Correlation between Fall Dormancy and Production Performance of Alfalfa (Medicago sativa)

Alfalfa (Medicago sativa) is the most widely distributed perennial leguminous forage in the world (Albayrak et al., 2018; Ventroni et al., 2010) but also the high-quality forage in China, alfalfa occupies the largest planting area with the highest forage value (Liu et al., 2021; Shi et al., 2017). Alfalfa is rich in nutritional value, especially high in crude protein (CP) content, which can replace the conventional concentrated feed to feed livestock. Alfalfa can also help to solve the problem of insufficient supply of high protein forage during winter in Northeast China. Currently, alfalfa varieties introduced from abroad have the advantage of good quality and high dry matter yield (DMY) (Wang et al., 2021). Although, autumn harvest time (Berti et al., 2012), snow cover (Leep et al., 2001), soil moisture (Ryswyk et al., 1993) and soil nutrient content (Macolino et al., 2013) influences the DMY and winter survival rate (WSR) of alfalfa, whereas it is unclear whether fall dormancy (FD) also affects the production persistence and WSR of alfalfa. Therefore, it is necessary to understand the correlation among production persistence, WSR and FD of alfalfa varieties. Due to the importance of FD in adaptability and DMY, it is often used as an important reference index for alfalfa variety selection (Fairey et al., 1996; Volenec et al., 2002). The FD ratings are usually divided into three groups: dormancy (FD rating <4), semi-dormant (FD rating 4-6) and non-dormant (FD rating >6) (Barnes et al., 1979). Dormancy type varieties have short branches and extroversion in autumn and slow stem elongation after harvest in summer. Therefore, dormancy type varieties have higher WSR (Haagenson et al., 2003). In contrast, non-dormancy type varieties thrive in autumn and the branches stretch rapidly after mowing in summer and autumn, forming long and upright branches (Brummer et al., 2002; Haagenson et al., 2003). Therefore, FD is not only related to the WSR of alfalfa, but also closely related to its yield and quality. Although there are many factors affecting the DMY and quality of alfalfa during its growth, such as mowing period, cutting frequency, climatic conditions and others (Djaman et al., 2020), the relationship among FD, DMY and nutrient components still remains unclear. Therefore, this study aims to explore the correlation between WSR, DMY, nutrient composition and FD of alfalfa varieties and screen alfalfa varieties with greater yield, better quality and stronger winter survival ability, so as to provide reference for introducing the best alfalfa in Heilongjiang Province, China and similar upper-latitude semiarid environments.

A total of eight alfalfa varieties were collected, the China (Zhaodong and Gongnong No. 2), the United States of America (Gold Empress, Sanditi, Sardi and WL525) and Canada (Caribou and Adrenalin). Zhaodong and Gongnong No. 2 (FD rating 1), Caribou (FD rating 2) and Gold Empress (FD rating 2-3) are all dormancy type varieties, Adrenalin (FD rating 4) and Sanditi (FD rating 5-6) are semi-dormancy type varieties and Sardi (FD rating 7) and WL525 (FD rating 8) are non-dormancy type varieties. The FD ratings are provided by the breeding company and Fang et al., (2015). The test plot was conducted at the Branch of Animal Husbandry and Veterinary of Heilongjiang Academy of Agricultural, Qiqihar, Heilongjiang, China (47°15'N, 123°41'E). The climate of the experimental site is mainly characterized by drought and cold. The average temperature and total rainfall were 5.9°C and 559 mm in 2019, 2.6°C and 625 mm in 2020 and 5.2°C and 628 mm in 2021, respectively. Eight alfalfa varieties were planted in May 2018. The soil was aeolian sandy soil and contained (0-30 cm depth) 19.89 g kg-1 of organic matter (OM), 1.19 g kg^-1 of available nitrogen (AN), 124.8 mg kg^-1 of available potassium (AK) and 10.6 mg kg^-1 of available phosphorus (AP) and had a pH of 7.4. The test plots were designed as a randomized complete block with three replicates. Each test plot was 5.0 m long and 3.0 m wide, with row spacing 0.3 m. The alfalfa cultivar Zhaodong was planted as a protective plant around the test plot. The seeding rate was 15 kg ha^-1. No fertilizer was applied to the test plots before sowing and compaction was performed after sowing. The test plot irrigation was carried out once in the regreening stage, the squaring stage and the pre-wintering period. The irrigation technology adopts sprinkler irrigation and the irrigation quota is 225~375 m³ hm^-2. The test plots were hand weeded during the alfalfa growing period. Three mowing per year were carried out during the early flowering stage from 2019 to 2021 (June 12^th -June 18^th, July 17^th-July 22^th, August 25^th -August 31^st).
       
To measure the DMY, mowing was carried out during the early flowering stage (10% flowering). The fresh alfalfa yield of test plot was measured, stubble height was 5 cm and repeated 3 times. Fresh alfalfa (2.0 kg) was dried in a drying oven at 65°C until the weight was constant and the DMY per ha was calculated. Fresh sample (1.0 kg) was taken from each test plot and the leaves and stems were separated. The leaves and stems were weighed after drying and then the leaf to stem ratio (LSR) was calculated. From October 20^th to November 1^st of every year, three replicates of a 1.0 m long sample segment were randomly selected in each test plot. The number of surviving plants in each sample segment was investigated and then the number of surviving plants were recorded in the next year. The winter survival rate.
 
