**1. Introduction**

Stress in organisms can be induced by either abiotic or biotic factors [1]. In agro-ecosystems, of all the abiotic factors, drought is the main determinant that limits the development of plants and, consequently, their yielding [2]. Central Europe, including Poland, is located in a temperate climate [3], where the relevance of drought is often underestimated [4]. However, in recent years, with global climate change, the problem of drought has become increasingly serious in the region [4]. Rising atmospheric temperatures have resulted in increasing evapotranspiration rates [4], and seasonal and monthly distributions of precipitation have also been changing [5]. Drying trends were observed for spring and less pronounced for summer, i.e., for a large part of the vegetation period [5]. In general, drought reduces the uptake of minerals and their transport from the roots to the above-ground parts, which a ffects the rate of plant physiological processes [6]. Tolerance to water stress is a very important feature of plants during drought. Knowledge of species' sensitivity and response to water deficit can be helpful while selecting plants for cultivation, particularly in regions at risk of drought. Numerous studies indicate that resistance to stress is a genotypic trait [7–10].

Catch crops undersown in small grains (main crop) is a form of mixed cropping which offers numerous environmental benefits [11,12], and is considered as a sustainable agricultural practice [12,13]. Catch crop residues left in the field are a significant source of nutrient-rich organic matter [14]. After harvesting the main crop, they serve as ground covers and reduce nitrogen and phosphorus (P) leaching from the soil, as well as direct and indirect greenhouse gas emissions [15–19]. For the latter reason, the cultivation of catch crops is claimed to make an important contribution to climate change mitigation [20,21]. However, underplanted catch crops may compete for nutrients with the main crop [22,23], especially in unfavorable conditions such as water deficit [24]. Italian ryegrass (*Lolium multiflorum*) is one of the most popular catch crops, often undersown in spring barley (*Hordeum vulgare*) [25,26].

*Hordeum vulgare* is cultivated in many countries worldwide, with the grains intended mainly for animal feed purposes [27]. It is usually cultivated as the main crop. It is ranked fourth, following wheat, rice and maize, in terms of the area under cultivation [28]. Similarly to other cereals, it is also included in mixtures with leguminous plants [29–32], and is used as a protective plant for underplanted catch crops [25,33–35]. The species is distinguished by natural tolerance to drought [36]. This tolerance is determined by early flowering, which ensures optimal pollination, seed development and ripening in an optimal time period. In cereals, the consequences of water deficiency are determined by both the plant's development stage during which the stress occurs [37], and the frequency of drought occurrence during plant development [38].

*Lolium multiflorum* is a fast-growing annual or perennial grass originating from Europe [39]. *L. multiflorum*, similarly to *L. perenne*, is a valuable fodder plant cultivated in many regions of the world, in both dry and rainy areas [33,40–42]. *L. multiflorum* is cultivated as the main crop, but it can also be cultivated as an underplanted catch crop, similarly to other grasses, papilionaceous plants and their mixtures [15,43,44].

Phosphorus (P) is the second macronutrient after nitrogen, whose deficiency most frequently inhibits plant growth [45]. Poor water availability reduces the uptake of macronutrients from the soil [46,47], and may also affect their content in plants [48–51]. The more severe the drought, the more adverse the effect on the component ratio in the plant [52]. The literature offers many articles on the competition between the cultivated plants, particularly cereals and papilionaceous plants, for habitat resources [53–57]. However, no models have ever been developed to fully explain how plants compete for nutrients under water deficit conditions. Various indicators are used to assess this interaction, e.g., relative yield, relative competitive capacity [58], which are based not only on plant biomass [59], but also on the accumulation of macroelements [23,60]. Relatively more is known about nitrogen accumulation, while there are few studies on phosphorus [61,62]. This study may complement this information.

The study aimed to determine the influence of water deficit on P content and accumulation in the above-ground biomass of spring barley and Italian rye-grass growing separately and in the mixture (rye-grass undersown in barley), and on the inter-species interactions between these crops at different plant development stages.

