Morphological Differentiation, Yield and Cutting Time of Lolium multiflorum L. under Acid Soil Conditions in Highlands

1. Introduction

Grasslands constitute over 70% of the world's agricultural land and play a pivotal role in food security, the economy, and global ecology due to their adaptability and functionality [1]. It is anticipated that climate change will intensify weather events, which will, in turn, lead to an increase in the frequency of droughts. This will harm forage production while exposing agricultural and silvopastoral systems to a heightened risk of imminent hazards [2]. Implementing measures designed to enhance genetic heterogeneity, topography, and grassland diversity can improve stress tolerance, mitigate damage, and facilitate recovery following periods of drought [3].

Lolium multiflorum, a grass species cultivated annually worldwide, stands out for its exceptional adaptability to diverse soil types and various climatic conditions. Its rapid establishment, high palatability, good digestibility, high yields of quality forage, and tolerance to diverse grazing methods make it a preferred choice for many farmers and producers. Its ability to reseed reduces costs and facilitates soil restoration, providing a reassuring solution in the face of climate change [4,5].

Ryegrass, in conjunction with other forage species (clover, vetch), is the primary source of feed for livestock. This is particularly true in hillside areas, where prolonged droughts impact pasture production, leading to a reversible or irreversible disruption of the plant's functioning and structure. This, in turn, affects the economy and livelihood of the livestock community in the northern highlands of Perú [6,7,8].

Lolium multiflorum L. is a grass species widely used in forage production due to its rapid growth and high productivity. This plant is adapted to altitudes between 2400 and 3200 m asl and temperatures between 12 and 18 °C. In addition to exhibiting resistance to trampling, it is considered a primary source of winter forage [9]. However, in highland areas, where acid soils are prevalent, the yield of Lolium spp. Varieties are adversely affected by low nutrient availability, yield deficit, and environmental stress [10]. The ryegrass-soil interaction is significant due to the exposure to such soils, given the grass's high adaptability, absorption, and fixation capacity. This substantially balances crowns and roots, thereby ensuring the grass's vigour against these challenging soils [11]. The Lolium family has demonstrated remarkable resistance and adaptability, enabling grasses in the northern highlands of Peru to thrive in low temperatures. This suggests that they possess a valuable source of frost and drought tolerance genes, as evidenced by their ability to activate the cold acclimatisation process [7].

Genetic improvement of Lolium spp. has resulted in the generation of specific genetic mutations, which have enabled the species to behave as a perennial under managed conditions due to its high competitiveness, genetic diversity, and seed production [12,13]. In recent decades, research has concentrated on enhancing yield, dry matter, and forage quality, thereby facilitating more effective crop management and enabling a more significant number of harvests per year. These productivity gains are contingent upon optimal humidity and temperature, an adequate pH level, organic matter, potassium, and phosphorus, which are essential for plant development and maturity. Nitrogen is also a crucial element, as it is vital for seedling production [14,15].

Understanding the germination behaviour of ryegrass seeds is crucial for effective integrated weed management strategies. The type of dormancy enriches seed ecology, the species' life cycle, and seasonal changes in environmental conditions, including temperature, precipitation, and soil salinity, from dispersal to germination [16]. Ryegrass seeds initially exhibit dormancy, which may be broken and result in germination occurring at different times throughout the growing season. This knowledge empowers effective weed management strategies [17].

Lolium multiflorum L., a species with a rapid growth rate, is characterised by its long and thin foliage with a glossy appearance and a distinctive erect and hollow stem. The root system is characterised by high fibrousness and superficiality, which enables rapid absorption of nutrients and water. In response to environmental cues, the plant initiates an adaptive process to prolong its vegetative stage, manifested in various morphological changes. These include a reduction in the height of the panicle, a decrease in the length of intact leaves, and an increase in the number of growing leaves [18].

A reduction in grazing intensity was associated with an enhanced leaf elongation rate, reaching 3.5 cm.day, and a diminished tillering density, with 150 tillers.m². Additionally, plant height, green forage yield, and dry matter ranged from 115.0 to 130.7 cm, 1407 to 3240 kg. ha, and 392 to 976 kg. ha, respectively. These results significantly impacted the attributes of ryegrass (Lolium multiflorum L.), indicating that moderate to light grazing increases spatial heterogeneity and productivity [19,20].

