Crops of alfalfa genotypes in the soil with very low and very toxic concentrations of mobile aluminium

Abstract

Alfalfa adapts well to various climatic conditions, however, is sensitive to acidic soils. The aim of this study was to estimate the agro-biological traits in the acidic soil with different toxicity of mobile Al concentrations. The field experiment was carried out at the Vėžaičiai Branch of Institute of Agriculture of LAMMC. The crops of alfalfa genotypes (19 cultivars and 11 populations) were established on a Balthygleyic Dystric Retisol in 2018 and 2020. The agro-biological traits were evaluated in the seasons of 2019–2020 and 2021–2022. The results showed that the crops of alfalfa genotypes had the best agro-biological traits in the least toxic concentration of mobile Al (0.00–25.26 mg kg⁻¹). In the soil with 26.79–50.25 mg kg⁻¹ mobile Al, alfalfa genotypes differently varied by all agro-biological traits, these genotypes could be used to select the most specific genotypes with individual traits resistant to acidic soil. The soil with 52.14–74.08 mg kg⁻¹ mobile Al can be used to select genotypes from single plants that survive successfully after wintering. In soil with 76.86–97.00 mg kg⁻¹ mobile Al, the crops of genotypes died very quickly, due to very low resistance to acidic soil with mobile Al concentration.

IMPACT STATEMENT

Acidic soil accounts for about 40% of arable land and 70.0% of nonagricultural soils of the world's cultivated land, and aluminium toxicity is the main factor affecting crop growth and yield improvement in acidic soil. Alfalfa, the most important leguminous forage, plays an important role in livestock production, but it is very sensitive to acid-aluminum stress. However, direct selection of alfalfa genotypes for resistance to mobile Al during several selection cycles under natural acidic soil conditions with different concentrations of mobile Al is more effective because it is possible to select genotypes that produce higher or stabile yields and are more adapted to the local climate conditions. The study showed that the genotypes of alfalfa were the most productive under the lowest concentrations of mobile Al (0.00–25.26 mg kg⁻¹). In 26.79–50.25 mg kg⁻¹ mobile Al, the crops of the genotypes differently varied by individual plants diversity in agro-biological traits.

Keywords:

• Alfalfa
• mobile Al toxicity
• agro-biological trait
• concentration
• acidic soil

1. Introduction

The high phenotypic and genetic diversity of alfalfa (Medicago sativa L.) allows it to be grown in a wide range of climatic and soil conditions (Otero & Castro, 2019). As a widely cultivated leguminous fodder crop, it improves soil fertility and produces high yields of high nutritional quality (Annicchiarico et al., 2015; Arshad et al., 2017; Feng et al., 2022; Wenxu et al., 2019). Alfalfa is adapted to soil of neutral pH, but acidic soils containing mobile Al toxicity are stressful for the plants (Khu et al., 2012; Yang et al., 2013). At low pH, the mobile aluminium in the soil dissolves in toxic forms and is absorbed by the plant. The toxic effects of aluminium severely affect the development and growth of alfalfa plants, resulting in a significant reduction of forage yield (Dogbatse et al., 2020; Grevenstuk & Romano, 2013). In addition, the stress condition manifests itself in symptoms such as low growth rate, poor tillering, crop thinning and inhibition of root growth (Goulding, 2016). Soil acidity is considered to be a major problem, especially as it interferes with symbiotic relationships with native or inoculated rhizobia strains (Newman et al., 2007; Scott et al., 2008). The direct effects of Al toxicity can be evaluated by assessing plant parameters in terms of short-term and long-term response (Chen & Liao, 2016). The responses of plants to Al toxicity may vary between species and between genotypes within the same species. Genetic diversity is very important for the development of improved plant cultivars (Ambati et al., 2020; Jin et al., 2019). The selection of breeding material with improved traits has become a common and effective research method, as the selection and identification of the most promising genotypes or cultivars is based on agronomic and phenotypic traits (Hakl et al., 2021; Ta et al., 2020). The agronomic traits of many plants are quantitative and have a broad and complex genetic basis. Agro-morphological traits have been used to assess the genetic diversity of alfalfa collections and to select the most perspective genotypes (Nan et al., 2019; Tucak et al., 2014). Cultivars with the greatest traits can be screened starting with their phenotype. The cultivars respond differently to different selection methods and different in the degree of tolerance to soil acidity, as low concentrations of mobile Al are not dangerous to plants and lead to better plant growth or other desirable effects (Stevović et al., 2019; Kisnieriene & Lapeikaite, 2015). Through several cycles of phenotypic selection of genotypes for resistance to mobile Al under field conditions, it is possible to select cultivars or populations, that produce higher or stabile yields and are more adapted to climate conditions (Idupulapati et al., 2016; Li & Brummer, 2012). Direct selection of resistant genotypes to mobile Al under natural field conditions is more efficient because the field trials are affected by many uncontrolled variables, such as the occurrence of climatic, biotic, or nutritional stress, which do not occur in controlled environments (Haling et al., 2011; Lakić et al., 2019; Sreenivasulu et al., 2007).

Previous research has been carried out to assess the resistance of alfalfa genotypes to lower concentrations of mobile Al under field conditions. The studies showed that the productivity and biological traits of alfalfa cultivars and populations varied significantly at different mobile Al concentrations. However, lower concentration was not dangerous for the plants in each of the cultivars and populations (Liatukienė & Skuodienė, 2021, 2023). We hope to select not only resistant genotypes but also compare the traits in low but also in very toxic concentrations of mobile Al by growing alfalfa directly in naturally acidic soil.

