Effects of Continuous Cropping on Seed Germination and Seedling Growth, Physiological Characters of Alfalfa

Alfalfa is an excellent perennial leguminous forage. Because of its high nutritional value, rich in protein, amino acid (Ayhan et al., 2016; Avci et al., 2018), this crop has already been as one of the important fodder crops in the herbivorous animal husbandry. However, alfalfa fields are often thinned out and have low yields in the consecutive production (Seguin et al., 2002). Previous studies have indicated the optimal cultivated years of alfalfa should be not more than 8 years (Li and Huang 2008; Wang et al., 2015). This might be caused by soil water depletion, plant diseases and autotoxicity (Seguin et al., 2002; Li and Huang 2008). And autotoxicity was increasingly found to be a major reason (Seguin et al., 2002; Zheng et al., 2019). 
       
Alfalfa autotoxicity is the harmful allelopathy to its growth and development (Seguin et al., 2002; Zhang et al., 2020; Sun et al., 2020). It is caused by the released water-soluble compounds (i.e. saponins, canavanine and phenolic acid) from organs and litters of alfalfa (Abdul and Habib 1989; Hall and Henderlong 1989; Wyman-Simpson et al., 1991; Miersch and Jiihlke 1992; Yang et al., 2009). These water-soluble compounds often inhibit the seedling survival rate, yield and nutritional quality of alfalfa during continuous cropping (Jennings 2001; Chon et al., 2002; Rong et al., 2016; Liang et al., 2021). The inhibiting effect would increase with the increased content of the water-soluble compounds (Chon et al., 2002; Rong et al., 2016).
       
Plant functions is guaranteed by cell viability and structure integration which are controlled by plant physiological characteristics. Plants often change osmo-regulatory and anti-oxidative status in response to various stress conditions (Wang et al., 2019). Continuous cropping also leads to increase in leaf malondialdehyde content, decrease in leaf activity of peroxidase and catalase (Liang et al., 2021). These researches showed a few of clues in finding possible links of seed germination and seedling physiological characteristics, seedling growth conditions. However, there were few researches to describe their possible links in the continuous cropping. So understanding the seed germination and seedling growth and physiological mechanisms of alfalfa under continuous cropping system are important to sustainable use of alfalfa grassland.
       
This study is aimed to test the hypothesis that continuous cultivation would affect seed germination, seedling growth and physiological characteristics. The specific objectives have been to find out above-described parameters.

Experimental design
 
The experiment was conducted in an artificial climate chamber (PGX-380C, Shanghai, China), where the temperature was maintained at 25°C, the light/dark time was 16 hours/8 hours. The fresh samples, i.e. leaves, stem, root, and rhizosphere soil of alfalfa (Medicago sativa L. cv ZhongMu No.1) were collected randomly in the harvest period on 18 May, 2019. According to the ratio of 1 g fresh samples soaked in 10 ml sterile water, the extract was filtered by a quantitative filter paper and the extract content was 0.1 g/ml. Then the 0.1 g/ml extract were diluted to 0.05 g/ml and 0.01 g/ml by sterile water. The sterile water was used as the control treatment (0 g/ml).
       
The matured seeds of Medicago sativa were picked up and sterilized with 0.1% KMnO₄ for 20 minutes, then washed with sterile water. The seeds were soaked in sterile water for 12 hours before processing. Two layers of wet filter paper were laid in the petri dishes (10 cm diameter) and 100 seeds and 10 ml extract were placed slowly in each dish. Subsequently germination, seedling growth and physiological characters were recorded as described.
 
Measurements
 
Germination rate (GR) and germination potential (GP) were estimated at day 7 and day 4 by radicle protrusion (i.e. appearance of a radicle ≥ 2 mm in length) as the criterion, then the germination index (GI) and vigor index (VI) were calculated (Li et al., 2020).
       
The malondialdehyde (MDA) was measured using a modified thiobarbituric acid method (Puckette et al., 2007). The proline was measured using an acid-ninhydrin colorimetric method (Zhang et al., 2014). Soluble sugar (SS) was measured using an anthrone method (Sánchez and others 1998). Soluble protein (SP) was measured using a coomassie brilliant blue method (Georgiou et al., 2008). Superoxide dismutase (SOD) was measured on the basis of its ability to inhibit the photochemical reduction of nitro blue tetrazolium (Stewart and Bewley 1980). The catalase (CAT) was measured by the method of Iodin titration (Kenten and Mann 1952). The peroxidase (POD) was assayed as the recorded absorbance increased at 470 nm by guaiacol method (Castillo et al., 1984).
 
