The Grain Protein Content of Polish Cereals Other than Wheat: Can It Be Increased by Combining a Crop Sequence System, Cultivar Selection, and Plant Protection?

Abstract

After legumes, cereals are the most important source of protein for humans and livestock worldwide. One way to meet growing nutritional demands is to increase the grain protein content (GPC) of cereals. Breeding advances in this regard should be supported by optimized agricultural practices. The GPCs of winter rye, winter triticale, spring barley, and spring oats grown in 2018–2022 in northeast Poland were evaluated to determine the influence of the crop sequence system (continuous monocropping, crop rotation), cultivar (two for each species), plant protection level (control treatment, herbicide, herbicide, and fungicide), and interactions among these factors. The cultivar selection was a significant GPC determinant in all cereals. Growing triticale in crop rotation after a legume increased its GPC compared to continuous monocropping, but decreased the GPC of rye and had no effect on the GPCs of spring cereal that followed non-legume crops. Using herbicides and herbicides combined with fungicides promoted the GPC of rye and oats, but not of triticale and barley. The heterogeneity of the interaction effects of the studied agricultural practices on the GPCs of the individual cereals prevents the identification of a universal combination that would ensure the highest GPC levels. The inter-annual weather variability played a significant role in shaping the GPCs of cereals and in modifying the influence of the controlled factors.

1. Introduction

Cereals are a basic element of global food and feed production, comprising 23.5% and 69.7% of these respective sectors [1]. Among the cereal crops, maize, wheat, and rice collectively account for the majority of global cereal production, with a combined contribution of nearly 90% [1]. Other cereal species also hold importance, especially in regions with specific climatic and soil conditions [1]. In Polish agriculture, wheat, maize, and triticale currently play a strategic role (77% of national cereal production), followed by rye, barley, oats, and cereal mixed for grain [2]. Worldwide, cereals constitute a major source of both energy and protein, representing a vital component of human diets and animal nutrition. At present, these crops provide 42.8% and 36.4% of the global calorie and protein supply, respectively [1]. Although cereals primarily contribute to energy intake as carbohydrates, their protein content is also of critical importance for maintaining humans’ nutritional balance [3]. Despite their lower biological quality compared to protein derived from animal and legume sources [4,5], cereal proteins are generally regarded as being of good nutritional quality; nevertheless, lysine is often the limiting amino acid in cereals’ production of protein [4]. At present, nearly 30% of the world’s population suffers from protein malnutrition, and increasing the grain protein content (GPC) of cereals is one of the most effective approaches to meeting nutrition requirements [6].

The GPC is a critical component of cereal quality [3,7,8]. It not only influences the nutritional value of grain but also its technological properties, which are important in the food industry [8,9]. Grain designated for consumption and feeding purposes is required to have a high protein content [8,10], unlike that utilized for alcohol and biofuel production [8]. Besides its nutritional role, protein also plays significant functions within the plant itself, participating in key biological processes that directly affect plants’ development and survival under changing environmental conditions [11].

Compared to legumes and oilseeds, cereal grains contain low levels of protein. As Shewry [8] reported, these contents vary between 5% and 24% of the dry matter of cereal grains, with many species falling within the range of 8–15%. According to Biel et al. [7], wheat (14.3%) and hulless oats (14.4%) have higher GPCs than barley (12.0%), hulled oats (11.5%), and triticale (9.5%), while Tomicić [12] claims that oats (13.22%) have the highest GPC, followed by wheat (12.35%), barley (11.64%), rye (9.47%), maize (9.11%), and rice (7.83%). According to Kowieska et al. [13] and Alijosius et al. [14], the GPC of triticale (10.79–11.70%) is nearer to that of wheat grain (11.13–13.85%) than it is to that of rye (9.37–10.37%). It is broadly recognized that the GPC of cereals is governed by a multifaceted interplay of genetic, environmental, and agronomic factors [8]. This presents challenges, but also opportunities for increasing the protein content of grain harvested from the field.

Recent advancements in breeding methodologies in cereals (i.e., molecular identification of the high-protein-content genes, marker-assisted selection, gene introgression, and gene pyramiding) have facilitated the development of cereal cultivars with elevated GPCs [9,15,16,17,18]. Furthermore, strategies have been identified to overcome the inverse correlation between grain yield and GPC [16,19]. Choosing the appropriate cultivar for a particular production purpose is thus the first step in achieving a yield with the desired GPC. Then, to capture the full genetic protein potential of the selected cultivar, it must be provided with the best possible growth conditions, particularly during the grain-filling stage [20].

As many studies have proven [20,21,22,23,24], the GPC of cereals is sensitive to environmental factors, such as the soil quality, weather conditions, and climate variability. Healthy soils that are rich in nutrients have been shown to promote protein accumulation in grains [21,25]. Conversely, water deficiency during critical growth stages such as heading and grain filling [26] can lead to a reduced GPC [20,23,24]. Cultivating crops on soils that meet their nutritional requirements is a fundamental component of good agricultural practice. However, weather conditions are inherently uncontrollable. Moreover, as global warming continues, farmers will face an increase in the unpredictability of weather anomalies and extremes (droughts, heat waves, excessive rainfall) [27]. The impact of the changing climate on the GPC of cereals is gaining substantial attention [20,22]. The selection of cultivars that are both protein-rich and tolerant to abiotic stresses and their variability is therefore a desirable strategy [22].