In this context, the aim of the study was to determine the agro-biological characteristics of alfalfa genotypes in the naturally acidic soil and compare the agro-biological traits between different mobile Al concentrations, which range from very low to very toxic.

2. Materials and methods

2.1. Experiment site and climate conditions during the seasons

The study was conducted at the Vėžaičiai Branch of Institute of Agriculture of Lithuanian Research Centre for Agriculture and Forestry in the western part of Lithuania (55°70 N lat., 21°49 E long.). The soil of the experimental sites is Retisol due to the low amount of clay particles and carbonates, which causes the environment without carbonates to become more acid in nature, the absence of clay particles makes it difficult to accumulate and retain moisture, and at the same time does not accumulate nutrients. Moreover, due to the different meteorologic conditions (higher precipitation amounts in west Lithuania), accumulated substances are washed out faster and are not retained (Vilkiene et al., 2023).

The average annual weather temperature according to the standard rate of climate (SRC) in the period of 1991–2020 in the west Lithuania was 7.4 °C (Figure 1A). The average precipitation in the year was 908 mm and was higher in the second part of the year (Figure 1B). Crop growing season (when the average air temperature of the day is >5 °C) was quite long (188–237 days). Meteorological conditions in the period of 2018–2022 were very diverse. Especially unfavourable year for the crops of alfalfa was in 2018 with 84.2% of precipitation, and after the alfalfa sowing in May and June – the amount of precipitation reached only 66.2%. Dry weather dominated also in 2020 and 2022: compared to the standard rate of climate through the year and plant growing season, the amount of precipitation reached respectively, 92,4 and 88.4%, 76.3 and 87.2% of the norm. Favourable conditions for the growth of alfalfa crops were in 2019 and 2021 when during the plant growing season and through the year the amount of precipitation was close to the SRC. In regard to warmth, all investigation years and periods of plant growing season were by 0.7–1.8 °C and 0.4–2.1 °C warmer compared to the SRC.

Figure 1. The weather conditions during experimental seasons from 2018 to 2022 (data from Vėžaičiai Automatic Meteorological Station), SRC–the standard rate of climate.

2.2. Plant materials of experiment, experimental design, and soil description

The nineteen cultivars from different origin were selected on the basis of their resistance to mobile Al toxicity used Petri dish screening and hydroponic methods (Liatukienė, 2012). Eleven populations were developed under laboratory and glasshouse conditions during the period of 2008–2014 (Skuodienė et al., 2023). The studies on resistance of alfalfa genotypes to mobile aluminium were carried out under field conditions by sowing alfalfa genotypes in two different experimental sites. In the first experiment was carried out in 2019–2020 growing seasons of sowing year 2018 and the second experiment was carried out in 2021–2022 of sowing year 2020 in the naturally acidic soil a Balthygleyic Dystric Retisol. Retisol is a soil with low buffering capacity and sorption, containing high level of toxic Al³⁺, with intermediate-humus content (2–3%) and higher retention of nutrients and water (Repšienė & Karčauskienė, 2016). The soil reaction of the first experiment field in 0–25 cm layer was naturally acidic (pH_KCl 4.14–5.68) with 0.0–23.41 mg kg⁻¹ concentration of mobile Al. Also, in the second experiment field, the soil reaction in 0–25 cm layer was naturally acidic (pH_KCl 4.02–4.31) with different 0.0–97.00 mg kg⁻¹ concentrations of mobile Al (Table 1). The experimental field was ploughed in autumn. In spring, the soil was levelled and rolled before sowing. The crops of alfalfa genotypes were planted on the 24th of April in 2018 and on the 4th of May in 2020. The genotypes of alfalfa were laid out in a randomised block design with four replications. The seeds of each genotype were sown at a rate 0.2 g of scarified seed in two rows of 3 m length. The distance between different genotypes of alfalfa was 1.0 m, and the field plot of one genotype was 1.5 m². Protection of alfalfa crops against weeds was carried out at the early stage of alfalfa seedling development by herbicide Basagran 480 (a.i. bentazon 480 g L⁻¹) 2 L ha⁻¹. Protection of alfalfa genotypes against pest was carried out before the flowering stage of alfalfa plants by insecticide Mavrik 2F (a.i.tau-fluvalinate 240 g L⁻¹) 0.15–0.20 L ha⁻¹.

Table 1. Soil characteristics of experiment sites.

Pedological indices of soil
Experiment site of 2018 and 2020 sowing year
Soil type	Retisol
Sand, %	46.9
Silt, %	45.1
Clay, %	8.0
Mobile P2O5 mg kg^−1	177.0–335.0
Mobile K2O mg kg^−1	195.0–234.0
Ntotal, %	0.12–0.14
Experiment site of 2018 sowing year
pHKCl	Mobile Al mg kg^−1
4.14–5.68	0.00–23.41
Experiment site of 2020 sowing year
pHKCl	Mobile Al mg kg^−1
4.31–5.69	0.0–25.26
4.16–4.23	26.79–50.25
3.96–4.21	52.14–74.08
3.87–4.02	76.86–97.00

The samples of the soil were taken for agrochemical analysis, 90 samples of the soil in 2018 and 180 samples of the soil in 2020. The soil sample shall consist of at least 15 probe punches, following the diagonal lines of the field. Before establishment the experimental plots of alfalfa, the agrochemical characteristics of the soil were determined taking samples from a depth of 0–20 cm with a drill from each plot. The pH of soil was measured by the potentiometric method in the extraction of 1 M KCl. The content of N was determined by the Kjeldahl method using a spectrophotometric measurement. Mobile P₂O₅ and K₂O in the soil were determined using the Egner–Riehm–Domingo (A-L) method. Mobile Al was determined according to the standard ISO14254:2018(Soil quality—Determination of exchangeable acidity using barium chloride solution as extractant).