The morphological variation of Lolium multiflorum L. is interpreted based on its high phenotypic plasticity, the ability of an organism to change its phenotype in response to changes in the environment. This is the ability to alter its physiological characteristics in response to environmental variations and its hybridization (such as L. multiflorum x L. perenne). This highlights the need for effective localised control methods for this genus's different species and hybrids, thanks to its competitive nature, which makes it prone to developing resistance to herbicides. Applying pesticides that are harmful to forage and soil while reducing the occurrence of crown rot under different management conditions has resulted in variations in height ranging between 115.0 and 130.7 cm and green forage yields ranging between 1407 and 3240 kg. Ha, and dry matter production ranging between 392 and 976 kg. Ha [13,21].

It has been demonstrated that annual ryegrass can exhibit enhanced quality when subjected to water stress. Landscape heterogeneity indicates that, in specific locations, adding nutrients and soil amendments can significantly regulate pH and create favourable conditions for increased forage production. This is supported by evidence from studies by Abraha et al. (2015) [22] and Bridges et al. (2019) [23].

The Poaceae, including Lolium multiflorum L., play a significant role in the remediation of degraded soils. It mitigates these processes' deleterious effects on the environment. It facilitates the restoration of soils that can be utilised for agricultural production in the context of progressive soil degradation and depletion [24,25]. Cultivated as a cover crop, they have been demonstrated to enhance soil fertility, mitigate erosion, and curtail nutrient loss [26,27]. The sustainable management of soil offers solutions to the conservation and reduction of pollution by utilising plants adapted to the local climate and specific pollutants. These plants demonstrate effective responses to drought and salinity stress at the morphological, physiological, and biochemical levels. Consequently, the Poaceae are indispensable not only for sustainable food production but also for protecting the environment and managing ecosystems [28,29].

The appropriate requirement is fertiliser in ryegrass ranging between 100-150 kg.ha of N, 40-60 kg.ha of P, and 60-80 kg/ha of K. The correct application is crucial to increase yields and improve soil quality by maximising the yield and nutrient absorption of Lolium multiflorum L. in acid soils (pH 5.1 - 6.1). These practical recommendations can be readily implemented, thereby optimising milk production and farmers' profits while reinforcing the significance of proper soil management for attaining more efficient and sustainable agricultural practices [30,31].

The findings of this study are of significant value as they provide invaluable insights into the productive performance of Lolium multiflorum L. 'Cajamarquino ecotype' under acidic soil conditions in the northern highlands of Peru. The knowledge gained in this study can be used to inform recommendations on genotype management practices and cutting times, as well as the importance of yield differentiation under different climatic conditions.

2. Results

2.1. Yield Performance

Table 1 shows the superiority of the LM-58 genotype over LM-43 in green forage yield (20.97 Mg. ha), dry matter or biomass (4.49 Mg. ha), seed yield (259.23 Kg. ha) and protein value (13.48%); the LM-58 genotype shows a higher production capacity in terms of forage quantity and nutritional value in most locations. 48%); the LM-58 genotype shows a higher production capacity in terms of forage quantity and nutritional value in most locations, which represents a more advantageous option for optimising both agricultural productivity and forage quality.

2.2. Cutting time

Table 2 shows the crucial interactions between cutting days and biomass and protein production, highlighting the significance of our results that by increasing the cutting interval from 30 to 60 days, biomass produced increases significantly from 18.87 to 49.10 Mg. ha. yr (p < 0.001); similarly, protein production increases from 2.8 to 4.9 Mg. ha. yr (p = 0.0405). These results underline the direct influence of cutting time in optimising biomass and protein production, suggesting that a 60-day cutting interval may be more beneficial for maximising both parameters in the crops evaluated.