Agronomic factors, defined as agricultural practices that are employed on specific farms, are fully controllable and contingent on farmers’ decisions, and these decisions are driven by their knowledge coupled with other factors (economic, organizational, social) [28]. Since the effect of agronomic treatments on the GPC of cereals is often genotype-dependent [4], implementing a genotype- (species-, variety-) appropriate set of agricultural practices appears to be crucial. Among agronomic factors, nitrogen (N) fertilization is considered a key element in determining the GPC of cereals [8,29,30] since N is a fundamental component of protein-building amino acids [31]. When supplied to cereal crops in the right form, rate, and time (i.e., plant development stage), N significantly increases the GPC of cereals [8,30]. Sowing density is another well-known factor that affects GPC [30,32,33,34]. It impacts the competition among plants for resources, notably nitrogen, as well as grain filling. A general decrease in GPC with an increasing sowing density has been reported in the literature [30,32,33,34]. Beyond the fertilization and sowing density, other agronomic factors, such as the adoption of a crop sequence system (e.g., crop rotation vs. continuous monocropping) [35,36,37] and plant protection against weeds, diseases, and pests [38,39], also have the potential to directly or indirectly influence the GPC of cereals.

Cropping cereals using crop rotation is generally beneficial for production and environmental reasons. This view is supported by numerous studies that have applied such a cropping system and compared it to growing cereals after cereals, up to a continuous cropping of one species (continuous monocropping, monoculture) [37,40,41,42,43,44,45,46]. However, the impact of these cropping systems on the GPC of cereals has not been clearly delineated [35]. Explaining this effect may involve a gross generalization because of existing interactions between the adopted cropping sequence and other crop-associated management practices, including fertilization [47,48]. A large body of evidence points to the importance of the previous crop in this context, specifically the biological potential of the soil after harvest (nutrients and water left, quantity and quality of post-harvest residues, allelopathic exudates, microbial activity, and more) [36]. Legumes are recognized as previous crops that support the GPC of cereals [36,37,41,42] by enriching the soil with biologically fixed N [48,49] and increasing its pool of N that is available to the subsequent cereals during their grain-filling period. Nevertheless, in some trials, better results were obtained after cereals than after legumes [43,50]. Some authors found no impact of crop rotation [35,51] or the choice of previous crop [52] on the GPC of cereals, while others observed the maximum GPC for continuously grown cereals [53]. The latter was probably a consequence of very low grain yields (inverse correlation). Such contradicting opinions confirm the complexity of the issue and justify the need for further research.

With few exceptions [40], cereals cultivated in diversified crop rotations exhibit an enhanced performance when pesticides are integrated into the control of weeds, pathogens, and pests [47,54]. This support is often needed when growing less weed-competitive genotypes [44] and when generalized diseases and pests occur [47]. By competing for resources (weeds) [55], feeding on living host cells (pathogens, pests) [56,57], or secreting cell-degrading enzymes and phytotoxins (pathogens) [56], these biotic stressors affect the availability of resources and the host plant’s physiology [56,58]. Thus, they can affect the GPC of cereals by modifying the relationship between N and carbohydrate accumulation, which determines this parameter [59]. Eliminating harmful organisms with pesticides usually facilitates plants’ functioning and thus increases their productivity; however, at the same time, a negative correlation between the grain yield and GPC is often exposed [60]. Furthermore, the pesticides used may have individual effects on the GPC. Depending on the mode of action, they may affect specific biochemical pathways in the protected crop [39]. New pesticides are released continuously [61], and all aspects of their action should be recognized. On the other hand, growing a biotic stressor-tolerant cultivar in a rationally planned crop rotation can lower the need for pesticide use [62].

Research on the effects of the interaction between the genotype, environment, and agronomic management on cereal performance, including the GPC, has been commonplace and ongoing for a long time [8,29,35,53]. However, interest in the issue persists, and the need for further efforts has not diminished. This ongoing interest can be attributed to a number of rationales that pave the way for sustainably managing protein production in the context of a growing demand for food and feed [8,63]. Among these rationales are: (i) the continuous improvement of cultivars to obtain the best possible set of traits (yield and quality, resistance to biotic and abiotic stresses), which generates the need to test them under different real agro-environmental conditions; (ii) the development of new agronomic measures, ways to exploit the potential of these cultivars (e.g., new pesticides), and their integration with old, reliable practices (e.g., crop rotation); and (iii) progressive climate change and the increasing variability of weather events. Together, they force an intensified search for means to increase the resilience of agroecosystems to their impacts. Long-term studies on the effects of cultivar selection and specific agricultural practices on the GPC of cereal can contribute to finding sustainable ways to improve the protein nutrition of both humans and livestock—starting at the field level.

In a multitude of European countries, wheat represents the most significant source of cereal protein [1]. It is not surprising, therefore, that the GPC of this cereal has been the most studied [8,9,15,16,17]. In Poland, in addition to wheat [1,64], other species, including rye, triticale, barley, and oats, merit consideration as well, particularly in the context of animal nutrition [64]. In the country, rye and triticale are predominantly cultivated as winter cereals, while the spring form of barley dominated until 2022, and oats have been cultivated exclusively as a spring cereal to date [2]. The literature on exploiting the genetic protein potential of the cultivars of these cereals through integrating agricultural practices is less abundant. This study aims to evaluate the effects of two opposing crop sequence systems (continuous monocropping and six-field crop rotation), two cultivars, three levels of plant protection, and the interactions between these factors on the GPC of winter rye, winter triticale, spring barley, and spring oats over a five-year period of the long-term experiment. The research was hypothesized to identify the optimal combination of the aforementioned practices, which would provide the maximum possible GPC of the four studied cereals grown under northeastern Polish conditions.