2.3. Determination of agro-biological traits in the alfalfa genotypes

During the seasons of 2019–2020 of sowing year 2018, the experimental trials of alfalfa genotypes were assessed in four replications under mobile Al (0.00–23.41 mg kg⁻¹) concentrations. Also, the experimental trials were assessed in four replications during the seasons of 2021–2022 of sowing year 2020, in the soil with mobile Al concentrations (Table 1). Wintering of alfalfa crops was evaluated in each year at the beginning of the growing season (W, 1–9 score, where 1–resistant; 9–plant dead).

The height of plants in each genotype in spring regrowth (SR) and also the height before flowering (FH) was measured on 30 plants (cm) in each field with different concentration of mobile Al for each genotype of alfalfa.

The aboveground mass was evaluated in each plot of alfalfa genotypes at the beginning of flowering (about 10% plants flowers). Aboveground mass was evaluated using the scale: 1–9 score, where 1 – the aboveground mass is the lowest; 9 – the aboveground mass is the highest. The number of stems (SN) was counted in each plot of alfalfa genotypes and the number of stems was converted to square meter (m²).

In each experimental year, the seed yield (SY– kg ha⁻¹) was harvested and measured from all plots of alfalfa genotypes in the end of September, when the pods in each plot of alfalfa had dark brown colour (90.0%).

Spring black stem leave spot (SBSLS) was evaluated during the period of May–August months in each experimental year. Disease severity was evaluated using the percentage scale: 0.0, 0.1, 1.0, 5.0, 10.0, 20.0, 40.0, 60.0 and 80.0%.

2.4. Statistical analysis

The significant differences among the treatment means were assessed by Tukey’s test, were p-value was calculated, and a value of p < 0.05 was considered statistically significant. One-way analysis of variance (ANOVA) was used to assess the data of all agro-biological traits. The interactions between the year × mobile Al concentrations in all traits were evaluated by means of two-factor ANOVA analysis. The correlation–regression analysis, between probability levels was p < 0.05 and p < 0.01. The experimental data on all traits are presented as mean and standard error (SE) and four replicates were used for calculations. For statistical analyses, we used the statistical program SAS Enterprise Guide, version 7.13 (SAS Institute Inc., Cary, NC, USA).

3. Results

3.1. The data of statistics

In seasons 2019 and 2020 of 2018 sowing year, the results showed, that the concentration of mobile Al in acidic soil had very low influence on all agro-biological traits of alfalfa cultivars and populations. In season 2021–2022 of 2020 sowing year, the results of variance (ANOVA) showed, that mobile Al concentrations influenced wintering, the height at spring regrowth, the height before flowering, aboveground mass, the stem number, the seed yield and spring black stem leaf spot of the cultivars and populations of alfalfa. The significant influence of the year was determined on the height before flowering, aboveground mass, and the seed yield. Mobile Al × year interaction was significant for the height at spring regrowth, the height before flowering, aboveground mass, stem number, the seed yield, and the spring black stem leaf spot (Table 2).

Table 2. The two-way ANOVA results by year and mobile Al concentrations (0.00–97.00 mg kg⁻¹) and their effect on agro-biological traits in seasons 2021–2022 of 2020 sowing year.

Source of variation	Degree of freedom	Wintering
		Mean squares	p Value
Al	3	29.0408	0.0000
Year	1	0.5513	0.1410
Al x Year	3	0.0788	0.8030
Source of variation	Degree of freedom	Spring regrowth
		Mean squares	p Value
Al	3	580.507	0.0000
Year	1	35.4903	0.0009
Al x Year	3	475.168	0.0000
Source of variation	Degree of freedom	Height before flowering
		Mean squares	p Value
Al	3	6620.24	0.0000
Year	1	4458.04	0.0000
Al x Year	3	2337.55	0.0000
Source of variation	Degree of freedom	Aboveground mass
		Mean squares	p Value
Al	3	67.244	0.0000
Year	1	3.4453	0.0000
Al x Year	3	6.9703	0.0000
Source of variation	Degree of freedom	Stem number
		Mean squares	p Value
Al	3	4898.8	0.0000
Year	1	3413.45	0.0006
Al x Year	3	3020.87	0.0000
Source of variation	Degree of freedom	Seed yield
		Mean squares	p Value
Al	3	91865.1	0.0000
Year	1	15295.0	0.0000
Al x Year	3	5888.38	0.0001
Source of variation	Degree of freedom	Spring black stem leave spot
		Mean squares	p Value
Al	3	271.618	0.0000
Year	1	0.32	0.5437
Al x Year	3	280.281	0.0000

3.2. Wintering and the height at spring regrowth of alfalfa genotypes

In seasons 2019–2020 of 2018 sowing year, the agro-biological traits of the genotypes differed very slightly in the soil with 0.00–23.41 mg kg⁻¹ mobile Al concentration. However, the agro-biological traits more differed between the experiment years (Figure 2). In seasons 2019–2020 of 2018 sowing year, the wintering of the genotypes was very similar (Figure 2A).

Figure 2. The agro-biological traits of alfalfa genotypes in the acidic soil with 0.00–23.41 mg kg⁻¹ mobile Al concentration in 2019–2020 of 2018 sowing year, (a) wintering and aboveground mass; (b) the spring regrowth and height before flowering; (c) the seed yield and stem number; (d) spring black stem leave spot. The differences between experimental years, different letters in each trait according to Tukey’s test are significant p-value < 0.05. Vertical dashes indicate the mean of standard error. Note: spring black stem leave spot compared between seasons.