The effect of the interaction between cutting time (C) and location (L) on biomass and protein of Lolium multiflorum L. Figure 1 shows in detail the interaction between C x L, highlighting their effect on biomass and protein indicators of Lolium multiflorum L. Panel (A) shows the biomass production expressed in Mg. ha. year. At the same time, panels (B), (C) and (D) show the protein ratios in percentage (%), Mg ha. cut and Mg ha. year, respectively, for the genotypes LM-58 and LM-43. This analysis shows that the Baños del Inca site had the best yields in terms of biomass and protein content in Mg. ha. yr. These results highlight the importance of taking into account both the timing of cutting to optimise yield and quality of Lolium multiflorum L. crops, providing relevant information for farm management and improvement of cultivation practices. Taking into account the interaction for protein percentage (p= 0.0036)

2.3. Morphological characteristics

Table 3 shows the differentiation and morphological characterization of Lolium multiflorum L. for genotypes LM-58 and LM-43. The results highlight the superiority of the LM-58 genotype, which showed better results in the evaluated characters compared to the LM-43 genotype, which obtained lower results in all these parameters.

Table 4 shows a comprehensive morphological characterization of the qualitative characteristics of the genotype LM-58 of Lolium multiflorum L. This analysis reveals the diversity and complexity of the morphological characters of the genotype, which provides a detailed and multifaceted view of its behaviour and physical attributes throughout its growth and development.

Table 5 shows the evaluation of the qualitative characteristics of the genotype Ryegrass LM - 58, which was carried out with meticulous attention to detail, the results showed total uniformity in several aspects, where no atypical plant showed a different colour for the "colour of the lemma", which indicates uniformity in this character. Similarly, we observed uniformity and absence of atypical seeds for the presence of "kernel ridge", with no variation in the level of expression. This assessment was carried out visually by observing 30 plants, confirming their homogeneous classification in both assessment seasons.

Table 6 shows that the vegetative cycle of Ryegrass LM-58 exhibits an impressively early expression at 105 days, with coefficients of variability of 2.80% and 2.73% for the first and second seasons, respectively. Similarly, the "time of flower initiation" demonstrated an early expression at 40 days, with coefficients of variability of 2.91% and 2.59% for the first and second seasons, respectively. The classification of the variety as uniform according to UPOV regulations is supported by the observation that both characteristics exhibit coefficients below the permitted limit of 3% for self-pollinated varieties.

The analysis carried out to identify the mitochondrial DNA sequences of Lolium multiflorum L., genotype LM-58, for which a bioinformatic analysis was carried out where the ITS1 sequence haplogroup (ITS1 = INIA LM-58) differs in three mutational steps from the reference sequence of Lolium multiflorum L. (ecotype Cajamarquino). As can be seen in supplementary material S1.

3. Discussion

In this study, we evaluated the morphological differentiation, productive yield, and cutting time of Lolium multiflorum' Cajamarquino ecotype' under the challenging acid soil conditions of the northern highlands of Peru. Our impressive results indicate that the Cajamarquino ecotype adapts to unfavourable conditions. This adaptability is reflected in its morphological characteristics and productive yield. Even when faced with the primary source of abiotic stress, drought, which can severely impact plant growth and development, the Lolium multiflorum ecotype Cajamarquino produced favourable results regarding green forage and dry matter yields.

3.1. Performance yield

Green forage yield is influenced and conditioned by several factors, such as climate, grazing frequency and intensity, and cutting time [32,33]. Ryegrass is grown in winter because of its adaptability to various soils; this cover crop is valued for its cold hardiness, extensive root system and high biomass production. It is essential to remember that plant productivity is closely linked to the mineralization of soil nutrients and their uptake and utilisation by plants. This process involves intense interactions between roots, microflora and soil fauna [26,34].

It is worth noting that Lolium multiflorum L. boasts a higher nutritional value than Lolium perenne. Its rich concentration of vitamins and antioxidants makes it a highly sought-after species in the market, offering significant health benefits to consumers [35].

The dry matter yield of ryegrass is influenced by several factors, including soil moisture level, type and method of planting, number of harvests and fertilisation. Also, the soil texture and the competitive ability of Lolium multiflorum to grow on different soil types, on which the dry matter productivity will depend [36,37].

