the reduction of Na<sup>+</sup> and Cl<sup>−</sup> ion levels by reducing the toxic levels of salt ions. *P. oleracea* gave promising results on strawberry plants grown at different NaCl stress levels (0, 30, 60, and 90 mmol L<sup>−</sup>1). It is important to note that peat has a high bulk density. For example, Nugraha et al. [46] stated that capillary water movement had a very critical role in supplying water to the rooting zones of crop plants or the top parts of the soil. They reported that the rate of capillary water movement progressively corresponded to the increase in bulk density. Farina et al. [47] also stated that NaCl accumulation in peat mulching was much lesser than that of soil. They stated that if the porosity in the surface layers became small enough, irrigation or raindrops could plug macropores in the surface. They either block main avenues for water and roots to move

through the soil or they form a cement-like surface layer when the soil dries. The rock-solid upper layer or salt crust then restricts water movement and plant emergence. In our study, evaporation in the greenhouse was not high enough to build up NaCl accumulation in the top part of the soil. Therefore, no salt crust formation, which would have affected the results of our experimental findings, was observed.

Mozafari et al. [48] stated that salinity negatively affected the growth parameters, pigment content, and membrane stability, as well as disturbing the ionic balance in plants. For example, Saied et al. [49] stated that strawberry was considered to be a saline-sensitive plant. Many physiological and biochemical parameters deteriorated. This both directly and indirectly led to diminished productivity in plants [50]. We determined that fresh weight, dry weight, stomatal conductance, fruit average weight, fruit total yield, chlorophyll (Chl-*a* and Chl-*b*), total soluble solids, lycopene content, vitamin C content, and leaf mineral content (K+ and Ca2+) of strawberry plants significantly decreased with increasing NaCl levels. Strawberry plants grown in companionship with *P. oleracea* improved the condition of plants, and much lesser reductions in terms of total yield and quality were evident. The positive effect on strawberry growth was quite remarkable. The leaf electrolyte, proline, malondialdehyde, H2O2 contents, catalase enzyme activities, nd peroxidase enzyme activities, and leaf mineral contents (Na<sup>+</sup> and Cl−) of strawberry plants increased with an increasing level of salinity. The companion plants helped strawberry plants by reducing toxic ion levels, antioxidant enzyme levels, and stress metabolites. With the improvement of those parameters, electrolyte leakage and stomatal conductance were also improved, and this was reflected in the quality of fruits in terms of lycopene and vitamin C contents. This study proved that mixed planting with *P. oleracea* in saline conditions was an effective phytoremediation technique that might significantly increase the yield production and quality of strawberry. Similar findings were also made for *S. soda* plants by Karakas [51] who suggested that the improvement of tomato plants via companion plants under salt stress (1.3 and 6.5 dS m−1) was achieved with the synthesis of substances used for fruit development instead of building up substances for mechanisms of stress tolerance. It is important to note that synthesizing stress metabolites and antioxidant enzymes is quite costly for plants to cope with abiotic or biotic stress factors [17]. Instead of generating crop plants that can combat stress factors, the strategy that involves removing stress factors would be much appreciated. Any genetic modifications or biochemical approaches that increase the removing capacity of toxic ions or compounds from the soil habitat would be an environmentally friendly approach and a safe strategy. For example, Grafienberg et al. [52] and Karakas et al. [51] stated that reductions in stress metabolites and the uptake of toxic ions enabled tomato plants to use more energy to build up organic components such as lycopene and proteins instead of producing substances for defense mechanisms. In this study, salinity stress resulted in a reduction in vitamin C content and lycopene contents in strawberry. Jamalian et al. [53] showed that salinity reduced the vitamin C content of strawberries, which was in line with the results of the present study. The decrease in the vitamin C content of fruits at high salinity levels can be attributed to the decrease in carbohydrate (sugar) production caused by the decrease in photosynthesis required for vitamin C biosynthesis.

Yaghubi et al. [44] reported that MDA concentration was also high in strawberry plants at salt stress conditions. They reported that reactive oxygen species (ROS) production was muck higher than the scavenging capacity of antioxidant enzymes. The dismutation of O2 –2 into H2O2 and O2 was reported to increase H2O2 concentration [54]. This was observed by a higher H2O2 content in salt-stressed strawberry plants than in control plants. Since H2O2 was accompanied by an increase in the key antioxidant enzymes such as CAT, POD, and superoxide dismutase (SOD), a reduction of H2O2 was achieved. In our methodology, we achieved the decrease of stress metabolites while suppressing the antioxidant enzymes via the use of *P. oleracea* plants. Though antioxidant enzymes such as CAT, POD, and SOD are known to substantially reduce the levels of O2 - and H2O2 in plants and play a vital role in plant defense against oxidative stress [55], the increase of these enzymes might

interfere with the chemical compounds involved in quality parameters such as lycopene and vitamin C. With the use of *P. oleracea,* we were able to reduce stress metabolites and toxic ions, reduced further damages to cell components, and increased the quality-related compounds without increasing defense-related antioxidant compounds. This saved the energy to be used for defense responses, and this saved energy could be used to increase metabolic functions and quality parameters.

#### **5. Conclusions**

Strawberry cultivation has become popular recently, and this has led to an increase in cultivated areas. These areas have become saline-polluted, saline-prone, and salineprevalent. Since strawberry is a salt-sensitive plant, it is easily affected by a mild or moderate level of salinity. A very low level of NaCl could reduce crop yield and reduce the quality of fruits.

In this study, strawberry seedlings were grown alone or in combination with *P. oleracea* under differing NaCl concentrations. Strawberry seedlings under increasing NaCl salinity were negatively affected in terms of physiological, morphological, and biochemical parameters. Defending plants synthesized various stress metabolites such as proline, MDA, H2O2, and antioxidant enzymes to ease the negative effects of NaCl toxicity. However, increases of these metabolites were negatively correlated with quality-related metabolites such as vitamin C and lycopene. The cultivation of strawberry plants with *P. oleracea* plants reduced the concentrations of stress metabolites and antioxidant enzyme levels, as well as indirectly contributing to increases of vitamin C and lycopene contents.

We suggest that the employment of *P. oleracea* would remediate the conditions of strawberry parameters by accumulating Na+ and Cl<sup>−</sup> ions, thus causing reductions in the synthesis of stress metabolites. The use of *P. oleracea* is a quite practical and environmentally friendly approach where salinity is prevalent. *P. oleracea* has a high potential that could be used in high saline and in other environmental stress conditions.

**Author Contributions:** Conceptualization, S.K., M.D. and I.B.; methodology, S.K., I.B. and M.D.; software, S.K. and M.D.; validation, S.K., I.B. and M.D.; formal analysis, S.K. and M.D.; investigation, S.K., I.B. and M.D.; resources, S.K., I.B. and M.D.; data curation, S.K.; writing—original draft preparation, S.K.; writing—review and editing, M.D. and I.B.; visualization, S.K., I.B. and M.D.; supervision, S.K.; project administration, S.K.; funding acquisition, S.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Harran University Scientific Research Project (HUBAP), grant number 17247.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

