Biostimulants Application in Horticultural Crops under Abiotic Stress Conditions

Abiotic stresses strongly affect plant growth, development, and quality of production; final crop yield can be really compromised if stress occurs in plants’ most sensitive phenological phases. Additionally, the increase of crop stress tolerance through genetic improvements requires long breeding programmes and different cultivation environments for crop performance validation. Biostimulants have been proposed as agronomic tools to counteract abiotic stress. Indeed, these products containing bioactive molecules have a beneficial effect on plants and improve their capability to face adverse environmental conditions, acting on primary or secondary metabolism. Many companies are investing in new biostimulant products development and in the identification of the most effective bioactive molecules contained in different kinds of extracts, able to elicit specific plant responses against abiotic stresses. Most of these compounds are unknown and their characterization in term of composition is almost impossible; therefore, they could be classified on the basis of their role in plants. Biostimulants have been generally applied to high-value crops like fruits and vegetables; thus, in this review, we examine and summarise literature on their use on vegetable crops, focusing on their application to counteract the most common environmental stresses.

constraints. In 1982, Boyer estimated that yield losses caused by unfavourable environments were as much as 70% . Farooq et al. reported that drought induced a reduction of yield between 13% and 94% in several crops, and negative outcomes on crop production could be balanced in the medium term, several studies agree that in the long term, the negative ones will prevail . Based on future scenarios, adaptation and mitigation are essential to increase the resilience capacity of agricultural systems and to ensure crops yield and quality. Since environmental conditions cannot be controlled, several strategies on different levels are required, such as agronomical techniques or breeding of more tolerant cultivars .
In 2010, at the society's annual conference, Vegetable Breeding and Stress Physiology working groups of the American Society for Horticultural Sciences focused particularly on the "Improvement of Horticultural Crops for Abiotic Stress Tolerance" considering the effects of climate change . Up to now, most studies on climate change impacts focus on major crops, and only few papers pay attention to fruit and vegetable in terms of production, quality, and supply chain . An important aspect to take into consideration is the effect of the combination of different stressful factors. Most of the time, crops are subjected to several abiotic stresses that occur simultaneously in the field. In these situations, studying the stresses separately is not enough because plant response is unique and cannot be predicted by the reply obtained when each factor is applied individually . Moreover, biotic and abiotic components typically interact in an ecosystem. For instance, environmental conditions affect plant-pest interaction in different ways, by decreasing plant tolerance or increasing the risk of pathogen infection .
Focusing on horticultural species, the tolerance to abiotic stresses is an important trait because their cash value is usually higher than field crops, they require more resources for farming and because they provide a source of many nutrients, fibre, minerals, and carbohydrates, which are essential in a healthy diet . Food and Agriculture Organization (FAO) reports that about 90% of essential vitamin C and 60% of vitamin A for human comes from vegetables. Indeed, low fruit and vegetable intake is a major contributing risk factor to several widespread and debilitating nutritional diseases. According to the Global Burden of Disease Study, 3.4 million deaths can be attributed to low consumption of fruit and 1.8 million to low vegetables diets worldwide . Therefore, growing high-quality vegetables becomes one of the most important goals of current agriculture, in order to meet the needs of the population and the increasing demand for fruit and vegetables. Abiotic stresses do not only affect the yield but also the quality of these products, triggering morphological, physiological and biochemical changes that can alter the visual appearance and/or the nutraceutical value in a way that the product could become unmarketable . Bisbis et al. investigated the double effects of elevated temperature and increased CO on the physiology of different vegetables.
They observed several responses according to plant species and severity of the stress, taking into consideration the possible adaptation strategies that could be implemented in order to mitigate the effects of climate change. Nonetheless, these mechanisms are still under-researched and should be studied in depth, because not only different species but different cultivars also could respond differently to the same environmental stress. For example, cultivars with low levels of antioxidants are particularly vulnerable to oxidative stress compared to those with high antioxidant activity . This aspect has a particular importance as selection criterion in the choice of appropriate cultivars for a specific situation. Oxidative stress is a common phenomenon caused by several adverse conditions; it generally occurs when the balance between the production of reactive oxygen species (ROS) and the quenching activity is upset by a stressful event . Low levels of ROS are normally produced by different reactions during physiological metabolisms like photosynthesis or respiration, and they play an important signaling role in plant growth and development. Their amount dramatically increases under abiotic stress conditions and, if not controlled could result in cellular damage and death. Besides their toxicity to proteins, lipids or nucleic acids, the increased production of ROS under stressful conditions plays a key role in the complex signaling network of plants stress responses. Their concentration is maintained at non-toxic levels by the activity of the antioxidant system: a wide range of enzymatic or non-enzymatic antioxidant molecules are accumulated in plant tissues to quench ROS induced by stress .
Moreover, the maintenance of this equilibrium is also dependent on numerous factors, such as the timing of stress application, its intensity and duration. Indeed, moderate or controlled stress conditions could have a positive effect on quality traits of several crops . For example, water deprivation might be a useful crop management strategy to improve the quality of lettuce and fleshy fruits in terms of nutritive and health-promoting value and taste, by stimulating the secondary metabolism and concentration of different phytochemicals such as α-tocopherol, β-carotene, flavonoid and so on . Besides the production of ROS scavenging compounds, plants also increase the biosynthesis and accumulation of compatible solutes with an osmoprotective role, like sugars and proline.