It is noteworthy that in the results obtained and as stated by the authors, a particular genotype of Lolium multiflorum L. exhibits superior performance in terms of green fodder yield. Although both genotypes, LM-58 and LM-43, are Cajamarquino ecotypes, the LM-58 genotype exhibits significantly higher yield compared to the LM-43 genotype. This dominance can be attributed to the superior characteristics exhibited by the LM-58 genotype, including greater leaf length and width, intense and dark leaf colour, and greater plant height after vernalisation, among others. These characteristics, which are corroborated in detail in Table 3 and Table 4, indicate not only a higher yield in terms of biomass produced, but also a higher efficiency in nutrient uptake. In addition, the LM-58 genotype has shown greater resilience to adverse climatic and soil conditions, which is vital for its adaptation and productivity in the locations evaluated. The above characteristics make the LM-58 genotype a more viable and promising option for farmers seeking to maximise green forage production.

As for the locations evaluated, despite being in geolocations with similar conditions, green forage yields varied from location to location; this could be due to differences in cropping seasons across years and evaluation periods. Factors such as microclimatic variations, specific agronomic practices, and soil quality may have influenced these results, however, the Baños del Inca location showed the highest yield in both production seasons, as detailed in Table 8 and Table 1. This consistency suggests that Baños del Inca offers more favourable conditions for growing Lolium multiflorum L. than the other locations. These findings highlight the importance of carefully selecting locations to maximise crop production and suggest that Baños del Inca could serve as a model for successful agricultural practices in the region.

3.4. Cutting time

The analyses revealed that the forage quality of the Cajamarquino ecotype is determined by the moment of cutting according to the dry matter (DM) yield per hectare and the percentage of protein. Three groups of analysis were formed with samples every 30, 45 and 60 days, showing that at 30 days, the best result was obtained with a protein content of 14.84%, as opposed to 45 days, while biomass increases in this period, protein content decreases to 12.75%, suggesting that the optimum cutting time to maximise forage quality is at 30 days to obtain better utilisation.

The study showed that 90% of ryegrass seedlings emerging on the first day compared to other species was due to the mowing period's ability to manage ryegrass dominance. The time to first clip and repeat clipping on seedling growth depends on the impact of mowing height, as defoliating according to soil height minimises damage to the tillers' growing points. It was also found that 22-50% of the variability in growth at 60 days was due to these initial treatments [38,39].

This study shows that cutting times are critical factors in determining the quality and quantity of Lolium multiflorum L. forage. A 30-day cutting interval is optimal for maximising protein content, while adequate cutting height can significantly improve grass recovery and growth. Furthermore, the variability in green forage yield between sites underlines the importance of taking local conditions into account in agronomic practices. Finally, it is noteworthy that the genotype LM-58, in interaction with the sites, obtained a significant value (p = 0.0036) for protein percentage. These data provide valuable information for genetic improvement and selection in forage production and show that this genotype outperformed the other local genotypes, as confirmed in Table 2.

3.3. Morphological differentiation

Morphological differentiation among local genotypes of Lolium multiflorum L. is essential to understanding its adaptation and performance under various environmental conditions. The study results indicate that genotype LM-58 has superior characteristics to genotype LM-43. In the parameters evaluated in growth habit, LM-58 exhibited semi-erect growth and a greater intensity of dark green colour in its leaves, contrasting with the more upright growth and light green colour of LM-43. In addition, LM-58 showed a greater width of the last leaf (0.8 cm vs. 0.6 cm for LM-43) and a higher number of seeds per spikelet (13 vs. 10 by LM-43). The growth rate of LM-58 was also higher, reaching 1.6 cm per day compared to 1.2 cm per day for LM-43, allowing faster recovery after cutting and higher forage production in a shorter period. Age at the fourth leaf was earlier in LM-58 (28 days) than LM-43 (35 days), indicating a higher earliness of LM-58, which may benefit faster production cycles.

Bioinformatic analysis of mitochondrial DNA sequences shows the identity of Lolium multiflorum L. and identified two distinct haplotypes. The LM-58 genotype clustered into one haplotype together with other samples under study, differing from the reference sequence of the LM-43 genotype. Cross-pollinated species, such as ryegrass, show high genetic variability within and between populations. Thus, L. multiflorum and the wind dispersal mating system promote high genetic variability within populations but low genetic differentiation between populations [40,41]. This genetic variability facilitates that the haplotypic network constructed shows two unique haplotypes: haplotype 1 containing the three samples under study and haplotype 2 containing the reference sequence, thus reinforcing the genetic differentiation of the LM-58 genotype, as can be seen in the supplementary material S1. Finally, the LM-58 genotype showed increased resilience to adverse climatic and soil conditions, vital for its adaptation and productivity in the locations evaluated. These characteristics make LM-58 a more viable and promising option for maximising green forage production.