2. Materials and Methods

2.1. Site, Soil, Climate

This study was based on a large, long-term field experiment that was established in fall 1967 at the Production and Experimental Plant “Bałcyny” Sp. z o.o. in Bałcyny (NE Poland, 53.60° N, 19.85° E). The experiment is situated in a slightly undulating terrain on Luvisols formed from light silty clays. The chemical properties of the soil from the entire experiment are shown in Supplementary Table S1. The region is distinguished by high weather variability and significant fluctuations throughout the seasons from year to year. Rainfall is fairly evenly distributed throughout the year, although there are irregular short periods of drought, as well as of heavy rainfall. In Bałcyny, periods of drought, most threatening to vegetation, occur in July and August [65]. The monthly precipitations (sums) and air temperatures (mean, minimum, and maximum values) for the studied vegetation periods, as recorded by the Bałcyny Meteorological Station, are presented in Table S2.

2.2. Experiment Design and Agronomic Management

The cereal grain material for the present study was obtained from selected fields from within a larger experiment. These fields included winter rye, winter triticale, spring barley, and spring oats. The history of the overall experiment and a detailed description of its design were presented in previous papers [66,67]. Briefly, starting in 1993, the experiment was executed as a 3-factor experiment comprising the following factors: (i) crop sequence system (continuous monocropping vs. crop rotation; 12 crops studied), (ii) cultivar (2 for each crop, contrasted as much as possible in terms of agronomic characteristics), and (iii) chemical plant protection (3 levels). Each treatment combination (crop sequence system × cultivar × chemical plant protection level) was represented by three plots, constituting three replications. With 12 crops being grown, the entire experiment included 432 plots of 27 m² each (about 1 hectare in total). The arrangement of fields and plots can also be found in Figures S1 and S2.

The data presented in this paper cover five consecutive growing seasons: the 2017/2018–2021/2022 seasons for winter rye and winter triticale, and the corresponding 2018–2022 seasons for spring barley and spring oats. The levels of the following experimental factors were evaluated in this study: (i) crop sequence system (CS): continuous monocropping (CM) of the cereals under study (winter rye, spring barley, and oats were grown on the same field since the beginning of the experiment; continuous winter triticale was initiated in the autumn of 1993) and growing of said cereals in a diversified crop rotation (CR; A: potato–spring oats–fiber flax–winter rye–faba bean–winter triticale, or B: sugar beet–corn–spring barley–peas–winter rape–winter wheat); (ii) cultivar (Cv): two distinct cultivars were utilized for each species (for winter rye, KWS Binntto and Dańkowskie Diament; for winter triticale, Trapero and Borowik; for spring barley, Radek and Skald; for spring oats, Elegant and Bingo); (iii) chemical plant protection (PP): no herbicide and fungicide protection—control treatment (CT), herbicide protection (H), herbicide and fungicide protection (HF). This study included 144 plots from the long-term experiment (four crops/cereals × two crop succession systems × two cultivars × three plant protection levels × three replications).

Moreover, the year of this study (Yr) in the five-year study period was designated as an additional, fourth (iv) factor representing an uncontrolled source of variability.

The selection of cultivars was made in accordance with the recommendations provided by the Research Center for Cultivar Testing (COBORU) in Słupia Wielka. The characteristics of the cultivars can be found in Table S3. Annually, certified sowing material of pure cultivars of the tested cereals was utilized. The herbicides and fungicides utilized in this study were chosen based on the recommendations of the Institute for Plant Protection—National Research Institute in Poznań. A list of the chemicals and application data is presented in Table S4. Other basic agrotechnical data of cereals can be found in Table S5.

2.3. Data Collection

Each year, during the combine harvest, samples measuring approximately one kilogram were collected from the harvested grain yield of each plot. These samples were placed in paper bags and transported to the laboratory for further preparation and grain protein content analysis. Thereafter, 200 g portions of each sample were weighed and purified to remove impurities and weed seeds. Grain protein content (GPC) was determined in these sub-samples using a near-infrared (NIR) grain analyzer (Infratec 1241, FOSS, Hillerød, Denmark). The Kjeldahl method was the reference method for each species and year.

2.4. Statistical Analysis

The GPCs of winter rye, winter triticale, spring barley, and spring oats were analyzed separately. A four-way analysis of variance (ANOVA) was performed for a completely randomized experiment. A linear fixed model with fixed effects of three agronomic factors and the year was used (Equation S1). The effects of the crop sequence system (CS), cultivar (Cv), chemical plant protection (PP), year (Yr), and their interactions (two-way: CS × Cv, CS × PP, Cv × PP, CS × Yr, Cv × Yr, PP × Yr; three-way: CS × Cv × PP, CS × Cv × Yr, CS × PP × Yr, Cv × PP × Yr; and four-way: CS × Cv × PP × Yr) were assessed for their individual and combined contributions to the GPC of each cereal species. The distribution normality of the variables and the variance homogeneity were confirmed using the Shapiro–Wilk W and Levene’s tests, respectively. Tukey’s HSD test was applied to evaluate the differences between the means for the treatments. Moreover, Pearson’s simple correlation coefficient (r) was employed to demonstrate the relationship between the GPC and grain yield of individual cereals. For all analyses, the criterion for statistical significance was a p-value less than 0.05. The STATISTICA 13.3 program [68] was used to perform statistical procedures.

