Alfalfa Autotoxicity: Role of Cultivar Renovation in Mitigating Germination and Seedling Growth under Continuous Cropping Systems

Alfalfa, a highly valued perennial leguminous forage crop, is cultivated across 30 million hectares worldwide, particularly in infertile areas (Teixeira et al., 2023). Simulated average alfalfa yields range from 4.5 to 28 tons of dry matter per hectare per year (Teixeira et al., 2023; Glowacki et al., 2023; Mohammed et al., 2023). Alfalfa cultivation not only enhances soil physiochemical properties (Sainju and Lenssen, 2011) but also provides high-quality forage for local animal husbandry (Du et al., 2020; Fan et al., 2024). However, consecutive production of alfalfa often results in field thinning and yield reductions due to factors such as soil water depletion, plant diseases and autotoxicity (Seguin et al., 2002; Li and Huang, 2008; Fan et al., 2023). Among these, autotoxicity has been identified as the primary cause of reseeding failure in alfalfa (Seguin et al., 2002; Zheng et al., 2019).
       
Autotoxicity in plants refers to the harmful allelopathic effects on their own growth and development (Seguin et al., 2002; Zhang et al., 2020). It occurs when a plant species releases specific chemical compounds that directly or indirectly inhibit the germination or growth of the same species (Wang et al., 2022; Wu et al., 2023). In alfalfa, autotoxicity is caused by water-soluble compounds released from leaves, roots and stems (Yang et al., 2009). These compounds include saponins (Wyman-Simpson  et al., 1991), canavanine (Miersch and Jiihlke, 1992) and phenolic acids (Abdul and Habib, 1989; Hall and Henderlong, 1989). Their concentrations are influenced by factors such as stand age (Yang et al., 2009), cultivar (Wyman-Simpson  et al., 1991), reseeding delay (Seguin et al., 2002) and plant organ (Rong et al., 2016), all of which impact the reseeding success of alfalfa.
       
To mitigate alfalfa autotoxicity, practices such as crop rotation with non-leguminous crops (Jenning and Nelson,  1998; Zhang et al., 2023) and seed priming (Tanha et al., 2017) have been recommended in previous studies. Moreover, Wyman-Simpson et al., (1991) observed that saponin concentrations varied among six alfalfa cultivars and higher saponin concentrations were associated with increased autotoxicity (Chuang and Miller, 1995). This raises the question of whether selecting alfalfa cultivars with low autotoxicity could serve as a novel and effective strategy to reduce the autotoxicity of alfalfa-a question warranting further investigation.
       
In this study, we evaluated the seed germination, seedling growth and physiological characteristics of alfalfa. We hypothesized that cultivar renovation could enable successful continuous cropping of alfalfa. The specific objectives were to determine: 1. How seed germination and seedling growth of four alfalfa varieties respond to different concentrations of alfalfa leaf extract; 2. The relationships between seed germination, seedling lengths and physiological characteristics of alfalfa; 3. Whether cultivar renovation could be a novel and effective approach to overcoming alfalfa autotoxicity in continuous cropping systems.

Seed material
 
The four alfalfa (Medicago sativa) cultivars were XunLu(XL), GongNong No.1(GN,), ZhongMu No.1(ZM) and JuNeng 995(JN).
 
Experiment design
 
The experiment was carried in College of Agriculture and Forestry Science, Linyi University, China. The senesced leaves of alfalfa (ZhongMu No.1) were collected randomly in the first harvest period in 24 May, 2023. And the leaves were cut into pieces for preparation of the extract. According to the ratio of 1 g of leaves soaked in 10 ml sterile water, the leaf extract was filtered by a quantitative filter paper and the extract concentration was 0.1%. Then the 0.1 g/ml extract concentration was diluted to 0.05% and 0.01% by sterile water. The sterile water was used as the control treatment 0.
       