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

#### *2.1. Experimental Design*

The study was based on a pot experiment conducted at a greenhouse laboratory of the Faculty of Biology and Biotechnology, University of Warmia and Mazury. The study was carried out on spring barley (Rastik cultivar) and Italian rye-grass (Gaza cultivar).

The experimental factors were as follows:

1. water supply of the plants: optimal (OW), and reduced by 50% in relation to the optimal one (LW), 2. sowing type: barley grown as a single species (BP), rye-grass grown as a single species (RP), barley in a mixture with rye-grass (BM), and rye-grass in a mixture with barley (RM).

An optimal dose of water was determined in a trial experiment, in which plants' irrigation requirements were established based on water loss estimated by daily measurements of pot weight. At the beginning of the trial experiment, the pots with plants were well irrigated, and the soil moisture content was maintained daily by re-watering with the water lost in the previous 24 h. Daily amounts of water supplied to the pots with barley, rye-grass and barley-rye-grass mixture were recorded during the successive growth stages. After finishing the trial experiment, based on the recorded data of water amounts used for daily irrigation, the pattern of plant watering with a higher dose for the proper experiment was established. A higher daily dose of water, common for the three types of sowing, was calculated as an average of barley, rye-grass, and barley-rye-grass mixture requirements at a given stage of plant growth. This dose was dynamic according to the plant development (changeable during vegetation), and it was slightly verified during each growing season. The reduced dose was always equal to one-half of the higher one. At the beginning of each experimental series of the experiment (sowing), the soil moisture was about 20% (measured by time domain reflectometry (TDR) method).

The plants were cultivated on proper brown soil formed from slightly loamy silty sand. The soil had a slightly acidic reaction (pH in 1 M KCl 5.6–6.1), average phosphorus (51–61 mg kg−1), potassium (98–117 mg kg−1) and magnesium (33–42 mg kg−1) content, and an organic carbon content of 7.1–11.1 g kg−1. Each pot, a week before sowing, was filled with 8 kg soil material previously mixed with mineral fertilizers, in a dose of pure component (g pot−1): N–0.5 (urea), P–0.2 (monopotassium phosphate), K–0.45 (potassium sulphate).

Plant kernels were sown into Kick–Brauckmann pots (diameter of 22 cm, depth of 28 cm). When preparing the mixture, the additive pattern was applied, as it assesses the species' interactions at early development stages better than the substitution pattern [63,64]. When single-species sowing was applied, 18 spring barley kernels or 18 Italian rye-grass kernels were sown, while for the mixed-species sowing, 18 spring barley kernels and 18 Italian rye-grass kernels were sown (pure sowing stand). The kernels were distributed using templates at an equal distance from each other over the soil surface, and placed at a depth of 3 cm.

From the kernel sowing to plant harvesting, the temperature at the laboratory was maintained at 20–22 ◦C. The exception was a 9-day period during the full plant emergence when the temperature was lowered to 6–8 ◦C to pass the vernalization process.

Three one-year cycles of the experiment were conducted. Each year (cycle), an experiment was set up according to completely randomized design in four replications, and comprised 120 pots: two levels of plant water supply x three levels of sowing type (two species sown separately and in a mixture) x five testing dates x four replications.

#### *2.2. Plant Sampling and Analysis*

The phosphorus content in the above-ground biomass of the plants was assayed in five developmental periods, determined by the developmental rhythm of barley sown as a single species and supplied with an optimum water dose. These included (according to BBCH scale): leaf development (10–13), tillering (22–25), stem elongation (33–37), heading (52–55), and ripening (87–91). When barley reached the appropriate stage, the plants were removed from pots (intended for a particular stage), and the shoots were separated from the roots. The material subjected to testing included the above-ground parts of barley and rye-grass plants. The plants were dried in the air and then weighed. For barley, beginning from the stem elongation stage, the shoots were separated into stems and leaves, and from the heading stage, into the spikes as well. For rye-grass, the shoots were separated into the stems and leaves, beginning from the barley stem elongation stage.

The phosphorus content was assayed by the spectrophotometric method (PN-ISO 6491:2000) [65], at the Chemical and Agricultural Research Laboratory in Olsztyn.