4. Materials and Methods

4.1. Place and duration of study

This research was conducted in the Cajamarca Region - Peru, which had two experimental stages. The first phase was developed in the agricultural campaigns 2017 - 2018; the second campaign was created in the periods 2018 - 2019, where the productive yield parameters (Green Forage, Dry Matter, and time of cutting) were evaluated in two local genotypes LM - 43 and LM - 58, as detailed in Table 8, which was developed in 6 locations located in the region of Cajamarca, department of Cajamarca - Peru, with average annual temperature of 15.6°C, average relative humidity of 79% and an average annual rainfall 2963 mm/year [42], this research was evaluated in December, a suitable time for installing this forage species.

4.2. Experimental units

To determine the productive yield of Lolium multiflorum L., a Completely Randomised Block Design (CRBD) was used in 6 locations in the Cajamarca Region. This design, known for its robustness and reliability, was the perfect choice for our experiment. Rye Grass LM - 58 and LM - 43 genotypes were evaluated; each variety was sown in 6 m x 5 m plots, installing six plots in each location, with two replications randomly.

The treatments were divided according to the phenological stage of the Rye Grass Lolium multiflorum L., where T1 Genotype LM - 58 and T2 = Genotype LM - 43. It is essential to mention that the sowing system was broadcast in the 6 locations with a sowing density of 30 kg. ha of seed. Fertilisation was carried out in two seasons, each with two key fertilisation moments; in the first fertilisation at sowing, agricultural Urea (N) = 80 kg.ha.year, Triple Calcium Superphosphate (P₂O₅) = 130.4 kg.ha.year, Potassium Chloride KCl (K₂O) = 66.4 kg.ha.year and in the second tillering fertilisation consisted only of Urea = 80 kg.ha.year, using the same fertilisation rates for both campaigns.

4.3. Experimental material

The experimental material was derived from a backcrossing process involving a population of previously selected elite genotypes. The experimental treatments comprised the genotypes LM-58 and LM-43 of Lolium multiflorum L., which have undergone two selection processes. The initial phase involved 620 accessions, while the subsequent phase encompassed 21 exceptional emerging lines selected over 25 years.

Table 8 presents the detailed periods and critical activities of the study, such as the initial selection based on growth habit, anthesis, and pest and disease resistance, followed by propagation and polycrossing to enhance desirable traits. The research, which began in 1995 with the collection and germination of seedlings, progressed through several phases of characterization, selection, and field trials. After numerous tests, the genotype LM-58 was identified as an elite of Lolium multiflorum L., Cajamarca ecotype, marking a significant milestone in our research.

4.4. Sampling

We evaluated the morphological differentiation, yield, and cutting time of the Lolium multiflorum L. genotypes LM—58 and LM—43 with utmost precision. We obtained the samples using a square metre, manually cut the forage with a sickle, and weighed it fresh on an electronic balance. A 100g sample was then taken to the laboratory for further analysis, including determining the percentage of dry matter and calculating Biomass yield.

The amount of seed harvested was recorded per square metre, while plant height was measured with a ruler, averaging the values for each after forage cutting. Six samples were taken in each treatment to determine the best cutting time according to the phenological stage of the association. These samples were left to wilt for 12 hours, reduced to 5 cm in size, and placed in 50 kg polyethene bags.

For the chemical analysis, we followed the rigorous AOAC methodologies (AOAC, 1990) to ensure the accuracy and reliability of our results. The samples were dried in a forced air oven, ground to a specific particle size, and then analysed for Crude Protein (CP), Crude Fibre (CF), Ethereal Extract (EE), ash, and Free Nitrogen Extract (FN).

For seed production, a uniform cut was made before flowering to achieve uniform and synchronised growth between flowering and fruiting.

4.5. Evaluation characteristics

Following the comprehensive guidelines of the Union for the Protection of New Varieties of Plants (UPOV), the evaluation process was conducted at a single location and over two similar vegetation periods. Under these stringent conditions, the local lines LM-58 and LM-43 underwent a thorough evaluation in two cycles of identification trials.