3. Results

The grain protein contents (GPCs) of the studied cereals ranged from 6.9% to 10.2% for the winter rye, from 7.5% to 12.2% for the winter triticale, from 7.6% to 11.9% for the spring barley, and from 8.0% to 12.3% for the spring oats, and showed varying dependence on the experimental factors that were adopted (

Table 1. Analysis of variance (F values) for grain protein content of the cereals under study.

Source of Variation	Winter Rye	Winter Triticale	Spring Barley	Spring Oats
Crop sequence system (CS)	6.82 *	7.31 **	0.0	2.8
Cultivar (Cv)	157.77 ***	58.72 ***	14.2 ***	8.8 **
Plant protection (PP)	10.62 ***	1.55	0.1	8.1 ***
Year (Yr)	18.81 ***	24.84 ***	202.5 ***	289.5 ***
CS × Cv	10.29 **	2.78	0.4	3.7
CS × PP	7.78 ***	0.97	6.0 **	5.1 **
Cv × PP	0.80	3.87 *	2.3	0.1
CS × Yr	14.71 ***	0.48	7.3 ***	2.9 *
Cv × Yr	1.56	6.37 ***	2.3	2.4
PP × Yr	2.51 *	2.88 **	1.6	2.5 *
CS × Cv × PP	16.62 ***	0.45	3.8 *	2.7
CS × Cv × Yr	1.44	0.90	0.3	0.7
CS × PP × Yr	0.92	2.58 *	1.1	1.9
Cv × PP × Yr	1.06	0.48	3.2 **	0.5
CS × Cv × PP × Yr	1.17	2.13 *	1.0	1.3

* p < 0.05, ** p < 0.01, *** p < 0.001.

). In the winter cereals (rye and triticale), the crop sequence system (CS) and cultivar (Cv) demonstrated a significant impact, with the level of chemical protection (PP) also exerting an influence in the case of rye. In the spring cereals (barley and oats), the cultivar (Cv) exhibited a substantial impact, with the level of chemical protection (PP) also displaying relevance in the case of the oats. The GPC of all cereal species was also found to be significantly influenced by the study year (Yr). The most influential factor for the winter cereals was Cv, while for the spring cereals it was Yr. Furthermore, an array of interactions among the main sources of variation was observed: for winter rye, CS × Cv, CS × PP, CS × Yr, PP × Yr, and CS × Cv × PP; for winter triticale, Cv × PP, Cv × Yr, PP × Yr, CS × PP × Yr, and CS × Cv × PP × Yr; for spring barley, CS × PP, CS × Yr, CS × Cv × PP, and Cv × PP × Yr; and for spring oats, CS × PP, CS × Yr, and PP × Yr.

As demonstrated in

Table 2. Effects of crop sequence system, cultivar, plant protection, and year on grain protein content of the cereals under study (means and standard errors).

Source of Variation	Winter Rye	Winter Triticale	Spring Barley	Spring Oats
Crop sequence system (CS)
Continuous monocropping (CM)	8.32 ± 0.06 a 3	9.46 ± 0.09 b	9.91 ± 0.11 a	10.28 ± 0.11 a
Crop rotation (CR)	8.17 ± 0.08 b	9.68 ± 0.08 a	9.90 ± 0.09 a	10.37 ± 0.10 a
Cultivar (Cv) 1
A	7.89 ± 0.06 b	9.88 ± 0.08 a	9.79 ± 0.09 b	10.24 ± 0.11 b
B	8.60 ± 0.06 a	9.26 ± 0.08 b	10.02 ± 0.11 a	10.40 ± 0.11 a
Plant protection (PP)
CT 2	8.07 ± 0.08 b	9.61 ± 0.13 a	9.90 ± 0.13 a	10.17 ± 0.12 b
H	8.37 ± 0.10 a	9.63 ± 0.09 a	9.90 ± 0.12 a	10.37 ± 0.14 a
HF	8.29 ± 0.07 a	9.47 ± 0.09 a	9.92 ± 0.12 a	10.43 ± 0.13 a
Year (Yr)
2018	8.42 ± 0.09 a	9.49 ± 0.09 b	8.97 ± 0.09 c	10.50 ± 0.09 c
2019	7.99 ± 0.12 b	9.71 ± 0.16 ab	9.73 ± 0.08 b	9.91 ± 0.07 d
2020	8.51 ± 0.12 a	10.06 ± 0.09 a	10.78 ± 0.10 a	11.01 ± 0.07 b
2021	7.92 ± 0.09 b	9.76 ± 0.13 ab	10.98 ± 0.06 a	11.44 ± 0.06 a
2022	8.39 ± 0.10 a	8.84 ± 0.10 c	9.06 ± 0.06 c	8.75 ± 0.07 e

¹ winter rye: A—KWS Binntto, B—Dańkowskie Diament; winter triticale: A—Trapero, B—Borowik; spring barley: A—Radek, B—Skald; spring oats: A—Elegant, B—Bingo; ² CT—control treatment within the chemical plant protection factor; ³ different letters indicate significant differences at p < 0.05.