In 2019, the height of the genotypes at spring regrowth was 1.1-fold greater than in 2020 (Figure 2B). In 2021 of 2020 sowing year, the wintering of alfalfa was similar in the soil with 0.0–50.25 mg kg⁻¹ mobile Al. The wintering of alfalfa genotypes in the soil with 26.79–50.25 mg kg⁻¹ mobile Al was more resistant to wintering compared to the genotypes in the soil with 52.14–74.08 mg kg⁻¹ mobile Al – 1.4-fold, and with 76.86–97.0 mg kg⁻¹ mobile Al – 1.6-fold (Figure 3A). In 2022 of 2020 sowing year, also the wintering of alfalfa genotypes was similar in the soil with 0.0–50.25 mg kg⁻¹ mobile Al. The wintering of alfalfa genotypes in the soil with 26.79–50.25 mg kg⁻¹ mobile Al concentration was better, than the genotypes in the soil with 52.14–74.08 mg kg⁻¹ and 76.86–97.0 mg kg⁻¹ mobile Al, 1.3-fold and 1.7-fold, respectively (Figure 3A).

Figure 3. The agro-biological traits of alfalfa genotypes in the acidic soil with different mobile Al concentrations in 2021–2022 of 2020 sowing year, (a) wintering and aboveground mass; (b) the spring regrowth and height before flowering; (c) the seed yield; (d) the stem number; (e) spring black stem leave spot. The differences between concentrations of mobile Al with different letters in each trait according to Tukey’s test are significant p-value < 0.05. Vertical dashes indicate the mean of standard error. Note: spring black stem leaf spot compared between seasons.

In 2021, in the soil with 0.00–25.26 mg kg⁻¹ mobile Al, the height of alfalfa genotypes at the spring regrowth was higher than the genotypes in the soil with 26.79–50.25 mg kg⁻¹ – by 1.3-fold, 52.14–74.08 mg kg⁻¹ – by 1.7-fold and 76.86–97.0 mg kg⁻¹ – by 1.2-fold. In 2022, the height of alfalfa genotypes at spring regrowth in the soil with 0.00–25.26 mg kg⁻¹ mobile Al was significantly greater than the genotypes in the soil with 26.79–50.25 mg kg⁻¹ mobile Al concentration – 1.2-fold and with 52.14–74.08 mg kg⁻¹ mobile Al – 1.4-fold (Figure 3B).

3.3. Plant height before flowering and aboveground mass of alfalfa genotypes

In 2020 of 2018 sowing year, the genotypes were better by 1.2-fold for aboveground mass compared to 2019 (Figure 2A). In 2020, the plant height before flowering of the genotypes was 1.1-fold greater than in 2019 (Figure 2B).

In 2021, the aboveground mass of cultivars and populations was significantly greater in the soil with 0.00–25.26 mg kg⁻¹ mobile Al compared to the genotypes in the soil with 26.79–50.25 mg kg⁻¹ – by 1.5-fold, 52.14–74.08 mg kg⁻¹ – by 1.9-fold and 76.86–97.0 mg kg⁻¹ mobile Al – by 4.2-fold. In 2022, the genotypes in the soil with 0.00–25.26 mg kg⁻¹ mobile Al were better for aboveground mass than the genotypes in the acid soil with 26.79–50.25 mg kg⁻¹ and 52.14–74.08 mg kg⁻¹ mobile Al – 1.2-fold, respectively (Figure 3A).

In 2021, the height of the genotypes was higher before flowering in the soil with the lowest concentrations (0.00–25.26 mg kg⁻¹) compared to the genotypes in the soil with 26.79–50.25 mg kg⁻¹ – 1.3-fold, 52.14–74.08 mg kg⁻¹ – 1.9-fold and 76.86–97.0 mg kg⁻¹ – 2.2-fold. In 2022, the heights of the genotypes of alfalfa were the highest before flowering in the soil with 0.00–25.26 mg kg⁻¹ mobile Al. Also, the height of these genotypes was higher before flowering than the genotypes in the soil with 26.79–50.25 mg kg⁻¹ mobile Al – 1.2-fold, with 52.14–74.08 mg kg⁻¹ mobile Al concentration –1.4-fold (Figure 3B).

3.4. Seed yield and stem number of alfalfa genotypes

In 2019 of 2018 sowing year, the cultivars and populations were more (by 48.6%) yielding for the seed yield, compared to 2020. Also, in 2019, the genotypes had by 3.7% higher stem number compared to 2020 (Figure 2C).

In 2021 of 2020 sowing year, the genotypes of alfalfa were more yielding for the seed yield in the soil with 0.00–25.26 mg kg⁻¹ mobile Al than the genotypes in the soil with 26.79–50.25 mg kg⁻¹, 52.14–74.08 mg kg⁻¹ and 76.86–97.0 mg kg⁻¹ mobile Al concentrations – 46.4%, 87.2% and 99.9%, respectively. In 2022 of 2020 sowing year, the genotypes were more yielding for the seed yield in the soil with 0.00–25.26 mg kg⁻¹ mobile Al than the alfalfa genotypes in the soil with 26.79–50.25 mg kg⁻¹ (by 49.6%) and with 52.14–74.08 mg kg⁻¹ (by 78.4%) mobile Al (Figure 3C).