Statistical analysis
 
The differences in germination parameters of alfalfa seed, and the seedling growth and physiological characteristics were analysed using One-Way ANOVA. The effects of extract content (Co), extract part (Pt) and their interactions (Co×Pt) were analysed using Two-Way ANOVAs. The linear correlations were analysed with the model y = ax + b using SPSS 17.0.

Changes in GR, GP, GI and VI
 
The GR, GP, GI and VI of alfalfa seeds were all significantly affected by extract content, part and their interaction (P<0.01, Fig 1). Generally, GR, GP, GI and VI decreased with the increased extract contents, which were also found in the previous studies (Rong et al., 2016; Tanha et al., 2017; Ghimire et al., 2019). But they were shown differently on the extract parts of alfalfa. Overall, the negative effects of extract parts showed leaf > stem > root > rhizosphere soil on seed germination. This finding has been consistent with the finding of (Rong et al., (2016). These indicated alfalfa continuous cropping induced the autotoxicity of alfalfa, and firstly and negatively affected the seed germination of subsequent alfalfa. The seed germination has also been found to be affected most adversely in the leaf extract among the four extract parts.
 

 
Changes in MDA content
 
The MDA content in alfalfa seedling was significantly affected by extract content in using two-way analysis of variance at P<0.05 (Fig 2). But it was not affect by extract content or parts in using one-way ANOVA at P<0.05. This might be that the extract inhibit the germination of weak seeds, and the remaining germinated seeds have high resistance to autotoxicity.
 

 
Changes in osmotic adjustment substances (Proline, SS and SP) and enzyme activities (POD, CAT and SOD)
 
The proline and SP contents in alfalfa seedling were all significantly affected by extract content, part and their interaction (P<0.01, Fig 3), while the SS content in alfalfa seedling was only significantly affected by extract content (P<0.01).
 

       
The SOD activity of alfalfa seedling was significantly affect by extract content, part and their interaction, POD activity was significantly affect by extract content and part, and CAT activity was significantly affect by extract part and Co×Pt (P<0.01, Fig 4). Generally, the extract decreased the enzyme activities and the reduced conditions were different in the four parts, i.e. the POD and CAT activities were higher in the extracts of root than other parts.
 

       
Generally, MDA content is known to increase when a plant encountered an adverse condition (Cavalcanti et al., 2004; He et al., 2012), while there were no significant changes in the extract treatments of this study. But the reductions of osmotic adjustment substances (Proline, SS and SP) and enzyme activities (POD, CAT and SOD), which would weaken the abilities of scavenging radical oxygen species (ROS) (He et al., 2012; Usha and Dadlani 2016). Furthermore, the osmotic adjustment substances, POD and CAT were highest in the extract of root in the study, suggesting the seedling was affected less in the extract of root than in other extract parts of alfalfa.
Changes in the lengths of root, bud and whole seedling
 
The lengths of alfalfa root, bud and whole plant were significantly affect by extract content, part and their interaction (P<0.01, Fig 5). Generally, the extract enhance the lengths of root, bud and whole plant. During earlier studies (Rong et al., 2016; Tanha et al., 2017), a reduction in root length, bud length and in the soil extract was found (Yang et al., 2009) was noticed but during present studies enhancement was noticed in each aspect. These might be caused by result of the promoting effect of allelopathy in the extract (Li et al., 2021) and the effect was shown the most in the root extract in this study.
 

 
Liner correlations of germination parameters with seedling growth, physiological characters of alfalfa
 
Few correlations were found between germination parameters and seedling physiological characters of alfalfa. And there were negative correlations of germination parameters with seedling physiological characters of alfalfa, expecially the correlations were significant between GI and the length of root, bud and whole plant (P<0.05, Table 1). These indicated that the extracts limited the germination and promoted the seedling growth and seed germination would negatively affect the seedling growth.
 

This work was jointly supported by the Research Program of Science and Technology at Universities of Inner Mongolia Autonomous Region (2019ZD997) and the grants from the Undergraduate’s Innovation, Entrepreneurship Training Program of Shandong Province (S202010452050), the Natural Science Foundation of Shandong Province (ZR2020QC184), the Forage Industrial Innovation Team, Shandong Modern Agricultural Industrial and Technical System, China (SDAIT-23-10) and the Sheep Industrial Innovation Team, Shandong Modern Agricultural Industrial and Technical System, China (SDAIT-10-14).

None.