, the GPC of winter rye cultivated in the CM system was higher compared to that grown in the CR system. The cultivar Dańkowskie Diament demonstrated a greater GPC compared to the KWS Binntto. Protection strategies H and HF resulted in an increase in GPC relative to the CT treatment. Furthermore, the rye grains harvested in 2018, 2020, and 2022 exhibited a higher GPC than those harvested in 2019 and 2021. The GPC of the KWS Binntto cultivar grown in the CM system was higher than that grown in the CR system, while no significant differences between the CM and CR systems were observed for the Dańkowskie Diament cultivar (Figure 1a). Protection with H and HF against CT resulted in an enhanced rye GPC only in the CM system (Figure 1b). In 2018, 2019, and 2021, the rye cultivated in the CM system exhibited a higher GPC compared to that grown in the CR system (Figure 1c). As demonstrated in Figure 1d, in 2022, the H treatment resulted in a higher rye GPC compared to the CT treatment. In other years, the impact of PP on the rye GPC was found to be insignificant. Details of the effect of the CS × Cv × PP interaction on the GPC of the winter rye are shown in Table S6.

Contrary to the outcomes observed in winter rye, the cultivation of winter triticale in the CR system resulted in a higher GPC than in the CM system (Table 2). The Trapero cultivar exhibited a greater GPC compared to the Borowik cultivar, and the triticale demonstrated the highest GPC in 2020 and the lowest in 2022. The GPC of the triticale decreased in response to HF protection exclusively in the case of the Trapero cultivar (Figure 2a). During the five-year study period, the Trapero cultivar generally accumulated more protein than the Borowik cultivar; however, in 2019, the differences between the cultivars were significant (Figure 2b). The influence of PP was observed only in 2019, when the H- and HF-protected triticale exhibited lower GPCs than under CT treatment conditions (Figure 2c). Details of the effects of the interactions of CS × PP × Yr and CS × Cv × PP × Yr on the GPC of winter triticale are presented in Tables S7 and S8, respectively.

The Skald cultivar of spring barley exhibited a higher GPC compared to the Radek cultivar. During the five-year study period, the highest barley GPC was observed in 2020 and 2021, while the lowest was recorded in 2018 and 2022 (Table 2). In the CM system, H treatment led to a decrease in the GPC of barley, while, in the CR system, it resulted in an increase (Figure 3a). However, HF treatment in both crop sequence systems did not significantly alter the GPC in comparison to the CT treatment. In the years 2018, 2019, and 2021, no differences in the GPC of barley were observed between the CM and CR systems (Figure 3b). However, in 2020, a higher GPC was found in the CM system than in the CR system, and in 2022, the reverse result was observed. Details of the effects of the interactions of CS × Cv × PP and Cv × PP × Yr on the GPC of spring barley are presented in Tables S9 and S10, respectively.

The Bingo cultivar of spring oats had a higher GPC than the Elegant cultivar (Table 2). The application of H and HF protection resulted in an augmented oat GPC in comparison to the CT treatment. Between 2018 and 2022, aligning with the observations in barley, the highest oat GPC was observed in 2021, while the lowest was recorded in 2022. The analysis of variance revealed that the enhancement of the oat GPC due to H and HF treatments was exclusively observed in the CM system (Figure 4a). In 2020, oats grown in the CR system exhibited a higher GPC than those grown in the CM system, and in the other years, the CS factor did not differentiate this parameter (Figure 4b). The impact of the PP level on the GPC of oats exhibited variability across the five-year study period, except for 2022, where the PP factor proved to be statistically insignificant (Figure 4c). In 2018, the oat GPC was observed to be promoted by H protection, while HF treatment did not demonstrate a favorable response compared to CT treatment. In 2019 and 2020, an increase in the GPC of oats was exclusively observed under HF treatment, with no response to H treatment being noted. In contrast, in 2021, oats protected with H exhibited GPCs that were comparable to those observed with HF treatment and higher than those observed under CT conditions, with no differences being detected between the CT and HF treatments.

An investigation of the correlation between the GPC of winter rye grain and the yield of this cereal revealed a negative correlation (r = −0.3071, p = 0.000). However, no significant relationships between the GPC and yield were observed for winter triticale (r = 0.1004, p = 0.206), spring barley (r = 0.0637, p = 0.424), and spring oats (r = −0.0813, p = 0.307).

4. Discussion

The climatic conditions in northeast Poland, where this study was located, are considered favorable for growing winter rye, winter triticale, spring barley, and spring oats, as confirmed by the results of post-registry testing at the national level [69]. However, the cereal GPC values obtained in the present study tended to be lower than the maxima reached by other authors (

Table 3. Selected comparison data for the grain protein contents of the studied cereals.