In 2021, the genotypes in the acid soil with 0.00–25.26 mg kg⁻¹ mobile Al concentration had a higher stem number compared to the genotypes in the soil with 26.79–50.25 mg kg⁻¹ mobile Al concentration by 34.5%, with 52.14–74.08 mg kg⁻¹ mobile Al – by 64.7% and with 76.86–97.0 mg kg⁻¹ mobile Al – by 81.7%. In 2022, the genotypes had a higher stem number in the soil with 0.00–25.26 mg kg⁻¹ mobile Al than the genotypes in the soil with 26.79–50.25 mg kg⁻¹ mobile Al concentration by 39.7% and with 52.14–74.08 mg kg⁻¹ mobile Al – by 48.6% (Figure 3D).

3.5. Development of spring black stem leave spot in the crops of alfalfa genotypes

In 2019 of 2018 sowing year, the spring black stem leave spot developed less in the crops of alfalfa genotypes, due to the dry and slightly rainy weather conditions during the May–August months. The disease severity was the highest in August month, and the severity was 31.2% higher compared to severity in May. In 2020 of 2018 sowing year, the disease developed very quickly in all alfalfa crops, due to very rainy and hot meteorological conditions during the May–August months. In August month, the disease severity was 43.8% higher compared to the severity in May month (Figure 2D).

2021 of 2020 sowing year, the spring black stem leave spot developed more slowly in the crops of alfalfa genotypes, due to the hotter and slightly rainy weather conditions during the May–August months. The disease severity was the highest in August, and the severity was 24.3% higher compared to severity in May. In 2022 of 2020 sowing year, the disease developed very quickly in all crops of alfalfa genotypes, due to very rainy and hot meteorological conditions during the May–August months. The disease severity was 37.6% higher in the August month compared to the severity in May (Figure 3E).

4. Discussion

4.1. Wintering and the height at spring regrowth of alfalfa genotypes

Successful winter survival of alfalfa, which is one of the main features affecting its growth and productivity, is very dependent on how well it establishes during the year of sowing (Djaman et al., 2021; Wang et al., 2009). However, winter hardiness is a complex trait determined by resistance to several abiotic and biotic factors, such as ice encasement, oxygen deficiency, snow mold and frost as a key determinant in surviving over the winter. Plant tolerance to frost can be increased during a process termed cold acclimation when the low, non-freezing temperatures induce reduction and halt of plant growth allowing plants accumulate various metabolites and prepare for winter stress. Growth is known to be temperature-dependent under optimal conditions, it was used for characterising the genetic variability under unfavourable conditions. In the recent study on the crop growth during acclimation, authors revealed that leaf growth rates in autumn can predict freezing tolerance and thus uncover genetic adaptation of different cultivars and their potential (Jaškūnė et al., 2022). The survival, high yield and quality of alfalfa crops depend on a well-developed root system. Spring regrowth is a very important factor as the alfalfa canopy is closely linked to the morphological structure of stems and roots (Bélanger et al., 2006; Xu et al., 2020). A well-developed root system is important for the high yield, quality, and survival of alfalfa (Xu et al., 2020). The persistence of the alfalfa crops depends on the nitrogen supply in the roots, the soil temperature and snow content in winter, the moisture and nutrients content of the soil and the age of alfalfa stand (Avice et al., 1997; Dhont et al., 2003; Hendershot & Volenec, 1993; Leep et al., 2001). The results of this study showed that the crops of alfalfa genotypes were affected the by concentration of mobile Al during the growing season in the year of establishment. In all years of experiment, the weather conditions were not so critical for wintering of alfalfa genotypes. Statistical analysis showed that mobile Al in the soil had a significant effect on wintering (Table 2). In experimental seasons 2019–2020 of 2018 sowing year, the wintering of genotypes in the soil with 0.0–23.41 mg kg⁻¹ mobile Al was similar and the least damaging by mobile Al and the crops of all genotypes showed high survival rate. In 2021–2022 of 2020 sowing year, the plants of alfalfa genotypes were more affected by mobile Al concentrations, especially at the highest concentrations of mobile Al, and the crops of alfalfa genotypes were more affected by thinning or death during wintering seasons. This fact is confirmed by the strong positive correlation coefficient between concentrations of mobile Al and wintering r = 0.758** and r = 0.745** (Tables 3 and 4). Our study showed, that wintering influenced the growth and development of alfalfa genotypes, especially in the soil with the highest concentrations of mobile Al, the significant negative correlation coefficients were found between the wintering and spring regrowth, the height before flowering, the stem number, the seed yield and aboveground mass, correlation coefficients ranged from r = −0.348** to r = −0.744** in warm and dry growing seasons, and from r = −0.328** to r = −0.782** in warm and rainy growing seasons (Tables 3 and 4).

Table 3. Correlation analysis between the agro-biological traits and concentrations of mobile Al, in warm and dry growing seasons year of alfalfa crops (2019 of 2018 sowing year and 2021 of 2020 sowing year).

Trait	Mobile Al	W	SR	HF	SN	SY	SBSLS	AM
W	0.758**	–
SR	−0.432**	−0.348**	–
HF	−0.761**	−0.716**	0.706**	–
SN	−0.722**	−0.687**	0.638**	0.917**	–
SY	−0.798**	−0.642**	0.435**	0.68**	0.618**	–
SBSLS	−0.336**	−0.274**	0.325**	0.359**	0.372**	0.249**	–
AM	−0.961**	−0.744**	0.436**	0.762**	0.734**	0.805**	0.328**	–

Note: W: wintering; SR: spring regrowth; HF: the height before flowering; SN: the stem number; SBSLS: the spring black stem leaf spot, severity %; AM: aboveground mass; **—significant at p < 0.01.