Plants generally reply to non-optimal environmental conditions both with short-and long-term adaptation strategies, by the activation and regulation of the expression of specific stress associated genes .
Since plants are sessile organisms and they have to cope with adverse external conditions; all these mechanisms are essential for their survival. These strategies are effective if they are activated in time, in order to set a defense response and anticipate the environmental changes that might affect plant growth irreversibly. The trade-off between growth and acclimation metabolisms results in a sort of fitness cost for plants, since energy and nutrients normally destined to growth and production are intended for stress responsive mechanisms .
Agronomic management conducted in order to enhance plant tolerance towards abiotic stresses evolved over the centuries due to the technologic progress, climate change, scientific knowledge, and farmers' experiences. The choice of the correct cultivar, the best growing period, the sowing density, and the amount of water or fertilizers are some of the most common strategies applied to mitigate the negative effects of abiotic stresses . Protected cultivation is a cropping technique adopted to preserve plants from unfavourable outdoor conditions. It is mainly suited to vegetables and floriculture production in a non-optimal environment, through the control of temperatures, radiation or atmospheric composition. Another agronomical strategy, especially applied in vegetable crops, is soilless cultivation. This approach allows controlling of water and nutrients, avoiding the use of soil for cultivation and all the problems related to it, like poor quality or contamination.
Grafting is an additional tool adopted to counteract environmental stresses and increase tolerance in vegetable crops.
This technique is applied especially to high-yielding fruits and vegetables such as cucurbits and solanaceous to enhance tolerance against saline soil, nutrient or water deficiency, heavy metals or pollutants toxicity .
Agronomical strategies are essential in mitigating the negative effect of several abiotic stresses, but sometimes their application is not enough. Moreover, current experiments aim to transfer one or more genes involved in signaling or regulatory pathways, or genes encoding to molecules, such as osmolytes and antioxidants, conferring tolerance to a specific abiotic stress . Several functional and regulatory genes involved in abiotic stress tolerance have been identified and studied. Results of these studies can be exploited for genetic improvement aiming to introduce tolerance traits in cultivated crops. Since different physiological traits related to stress tolerance are under multigenic control, the manipulation of a single gene generally is not enough. Hence, scientists have paid more attention to regulatory genes, including transcription factors, due to their ability to regulate a vast array of downstream stress-responsive genes at a time .
However, the huge existing genetic variability among vegetable species, the lack of knowledge about minor cultivars genome, the complex responses triggered by abiotic stress conditions and the limited strategies currently available make genetic improvement really difficult and often inefficient. Moreover, besides the wide diversity of germplasms available, plant tolerance to stress depends both on stress features such as duration, severity, and frequency, as well as the affected tissues and development stages of crops .
Additionally, the increase of crop tolerance through genetic improvements requires many years of work and different cultivation environments that cannot be always taken into consideration. In addition to these strategies, it has been observed that stress tolerance can also be induced by biostimulants or specific bioactive compounds, if they are applied on vegetable crops when they really need to be protected .
Biostimulant application on horticultural crops under environmental stress conditions will be discussed in detail below. One of the key points of the discussion is about the application of these products in stressful conditions and their role as nutrients, not with a curative function. In particular, if a product has a direct effect against biotic stresses, it should not be included in the biostimulant category but should be registered as plant protection products. Table 1 is a summary of biostimulant products or bioactive molecules from different origins that have been evaluated for amelioration of abiotic stresses in several vegetables species. The biostimulants effectiveness to counteract the stressful condition depends on several factors, such as timing of application and their mode of action. The application of biostimulants can be carried out with different timings: before the stress affects the cultivation, during the stress, or even after. They could be applied on seeds, when plants are in early stages of growth, or when crops are fully developed, depending on the desired results . As general consideration, biostimulants that contain anti-stress compounds, such as proline or glutamic acid, can be applied when the stress occurs or during stress conditions. On the contrary, those that are involved in the activation of bioactive compounds biosynthesis must be applied before the stress occurs. Proper timing of application during crop development differs from species to species and it also depends on the most critical phases for crop productivity. Thus, the identification of the right time of biostimulant application is as important as the determination of the exact dose, in order to avoid waste of product, high production costs, and unexpected results. Biostimulants can be applied as foliar spray or to the roots, at sowing for protecting the seedling in the early development stages, in a floating system nutrient solution or during blooming or fruit setting. There is no general recipe that works for a crop species and in each stress situation.

Biostimulants and Crop Tolerance to Abiotic Stresses
The protective role of biostimulants on plants has been increasingly studied. These products are able to counteract environmental stress such as water deficit, soil salinization, and exposure to sub-optimal growth temperatures in several ways . They improve plant performance, enhance plant growth and productivity, interact with several processes involved in plant responses to stress, and increase the accumulation of antioxidant compounds that allow decrease in plant stress sensitivity.