Cereal Species	Cultivar	Grain Protein Content, %	Year	Reference
Winter rye	KWS Binntto	6.9–9.4	2018–2022	this study
	KWS Binntto	12.2–12.7	2019	[73]
	Dańkowskie Diament	7.3–10.2	2018–2022	this study
	Dańkowskie Diament	13.5–13.8	2019	[73]
	different	12–15 1	no data	[8]
	different	8.6–11.4	2019–2020	[74]
	different	11.1–14.3	2019	[73]
Winter triticale	Trapero	7.8–12.2	2018–2022	this study
	Trapero	15.2	2013	[75]
	Borowik	7.5–11.3	2018–2022	this study
	different	5.9–19.3	1982–2010	[76]
	different	7.9–19.7	2011–2019	[77]
	different	11.8–15.2	2013	[75]
Spring barley	Radek	7.6–11.6	2018–2022	this study
	Radek	9.7	2017	[78]
	Radek	10.6	no data	[79]
	Skald	8.1–11.9	2018–2022	this study
	Skald	10.5–13.0	2009–2010	[80]
	different	8–15 1	no data	[8]
	different	10.3–12.6	no data	[79]
	different	9.8–13.9	2011–2013	[81]
Spring oats	Elegant	8.0–11.8	2018–2022	this study
	Bingo	8.3–12.3	2018–2022	this study
	Bingo	11.4	2010–2011	[82]
	Bingo	10.7	no data	[83]
	Bingo	10.4–13.1	2016–2018	[84]
	different	12–24 1	no data	[8]
	different	9.4–16.7	2009–2011	[85]

¹ Data for species, irrespective of form and hulling.

), and were particularly low for winter cereals. This may be attributed to the N fertilization regime adopted in the long-term experiment on which the present study is based (Table S5). Nitrogen (N) is the most powerful GPC-determining agro-environmental factor [8], playing an instrumental role in protein synthesis and the activation of pivotal metabolic enzymes [70]. Another study showed that N requirements increased linearly with increasing grain protein [71]. The total GPC of plants is linked to their N uptake and remobilization post-flowering. Nitrogen absorbed before flowering supports photosynthesis, and in the late reproductive stage, it aids grain growth and development, enhancing grain protein accumulation [70]. Suboptimal N fertilization may reduce the N uptake of plants after flowering and limit N accumulation and the protein concentration of cereals’ grains [72]. In the present study, the level and timing of N fertilization established for cereals in the long-term experiment may have limited full exploitation of the protein potential of the tested cereals. Another limitation of the present study is the small number of cereal cultivars that were tested along with their maintenance over the years of the rotation cycle, which also comes from the general assumptions of the basic long-term experiment.

In the present study, the GPC of rye exhibited a decrease, while the GPC of triticale demonstrated an increase in the CR system compared to the CM system. In spring cereals, the CS factor proved to be insignificant. The observed effects can be attributed to the influence of the previous crops of these cereals, as has been shown in other studies on cereal and non-cereal previous crops for cereals [36,50,86,87]. Studies by other authors have shown that cultivating cereals in the CR system, specifically following legumes (field pea, mung bean, faba bean), leads to an increase in their GPCs [36,48,88,89]. In the present study, only triticale was cultivated after a legume (faba bean), which may have led to an augmentation in the GPC obtained in the CR system relative to the CM system. The remaining cereals, namely rye, spring barley, and oats, were sown in the CR system following fiber flax, corn, and potato, respectively. The decrease in the GPC of rye in the CR system compared to that in the CM system can be attributed to the higher rye yield in the former system. As previously demonstrated, winter rye reacts strongly to cultivation in the CM system, resulting in yield loss [40]. Conversely, cultivation in the CR system, even without weed control, results in a high yield of this cereal. Higher cereal yields are often associated with the effect of N dilution in grain due to photosynthetic activity during grain filling [90,91], which consequently leads to a reduction in the GPC. Numerous studies have demonstrated a negative correlation between the GPC and grain yield of cereals [16,19,53,92]. The present study confirmed such a negative relationship for rye, while no significant correlation was observed for other cereals.

As evidenced by numerous studies, the GPC of cereals is a cultivar (genetic) trait [77,93,94,95,96], with a low coefficient of variation [75,95]. In the present study, the significant impact of the Cv factor on the GPC was confirmed for all the species under study. In winter cereals, it emerged as the most influential source of variability, a finding consistent with other studies [93]. Among the rye cultivars that were examined, irrespective of the crop sequence system (CM or CR), the Dańkowskie Diament cultivar exhibited a higher GPC compared to the KWS Binntto cultivar, which aligns with the outcomes of the study by COBORU [97]. Furthermore, the GPC of the KWS Binntto cultivar was found to be lower in the CR system compared to that in the CM system. This phenomenon can be attributed to the higher yield potential exhibited by the hybrid cultivar KWS Binntto in comparison to the population cultivar Dańkowskie Diament. In optimal conditions within the CR system, there was a greater possibility to exploit the yield potential of KWS Binntto, which was associated with the N dilution effect seen in grains [91] and the subsequent reduction in GPC.

In the present study, the Trapero cultivar of winter triticale was found to have a higher GPC than the Borowik cultivar, a finding that aligns with the results of the study by COBORU [97]. Unexpectedly, the “older” cultivars of spring cereals, registered in the Polish National List of Varieties of Agricultural Plant Varieties until 2010 (the Skald cultivar of barley and the Bingo cultivar of oats), demonstrated higher GPCs than newer ones. The findings of the COBORU study on barley identified the Radek cultivar as having a higher GPC than the Skald cultivar [97]. The same studies reported no substantial differences in GPC between the oat cultivars Elegant and Bingo. The variability in GPC among cultivars may be driven by differing weather conditions, such as precipitation and temperature [98].