Table 4. Correlation analysis between the agro-biological traits and concentrations of mobile Al, in warm and rainy growing seasons year of alfalfa crops (2020 of 2018 sowing year and 2022 of 2020 sowing year).

Trait	Mobile Al	W	SR	HF	SN	SY	SBSLS	AM
W	0.745**	–
SR	−0.717**	−0.500**	–
HF	−0.851**	−0.782**	0.707**	–
SN	−0.754**	−0.769**	0.433**	0.77**	–
SY	−0.423**	−0.328**	0.448**	0.455**	0.322**	–
SBSLS	0.423**	0.511**	−0.213**	−0.519**	−0.595**	−0.204**	–
AM	−0.926**	−0.685**	0.613**	0.815**	0.771**	0.386**	−0.476**	–

Note: W: wintering; SR: spring regrowth; HF: the height before flowering; SN: the stem number; SBSLS: the spring black stem leaf spot, severity %; AM–aboveground mass; **—significant at p < 0.01.

Plant height at spring regrowth of alfalfa genotypes depended not only on the weather conditions in the early spring, but also on the mobile Al concentrations in the soil. ANOVA results showed a significant interaction between year and concentrations of mobile Al (Table 2). The height of plants may be related to differences between genotype, and the height of plants is the one of the main criterions for choosing better genotypes at the early stage of selection (Kebede et al., 2022; Tucak et al., 2008; Ullah et al., 2009). Fahad et al. (2017) suggested that differences in plant height among tested genotypes could be due to the genetics of genotypes and environments factors. The results in season 2019 of 2018 sowing year showed, that the genotypes had a higher height at spring regrowth compared to 2020, as the genotypes of alfalfa were not only affected by the warm weather conditions, but also by the lowest concentrations of mobile Al (Figure 2B). In season 2022 of 2020 sowing year, the height at spring regrowth of the genotypes was higher than in 2021, due to the warm and rainier weather conditions during the spring period. In 2022, the height at spring regrowth of alfalfa genotypes was higher compared to 2021 in the soil with 0.00–25.26 mg kg⁻¹, 26.79–50.25 mg kg⁻¹ and 52.14–74.08 mg kg⁻¹ concentrations of mobile Al, 1.4-fold, 1.5-fold, and 1.7-fold, respectively (Figure 3B).

At different stages of development, crops growth depends on the environment (Fahad et al., 2017). Smith et al. (2017) argued that the height of genotypes is better associated with the herbage yield and with the higher regrowth rate. In our study, the height at spring of genotypes varied differently, due to the weather conditions and concentration of mobile Al. Also, the aboveground mass of alfalfa genotypes varied differently during the spring regrowth period. It showed, correlation with the spring regrowth and aboveground mass (r = 0.436**) in warm and dry growing seasons. Correlation analysis was found with the spring regrowth and the height before flowering, stem number, seed yield and spring black stem leave spot (r = 0.706**, r = 0.638**, r  =  0.435** and r = 0.325**) (Table 3). In warm and rainy growing seasons (2020 and 2022 of 2018 and 2020), correlation was found with the spring regrowth and aboveground mass (r = 0.613**) and between the spring regrowth and height before flowering a strong positive correlation coefficient (r = 0.707**) was detected (Table 4).

4.2. Plant height before flowering and aboveground mass of alfalfa genotypes

Plant height differed significantly between cultivars and populations at different stages of growth and development (Diriba et al., 2014; Djaman et al., 2020; Tucak et al., 2014). In our study, the genotypes of alfalfa significantly differed in height before flowering due to the weather conditions during experimental seasons. The interaction between the mobile Al concentration and experimental year showed that the height before flowering differed among genotypes and mobile Al concentration. In 2020 of 2018 sowing year, the plant height before flowering was significantly higher than in 2019, due to warm and more rainy weather conditions. In 2022 of 2020 sowing year, the genotypes were taller before flowering due to the higher temperature and precipitation. In 2022, the plant height of alfalfa genotypes was higher compared to 2021 in the soil with 0.00–25.26 mg kg⁻¹ concentration of mobile Al – by 1.7-fold, with 26.79–50.25 mg kg⁻¹ concentration – by 1.8-fold and with 52.14–74.08 mg kg⁻¹ concentration – by 2.2-fold (Figure 3B). Plant height is closely related to the environment conditions, the genetic of individual genotypes and the height also influences the total biomass yield of crop (Acvi et al., 2010; Davodi et al., 2011; Djaman et al., 2020; Kavut et al., 2014; Rimi et al., 2010).

Hamd Alla et al. (2013) argued that the plant height varied between the individual plants within accessions and the height of plants depended on the genetic variability among individual plant. In our study, aboveground mass depended not only on the height before flowering but also on the weather conditions in each experimental year and mobile Al. In 2022, the aboveground mass was greater compared to 2021 in the soil with concentration 0.00–25.26 mg kg⁻¹ of mobile Al by 1.0-fold, in soil with 26.79–50.25 mg kg⁻¹ mobile Al concentration by 1.3-fold, and in soil with 52.14–74.08 mg kg⁻¹ concentration – by 1.6-fold (Figure 3A). The significant correlation coefficient was detected with the height before flowering and concentrations of mobile Al (r = –0.761** and r = –0.851**). Julier et al. (2000) and Tucak et al. (2008) detected the strong–positive significant correlation coefficients with the plant height and stem number as well as between the height and forage yield, r = 0.71** and r = 0.72**. In warm and dry growing seasons, the significant correlation was determined with the height before flowering and the stem number, the seed yield and aboveground mass (r = 0.917**, r = 0.680** and r = 0.762**, respectively) and in warm and rainy growing seasons (r = 0.770**, r = 0.455** and r = 0.815**, respectively) (Tables 3 and 4).