• − Cycle 1: In the 2017 - 2018 season.
• − Cycle 2: During the 2018 - 2019 campaign.

According to the regulations for generating new cultivars established in Decision 345 in Article 10° and as established by the Head Resolution N° 047-2000-INIA, Chapter VII, Article 12.- A variety is considered distinct if one or several distinctive characteristics are notoriously different from any other commonly known variety. It is sufficient that only one characteristic is distinct for the variety to be considered distinct, Article 21.- The Distinctness test, a crucial part of the evaluation process, must include the commonly known varieties with a more remarkable similarity to the variety for which protection is sought. For quantitative characteristics, the hypothesis of equality will be evaluated, and only if this hypothesis is rejected with a significance level of 5% probability will it be possible to conclude that the varieties are different. If the characteristic is qualitative, the variety to be protected must show a different characteristic status compared to other varieties.

The evaluations of the genotypes LM - 58 and LM - 43 were carried out according to the UPOV Codes: LOLIU_PER; LOLIU_MUL_ITA; LOLIU_MUL_WES; LOLIU_BOU; LOLIU_RIG. These codes, internationally recognized standards for the distinctness, uniformity, and stability (DUS) testing of plant varieties, were applied through the parameters for cultivating semi-perennial and perennial forage grasses. The criteria established by the International Union for the Protection of New Varieties of Plants [43] were used as a reference, and the focus was on the morphological characterization of Lolium multiflorum L., local cultivars LM - 58 and LM - 43. The evaluations unveiled these grasses' fast-growing and robust nature, signalling their promising adaptability to various soil types and climatic conditions. They also demonstrated efficient nutrient uptake and soil anchorage, further bolstering their potential.

Table 9 presents a detailed evaluation of the qualitative morphological characteristics of the genotypes LM-58 and LM-43 of Lolium multiflorum L., covering various physical and developmental attributes of the genotypes evaluated. This characterization highlights both differences and similarities between the genotypes, providing valuable information for the implementation of breeding programs and optimised agronomic practices.

4.5. Statistical analysis

Prior to undertaking the statistical analysis of differences, a comprehensive test for normality of the data (Levene, p < 0.05) and a thorough check for homogeneity of variances (Kolmogorov-Smirnov, p < 0.05) were conducted. Subsequently, Duncan's test was employed to ascertain the statistical significance of the factors and to identify the most prominent line. The data were analysed using Infostat software (version 2020).

5. Conclusions

The results obtained in the research have fundamental implications for agriculture in regions with acid soils due to the extraordinary capacity of Lolium multiflorum "cajamarquino ecotype" to maintain an excellent productive yield under adverse conditions, which suggests that it is a viable option to improve forage production in the northern highlands of Peru. It will contribute to the sustainability of diverse agricultural practices and improve rural communities' sustainability, resilience and economy. Correctly managing cutting time and fertilisation is critical to maximise forage yield and quality. The study of Lolium multiflorum L., ecotype 'Cajamarquino,' under acid soil conditions in the northern highlands of Peru has yielded essential results with important implications for agriculture in these regions. The ecotype 'Cajamarquino' LM-58 showed remarkable adaptability to the different geographical conditions of the six sites studied, maintaining high green fodder and dry matter yields and suggesting its viability for improving fodder production in these areas. The detailed analysis of the morphological characteristics of the LM-58 and LM-43 genotypes revealed valuable differences and similarities for the implementation of breeding programs and optimised agronomic practices, highlighting the superiority of the LM-58 genotype for its greater resilience and efficiency in the production of significant biomass yields, with a protein content of 14.84% at 30 days and 12.75% at 45 days. In addition, bioinformatic analysis of mitochondrial DNA sequences identified two distinct haplotypes, reinforcing the genetic differentiation of LM-58 and consolidating it as a promising option for farmers seeking to maximise green fodder production. In conclusion, the LM-58 genotype of the 'Cajamarquino' ecotype of Lolium multiflorum L. is a species well adapted to local livestock production, capable of maintaining excellent productive performance, and represents a viable alternative for improving forage production in the highlands of northern Peru.