The present study demonstrated that the GPC of H-protected winter rye exceeded that of rye under CT treatment, which is consistent with the findings of Yanev et al. [99]. This can be attributed to herbicide reducing the density and biomass of weeds [40,45], and thus lowering weed competition for nutrients, including nitrogen (N). As a result, the availability of N to the crop was increased. However, with low herbicide efficacy, particularly against expansive resistant weed species, weed competition can persist, affecting the availability of space, water, and nutrients [46]. The GPC of chemically protected rye (H and HF levels) was higher in the CM system than in the CR system, which was attributed to the inverse correlation between grain yield and GPC [53]; despite increases in yields due to H or HF protection, the rye yields under the CM-H and CM-HF conditions were still lower than under the CR-H and CR-HF conditions (unpublished data), similar to the findings by other authors [40,45]. The present study found that the application of herbicide and fungicide had no effect on the GPC of winter triticale. Some other studies have shown that the chemical composition of winter triticale grain (including the content of N—the basic component of amino acids) does not change under the influence of different methods of weed control [100], nor with the dose of herbicide applied [101]. It is noteworthy that the Borowik cultivar did not exhibit a change in its GPC under the influence of plant protection, while the HF-protected Trapero cultivar exhibited a reduced GPC compared to that which underwent CT treatment. This result is challenging to explain. As the Trapero cultivar is considered to be less susceptible to fungal pathogens than the Borowik cultivar [97], the importance of fungicide protection for both its yield and GPC would be expected to be lower. In the present study, as in some others [102,103], the level of plant protection did not differentiate the GPC of spring barley. However, the application of H and HF treatments increased the GPC of spring oats compared to those under CT treatment conditions. Notably, Noworolnik and Leszczyńska [38] also reported an increase in the GPC of spring barley due to herbicide application. The present study demonstrated that the incorporation of a fungicide into a chemical protection program (HF treatment) did not result in a change in the GPC of any of the cereals that were tested, when compared to the effect of the H treatment. It can be assumed that herbicide application reduced weed infestation [40,44,45] and weed competition for N. Therefore, more soil N was available to the crops, which promoted protein accumulation in their grain. The inclusion of a fungicide in the plant protection program did not seem to affect these processes. This finding is consistent with the results of other studies [102,104]. Malalgoda and Simsek [39] suggest that, although fungicides can have a negative impact on grain quality, their mode of action is specific to eliminating pathogens and they are rather unlikely to target biochemical pathways in crops. The available literature also reports an increase in cereal grain protein content (GPC) due to improved nitrogen remobilization from vegetative tissues [105], as well as a decrease in the GPC in grain resulting from higher photosynthetic activity and starch accumulation following post-heading fungicide application [106].

The inter-annual variability in GPC observed in the present study was an expected consequence of the influence of weather conditions, as confirmed by many other studies [23,24,36,98]. The highest GPCs of winter cereals were observed in 2020, a year characterized by even rainfall distribution during grain development (in June) and scarce rainfall during grain maturation (39.7 mm in July) (Table S2). Concurrently, the average air temperature in June (17.9 °C) exceeded the typical June average (16.4 °C based on data from 1991 to 2020). High temperatures after heading and moderate rainfall were shown to promote protein accumulation [23]. In contrast, cool and rainy days without sunshine were found to inhibit the rate of protein synthesis and delay maturation [24]. In case of the spring cereals, the highest GPCs were documented in the 2021 growing season, concurrent with the lowest yields of barley [67] and oats (unpublished data). This may be attributable to the impact of elevated temperatures during grain development (21.1 °C in July) and unfavorable moisture conditions during heading (only 31.3 mm of rainfall in June) and grain filling (as much as 128.4 mm of rainfall in July) (Table S2). Lower GPC values of winter rye were documented in the years that resulted in high yields (2019 and 2021) (unpublished data). The inverse relationship between the GPC and rye yield was confirmed by a negative correlation coefficient. Nevertheless, this explanation did not apply to other cereals.

The influence of the uncontrolled factor (Yr) overlapped with that of controlled factors (applied agricultural practices), and the effects of the interaction of these variability sources were specific in each species of cereal that was studied. The complexity of the observed phenomena makes it difficult to fully explain their mechanisms. The continuation of the research in this long-term experiment and the adoption of a longer-term view may facilitate a more profound understanding of this complexity. Studies over a sufficiently long period of time may allow correlations to be found between the GPC of cereals and intra-annual weather parameters across agricultural practices.