4.3. Seed yield and stem number of alfalfa genotypes

The seed harvest and quality of alfalfa genotypes depended on genetical background of genotypes and on the ambient conditions in the areas with favourable air conditions during the plant vegetation. Regions with sunny, warm weather and little or no rainfall are favourable for successful alfalfa seed production (Hossain et al., 2020; Jaškūnė et al., 2022). These ambient conditions are conducive for a good flowering of alfalfa and provide favourable conditions for bee pollination. The density of alfalfa genotypes is an important determinant of seed productivity, which depends on competition between genotypes and within plants, and the ability of plants to produce vegetative and reproductive material (Inal, 2023). In our study, the crop density of the genotypes depended on the stem number, climatic conditions, and mobile Al concentrations. The number of stems depends on the environmental conditions and soil moisture, if the environment conditions are favourable, stem production is constant and increases biomass production (Ventroni et al., 2010). In 2020 of 2018 sowing year, the stem number of the cultivars and populations was not much lower compared to 2019, due to warm and rainy weather conditions during spring–summer periods and low effect of mobile Al concentration (Figure 2C). In 2021 of 2020 sowing year, the stem number of the genotypes was lower compared to 2022, due to warm and dry weather conditions during periods of spring–summer and the plants of alfalfa genotypes were more stressed under mobile Al concentrations. In 2022, the stem number was greater compared to 2021 in the soil with 0.00–25.26 mg kg⁻¹ concentration of mobile Al by 19.9%, with 26.79–50.25 mg kg⁻¹ concentration – by 13.1% and with 52.14–74.08 mg kg⁻¹ concentration – by 45.0% (Figure 3D). Lush vegetative growth of alfalfa is a problem for seed yield where the high ground weight of the alfalfa causes the stem falls, adversely affecting yield and seed quality (Pajčin et al., 2020). In our study, in a rainier 2020 and 2022 of 2018 and 2020 sowing year, the stem number determined greater aboveground mass and the crop of alfalfa fell down and the seed yield was lower compared to the warm and dry 2019 and 2021 of 2018 and 2020 sowing year in the soil with the lowest concentrations of mobile Al (0.00–25.26 mg kg⁻¹). Conversely, higher concentrations of mobile Al in the soil resulted in low aboveground mass of alfalfa genotypes but the crops were not dropped, and the seed yield was higher in the dry year (Figures 2C and 3C). In 2019 of 2018 sowing year, the seed yield was greater compared to 2020, due to the dry and warm weather conditions during the stage of ripening pods. Also, in 2021 of 2020 sowing year, the seed yield was higher than in 2022 in the soil with 0.00–25.26 mg kg⁻¹ mobile Al concentration by 37.7% and with 26.79–50.25 mg kg⁻¹ – by 39.9%, due to favourable weather conditions during knitting and ripening of alfalfa pods. However, the seed yield in 2021 was similar compared to 2022 in the soil with 52.14–74.08 mg kg⁻¹ mobile Al concentration and the seed yield in 2021 was very low in the soil with 76.86–97.0 mg kg⁻¹ mobile Al concentration due to not only the weather conditions, but also due to the lowest stem number in the crops (Figure 3C,D). Luo (2021) reported that lower plant density reduces the seed yield of alfalfa. Tucak et al. (2023), Naydovich and Popova (2015), Petkovic et al. (2017), Morante and Lira (2018) stated, that successful production of seed depends on the rainfall and temperature during the summer months, as weather conditions play a key role for the formation of pods and seed ripening. The seed yield varies very considerably according to the environment, climatic conditions, and the location of study sites. The seed yield varied under favourable conditions, ranged from 189–805 kg ha⁻¹ (Bolaños-Aquilar et al., 2002; Rashidi et al., 2009). In our study, in 2021 of 2020 sowing year under favourable conditions the seed yield in the soil with 0.00–25.26 mg kg⁻¹ concentration of mobile Al varied from 283–334 kg ha⁻¹, with 26.79–50.25 mg kg⁻¹ mobile Al concentration – from 126–196 kg ha⁻¹, with 52.14–74.08 mg kg⁻¹ mobile Al concentration – from 16–56 kg ha⁻¹ and with 76.86–97.0 mg kg⁻¹ mobile Al concentration, from 1–5 kg ha⁻¹. The significant correlation with the stem number and the seed yield was r = 0.618** in warm and dry growing seasons, and r = 0.322** in warm and rainy seasons. Also, the strong correlation was found with the stem number and aboveground mass and between the seed yield and aboveground mass in warm and dry, and warm and rainy growing seasons, r = 0.734**, r = 0.805** and r = 0.771**, r = 0.386**, respectively (Tables 3 and 4). Bolaños-Aquilar et al. (2002) reported, that the correlation between the seed yield and fertile stems was high and positive.