The full seeds of the four alfalfa cultivars were picked out and sterilized with 0.1% KMnO₄ for 20 minutes, then washed with sterile water. The seeds were soaked in sterile water for 12 h before processing. Two layers of wet filter paper were laid in the petri dishes (10 cm diameter) and 100 seeds were placed in each dish. 10 ml extract solution of different concentrations (concentration setting: 0%, 0.01%, 0.05%, 0.1%) was added slowly. Petri dishes were placed in an artificial climate chamber (PGX-38^oC, Shanghai, China), which the temperature was set at 25^oC and the light/dark time was 16 h/8 h. The germination parameters were counted from day 1 to day 10 and 0.5 g of whole plants (fresh weight) were weighed at day 10 to measure the physiological indexes of seedlings.
 
Germination test
 
Germination was estimated by radicle protrusion, which the appearance of a radicle ≥2 mm in length as the criterion. The germination numbers of seeds were counted from day 1 to day 10. The germination rate (%) was estimated at the day 7, i.e. the accumulated number of germinated seeds from day 1 to day 7 divided by 100. The germination potential (%) was estimated at the day 4, i.e. the accumulated number of germinated seeds from day 1 to day 4 divided by 100.
 

  

 
Where,
DT = Days of germination.
GT = Germinated number of seeds corresponding to DT.
 

 Vigor index (VI) = GI × W

 
Where,
W = Fresh weight of the seeds at day 7.
 
Measurements of seedling length and physiological index
 
The seedling lengths were measured by vemier calipers with accuracy of 2 mm. The malondiadehyde (MDA) concentration was measured by a modified thiobarbituric acid (Puckette et al., 2007). Soluble sugar (SS) was analyzed by a modified anthrone method (Sαnchezet_al1998). Soluble protein (SP) was analyzed by a modified coomassie brilliant blue method (Georgiou et al., 2008). The activity of superoxide dismutase (SOD) was assayed on the basis of its ability to inhibit the photochemical reduction of nitro blue tetrazolium (Stewart and Bewley, 1980). The activity of catalase (CAT) was assayed by the modified method of Iodin titration (Kenten and Mann, 1952). The activity of peroxidase (POD) was measured as the recorded absorbance increased at 470 nm by guaiacol method (Castillo et al., 1984).
 
Statistical analysis
 
The effects of cultivar, extract concentration and their interaction on the germination test and seedling physiological index were analyzed using Two-Way ANOVAs. The differences in germination test and seedling physiological index on the effects of cultivar or extract concentration were examined using One-Way ANOVAs with SPSS 17.0. The liner correlations between germination index and seedling physiological index were analyzed with the model y=ax+b using SPSS 26.0

Germination rate and germination potential
 
Germination rate of four alfalfa cultivars increased firstly, then stabilized as germination days under the four extract concentrations (Fig 1). However, the days to stabilized germination rate were delayed with the increased extract concentrations. These indicated the higher concentration of the extract had more seriously autotoxicity on subsequent alfalfa. Even though, the 0.1% leaf extract made four alfalfa little germination rate. This might because of the phenolic acid metabolites from alfalfa which inhibit the germination of later alfalfa in the continuous cropping (Wang et al., 2022).


       
The alfalfa cultivars, extract concentration and their interaction showed significant effects on both germination rate and germination potential (Fig 2). Seed germination of four alfalfa cultivars were significantly inhibited at 0.05% and 0.1% (P<0.05). Ma et al., (2024) found the same results which used root extracts as the treatments. Germination rate and germination potential of XunLu were lower than those of the other three alfalfa cultivars under the four extract concentrations (P<0.05). These indicated alfalfa germination was more inhibited in Xunlu and the 0.1% leaf extract made four alfalfa little germination rate.


 
Germination index and vigor index
 
The alfalfa cultivars, extract concentration and their interaction showed significant effects on germination index and vigor index (Fig 3). As the increased extract concentration, the germination index and vigor index showed the significantly decreased trends (P<0.05). Germination index and germination index of JuNeng showed better than other cultivars (P<0.05). Even it still showed a high germination index and vigor index at the 0.05% extract concentration. These indicated different cultivars of alfalfa might show autotoxicity-tolerant or autotoxicity-sensitive varieties (Zhang et al., 2021), JuNeng 995 would be select to renovation from the germination index and vigor index as autotoxicity-tolerant variety.