The effects of the CS × Yr interactions in different cereal crops may be related to the program of farmyard manure (FYM) application in the CM (15 t ha⁻¹ every three years; fall 2016 and 2019—Table S5) and CR (30 t ha⁻¹ every six years to root crops; fall 2016) systems, and consequently to the ability of cereals to benefit from the N released from FYM in subsequent growing years and under specific weather (temperature and moisture) conditions. Numerous papers have reported an increase in the GPCs of cereals under FYM fertilization [107,108,109]. The position of the cereal in the crop rotation (previous crop, temporal distance from FYM application and legume crop) may also be of importance. In the present study, the temporal distance from the FYM application may contribute to explaining the differences in rye GPC between the CM and CR systems in 2018, 2019, and 2021. In the CR system, the rye was always grown in the fourth year after FYM application. In the CM system, however, the rye plants could benefit from the N released from FYM in the second (2018 and 2021) and third (2019) years after its application. The mineralization of organic N of FYM and increased activity of ammonifiers and nitrifiers, especially due to available organic carbon, may have contributed to the higher N uptake [107,110] and GPC observed in the CM than in the CR system in these years. In turn, in 2022, the higher GPC in the CR system compared to the CM system may be attributed to the utilization of N that was not used by the previous crop (fiber flax) [111] or N retained by abundant weeds in the previous crop that was subsequently released [112].

The significant effect of the Cv × Yr interaction on the GPC was confirmed only in winter triticale. In general, the Trapero cultivar exhibited a higher GPC than the Borowik cultivar, which is consistent with COBORU’s findings regarding the protein potential of these cultivars [97]. Only in 2019 did the difference between the cultivars reach statistical significance; in all other years, the differences were merely tendencies. The 2018/2019 growing season was conducive to high yields of triticale, particularly of the Borowik cultivar in the CR system. This cultivar exhibited superior tillering and, even without chemical protection, it outperformed weeds in the competition for nutrients [44]. The enhancement of the yield of this cultivar led to a dilution of nitrogen in its grain [90,91], which was accompanied by a substantial decline in its GPC. As a consequence, the disparities among cultivars were exacerbated.

The interaction of PP × Yr in shaping the GPCs of individual cereals cannot always be explained by an inverse relationship between the GPC and grain yield [8,16,19,53,70]. Such a relationship was statistically confirmed in the case of rye (negative correlation). Herbicide application in rye resulted in an increase in its GPC in 2022, the same year in which H treatment contributed to a decrease in the rye yield in the CR system (unpublished data). Protein dilution was an explanation for the decrease in the GPC of the triticale under H treatment in the 2018/19 growing season, which resulted in a substantial increase in the triticale yield [44]. In oats, H treatment promoted the GPC in 2018 and 2021 and did not change this trait in the other years, which, however, was not related to the oat yield. The inclusion of the fungicide in the crop protection program (HF level) in the winter cereals did not change their GPCs compared to the H treatment in any year. In oats, there was no change under HF compared to H (2021, 2022), and both an increase (2019, 2020) and a decrease (2018) in GPC. In the case of barley, the significance of the PP × Yr interaction on GPC was not demonstrated, nor was the relationship between the GPC and grain yield. The observed variety of effects may be explained to some extent by crop-weed competition for N and water affecting the GPC [55]. Under varying environmental conditions, which are determined by the cereal species and cultivar, the implemented agricultural practices (including the application of herbicides along with their efficacy), and, ultimately, the moisture and temperature regimes during a specific growing season, different weed communities emerge [113]. Depending on the differences in weed communities in terms of their abundance, composition, diversity, and functional structure, their competition can take different manifestations, including those related to the protein content in crop yields [55]. Some changes may also be caused by the inhibition of peptide bond biosynthesis by certain pesticides, which results in disrupted protein synthesis in the crop [104]. Such a mechanism was suggested by Iwaniuk et al. [104] to explain the reduction in wheat GPC that was observed as a result of the herbicide sulfosulfuron and the fungicides cyproconazole + propiconazole and spiroxamine + tebuconazole + triadimenol. In our own research, the fungicide cyproconazole + propiconazole was used in the protection strategies for all cereals, but the results do not allow us to confirm such an effect of this active ingredient. Changes may also arise from the infestation of grains by pathogenic fungi, which reduce the protein content of grains during storage [114].

5. Conclusions

The agronomic factors employed in this study, i.e., the crop sequence system, cultivar, and chemical plant protection, exerted specific effects on the GPC of the cereals under study. Only the significance of the cultivar choice, that is, the genetic determinant of this yield quality parameter, was confirmed for all species. Moreover, the influence of the cultivar was modified by other controlled factors and inter-annual variability in weather conditions, yet it was controlled differently in individual species and to varying degrees. The crop sequence system only affected the GPC of winter cereals, with no effect being observed in spring cereals. Winter rye cultivated in crop rotation following fiber flax exhibited a reduction in GPC compared to that cultivated using continuous monocropping, contrasting with the case of winter triticale, which followed faba bean in the crop rotation. Crop protection with herbicide and combined herbicide and fungicide promoted the GPCs of winter rye and spring oats, while this phenomenon was not observed in winter triticale and spring barley. The interaction of the evaluated agricultural practices in shaping the GPC of cereals manifested itself uniquely in individual cereal species, preventing a definitive recommendation of a universal combination of the tested factors to ensure the highest level of this parameter. This study also confirmed the strong contribution of inter-annual weather variability in shaping the GPC of all cereals, as well as in modifying—although to varying degrees—the influence of the crop sequence system, cultivar, and crop protection strategy on this grain quality parameter.

The observed complexity of the effects of interacting experimental factors on the GPC of cereals argues for continued research into the possibilities of capturing genetic protein potential through skillfully selected combinations of agricultural practices. The development of new crop cultivars and crop protection products, as well as the need to cope with the unpredictable variability of weather conditions associated with climate change, provide additional arguments for continued scientific support to farmers seeking optimal solutions in this area.