4.4. Development of spring black stem leave spot in the crops of alfalfa genotypes

The causal agent of SBSLS (Phoma medicaginis) overwinters abundantly in plants debris and the disease agent spreads rapidly in very early spring period (Castell-Miller et al., 2007). In our study, the disease development varied according to the weather conditions, especially amount of rainfall and mobile Al concentration. In a study, Ellwood et al. (2006) and Kamphuis et al. (2008) suggested, that alfalfa, as a cross-pollinated plant, consists of individuals that differ in resistance. The severity of this disease damages susceptible plants and significantly reduces seed and grass yields. Highly susceptible alfalfa crops can be destroyed (Ellwood et al., 2006). Our investigation showed that the development of disease in the crops of alfalfa varied individually from genotype to genotype in the soil with different mobile Al concentrations. However, the severity of disease was very similar in each concentration of mobile Al. The development and spread of the disease depended on the meteorological conditions in spring and throughout the vegetation season of alfalfa crops. The disease severely affects alfalfa genotypes and completely destroys of crops when the disease spreads strongly in the crop during very rainy and warm weather conditions (Barbetti et al., 2007, 2020; Ellwood et al., 2006; Kamphuis et al., 2008). Our study showed, that in warm and dry year (2019 of 2018 sowing year), the crops of alfalfa were less damaged, and the disease developed very slowly in alfalfa crops and the severity of disease was 3.9% in May, 19.9% in June, 26.2% in July and 35.1% in August (Figure 2D). Also, in 2021 of 2020 sowing year, the damages of disease in crops of alfalfa were less due to the warm and dry weather conditions. The disease progressed very slowly during the growing season of alfalfa and the disease severity was 8.5% in May, 17.9% in June, 29.9% in July and 32.8% in August (Figure 3E). In warm and dry weather conditions (2019 and 2021of 2018 and 2020 sowing year) the disease developed and progressed very slowly, and the disease had very little influence on the height of plants at spring regrowth, height before flowering, stem number, seed yield and aboveground mass. It showed the significant correlation coefficient with the SBSLS and these traits, respectively: r = 0.325**, r = 0.352**, r = 0.372**, r = 0.249**and r = 0.328** (Table 3). The significant correlation coefficients with the SBSLS and mobile Al and between SBSLS and wintering were r = −0.336** and r = −0.274** (Table 3). In 2020 of 2018 sowing year and 2022 of 2020 sowing year, warm and rainy weather conditions were also favourable for the development and progress of the disease, and the alfalfa crops were not completely damaged. In 2020, the disease severity was 4.0% in May, 23.6% in June, 35.5% in July and 47.8% in August month (Figure 2D). In 2022, the disease severity was 12.0% in May, 26.5% in June, 39.8% in July and 49.6% in August month (Figure 3E). The disease very low influenced the spring regrowth, height before flowering, stem number, seed yield and aboveground mass. It showed a significant correlation between SBSLS and these traits, respectively: r = −0.213**, r = −0.519**, r = −0.595**, r = −0.204**and r = −0.476** (Table 4). The significant positive correlation between the SBSLS and mobile Al, and between the SBSLS and wintering were r = 0.423**and r = 0.511** (Table 4).

5. Conclusions

The results of this research showed that the crops of alfalfa genotypes highly varied by agro-biological traits in the soil with very low and very high toxicity concentrations of mobile Al. The crops of alfalfa genotypes had the best agro-biological traits in the acidic soil with the least toxic concentration of mobile Al (0.00–25.26 mg kg⁻¹). The genotypes of alfalfa showed stronger reaction by agro-biological traits under 26.79–50.25 mg kg⁻¹ concentration of mobile Al. In 26.79–50.25 mg kg⁻¹ mobile Al concentration, the crops of genotypes differently varied by all agro-biological traits, it showed, that these genotypes could be used to select the most specific plants by individual diversity in agro-biological traits, which showed resistance to acidic soil. These genotypes in the soil with 52.14–74.08 mg kg⁻¹ mobile Al concentration can be used to select individual genotypes from single plants that survive successfully after wintering, as the crops of these genotypes are highly exposed to aluminium toxicity. The crops of alfalfa genotypes are very sensitive to mobile Al containing 76.86–97.0 mg kg⁻¹ concentration and these genotypes die very quickly, due to very low resistance to acidic soil with very high mobile Al concentration. In the acidic soil with different toxicity concentrations of mobile Al, ranging from 26.79 to 74.08 mg kg⁻¹, the diversity of agro-biological traits in each alfalfa crop depended on the climate conditions and the resistance of the plants in each genotype to mobile Al concentrations.

Author contributions

Conceptualization, R.S. and A.L.; methodology, R.S, A.L.; software, A.L.; validation, R.S., A.L., V.B.; formal analysis, A.L., R.S.; investigation, R.S., A.L.; data curation, A.L., R.S., V.K.; writing–original draft preparation, A.L., R.S., V.B.; writing–review and editing, A.L. R.S., V.K.; visualization, A.L, R.S.; supervision, R.S. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

The paper presents research findings obtained through the long-term research programme ‘Genetics, Biotechnology and breeding for plant biodiversity and innovative technologies’ implemented by the Lithuanian Research Centre for Agriculture and Forestry.

Additional information

Notes on contributors

Aurelija Liatukienė

Dr. Aurelija Liatukienė main areas of interest are alfalfa breeding and investigation of the genetic resources ex situ and in situ. Investigations of alfalfa biotic and abiotic resistance make up considerable part of breeding work.

Regina Skuodienė

Dr. Regina Skuodienė is a chief researcher at Vėžaičiai Branch of LAMMC. Her research interests include biodiversity, natural grassland, productivity and quality of grassland ecosystem, and weed problems in different cropping systems.

Vilma Kemešytė

Dr. Vilma Kemešytė main areas of interest are breeding and investigation of the genetic resources of forage and turf grasses, cultivar plasticity/adaptability. She also investigates the benefits of combining forage grasses with legume such as clover and alfalfa.

Vasily Buhaiov

Vasily Buhaiov is a head of the Department for Feed, Grain and Technical Crops Breeding. The study interests are perennial leguminous and cereal grasses collections and creation of varieties with increased adaptive potential for different soil and climatic conditions of Ukraine, in particular resistance of plants to high acidic soils.