 
Lengths of alfalfa seedlings
 
The extract concentrations showed significant effects on the lengths of alfalfa seedlings (Table 1). The lengths of root and whole plants were highest in the 0.01% concentration and the lengths of bud increased with the extract concentrations. This might becaused by result of the promoting effect of allelopathy in the extract (Li et al., 2021). The cultivars showed significant effects on the lengths of alfalfa bud. GongNong showed the highest lengths of bud, JuNeng showed the higher lengths of bud and JuNeng showed the highest lengths of root and whole plants in the four cultivars under the 0.01% concentration. In summary, JuNeng was selected as the optimal cultivar to cultivate for continuous cropping alfalfa from the indicators of seedling lengths in this study. Wang et al., (2022) found that the metabolites from alfalfa could significantly decrease the root length of alfalfa seedlings.


 
Malondiadehyde (MDA), soluble sugar (SS) and soluble protein (SP)
 
The alfalfa cultivar, extract concentration and their interaction showed significant effects on the MDA, SS and SP concentration (Table 2). MDA increased with the extract concentration in Gongnong. and it was no significant change in the four cultivars under the 0% extract concentration, the MDA of Gongnong showed the highest in the four cultivars under the 0.05% and 0.1% extract concentration. The SS decreased with the extract concentration. The SS did not change significantly with cultivars in the 0% and 0.05% extract concentrations, but it showed the highest at ZhongMu in the 0.01% extract concentration. The SP decreased at GongNong and XunLu, increased at JuNeng and decreased firstly, then increased at ZhongMu with the extract concentration. The SP of JuNeng was lowest in the 0% extract concentration and highest in the 0.01% and 0.05% extract concentration in the four cultivars. These results indicated different alfalfa varieties would regulate different osmoregulatory substances to counteract the autotoxicity from the leaf extract (Zhang et al., 2021), i.e. JuNeng is mainly through the regulation of SP concentration.

​
 
Antioxidative systems (CAT, POD and SOD)
 
The alfalfa cultivar, extract concentration and their interaction showed significant effects on CAT (Table 3). The CAT was highest in 0.01% extract concentration (P<0.05). And the CAT of GongNong was highest in the four cultivars. The alfalfa cultivar and extract concentration showed significant effects on POD (Table 3). The POD activities significantly decreased in ZhongMu (P<0.05) and changed insignificantly in the other three cultivars of alfalfa (P>0.05) with the increased extract concentrations. And the POD of ZhongMu was highest in the four cultivars, basically. The SOD activities were highest in XunLu on the 0.01% extract concentrations (P<0.05, Table 3). and there were no significant changes in other conditions (P>0.05). Antioxidative enzymes were also a plant’s defense against adversity. These results indicated different varieties of alfalfa showed different antioxidant enzymes against autotoxicity (Zhang et al., 2021).

​
 
Relationship between lengths of alfalfa, physiological characters and seed germination
 
There were negative correlations of alfalfa lengths with the germination parameters (Table 4) and the significant correlations were shown between alfalfa lengths with GI (P<0.05). But there were less correlation between the physiological characters and the germination parameters. The results indicated seed germination would negatively affect the seeding growth. This might be that weak seeds were inhibited by the leaf extract, but the remaining germinated seeds resisted the autotoxicity and promoted the seedling growth of alfalfa (Yan et al., 2022).

​

Alfalfa autotoxicity is a generally phenomenon. The results in this study found that the alfalfa autotoxicity might be more severe as the increasing extract concentration of the leaves. Suitable cultivar renovation was found as an effective way to alleviates the alfalfa autotoxicity. And JuNeng 995 could be selected for planting after ZhongMu No.1 through the germination and seedling growth in this study.

The present study was supported by the grants from the Natural Science Foundation of Shandong Province (ZR2020QC184), the National Natural Science Foundation of China (31700553), the Forage Industrial Innovation Team, Shandong Modern Agricultural Industrial and Technical System, China (SDAIT-23-8) and the key technology research project of consolidation and enhancement of carbon sequestration capacity of ecological public welfare forest in the middle mountainous area of Shandong province (SDGP370000000 2024 02000946).
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.