The crude protein (CP) content was measured by the AOAC method (AOAC, 1990). Acid detergent fiber (ADF) and neutral detergent fiber (NDF) were determined by Ankom Fiber analyzer filter bag method (Anonymous, 1997). The digestible dry matter (DDM), dry matter intake (DMI) and relative feed value (RFV) were calculated as follows (Albayrak et al., 2018; Avci et al., 2017).
         
The experiment was conducted for three years (2019, 2020 and 2021) in one location. Years was treated as a fixed effect and two treatments (Years and FD Varieties) interactions were analyzed using Two-factors ANOVA. Analysis of variance (P<0.05, P<0.01) and correlation analysis were performed using SAS 9.0 software (SAS Institute Inc, 2002, NC USA). The data processing was performed with Excel 2007 (MicroSoft, Redmond, WA USA) and the figure was drawn with Sigma Plot 12.5 software (Systat Software Inc, 2003, San Jose, CA USA).
 

Dry matter yield
 
Results of correlation analyses are presented in Table 1. Main effect means and results of statistical analyses for year and FD variety are presented in Table 2. Year and variety were significant for all variables, while the year x FD variety interaction was significant only for DMY and LSR. In addition to the significant main effects of year and variety for DMY (Table 2), the significant year x variety interaction is shown in Fig 1. Previous studies have indicated that there was no definite relationship between FD rating and alfalfa yields (Chen et al., 2014; Rimi et al., 2012). Our results were contrast with the previous report. Based on the three-year average, FD rating 1 (Zhaodong and Gongnong No. 2) had the greatest DMYs. However, the alfalfa varieties with FD ratings between 2 and 8 (Caribou, Gold Empress, Adrenalin, Sanditi, Sardi and WL525) performed poorly in Heilongjiang, indicating that there was a negative correlation between the FD rating and DMY (Table 1). The DMYs of dormancy type and semi-dormancy type varieties were markedly higher than those of non-dormancy type varieties (Fig 1). One hand, this pattern may be related to low the WSR of alfalfa varieties, the long term evolution and selection for the local varieties maybe the main reason and the foreign varieties were also affected by the cold winter climate conditions in this region (Djaman et al., 2020). The other hand, the DMY may be closely related to genetic factors in varieties (Hill and Barnes, 1977; Albayrak et al., 2018). The FD rating should not be used as the main indicator for selecting alfalfa varieties in Heilongjiang, which was agreement with the results of Wang et al., (2021). Their studies confirmed that the FD rating was not related to the production performance of alfalfa in semi-arid climatic conditions. Previous studies have also reported that in areas with warmer winters, the establishment of suitable harvesting systems is the main factor affecting alfalfa production and FD rating is a secondary factor (Ventroni et al., 2010).
 






The DMYs of the first, second and third mowing of alfalfa are shown in Fig 2. The forage yield of each cutting accounted for 48%, 36% and 16% of the total yield in the whole year, respectively. Wang et al., (2009) found that the yield was significantly different with different cutting times and the yield of the first cutting was higher than that of the second cutting and the third cutting. These results of this study showed the same rule. This also in consistent with the typical growth/regrowth pattern of well-watered alfalfa that is driven by high summer temperatures (relative to FD adaptation) and declining daylength after summer solstice, which is exhibited by fall growth upon which fall dormancy categories are based. The yield of the first two cuttings accounted for more than 80% of the total yield in the whole year. In addition, there was a positive correlation between alfalfa yield and WSR (Table 1), which shows that the first cutting yield is very important to the total annual yield of alfalfa. Even though the growth of the first cutting alfalfa is affected by low temperature in winter and spring, it still contributes the most to the annual yield of alfalfa (Djaman et al., 2020). For less dormant varieties (i.e., Sardi and WL525), spring yields are reduced, compared to more dormant varieties, by attempted winter growth that reduces root carbohydrates (Liu et al., 2019). Therefore, good field management of the first cutting alfalfa is the key to achieve yield increase. The growing period of the second and third crops of alfalfa is in the season of rising temperature and increasing precipitation (June- August) and the plant grows rapidly. If the field management and protection can be strengthened during this period, alfalfa will have great potential for increasing yield (Chen et al., 2014).


 
Winter survival rate
 
Significant differences were found in the WSR among the alfalfa varieties (Table 2). The WSR of Zhaodong was highest from 2019 to 2020. The WSR of the alfalfa varieties decreased with the increase of the FD rating. The WSRs of Zhaodong (96.43%), Gongnong No. 2 (94.59%), Caribou (85.59%) and Gold Empress (75.10%) were greater than those of Adrenalin, Sanditi, Sardi and WL525 (FD rating 4-8). Correlation analysis showed that there was a negative correlation between the WSR and the FD rating (Table 1). Similar results were obtained in previous studies (Brummer et al., 2002; Wang et al., 2021). This indicated that the WSRs of dormancy type varieties (FD rating 1-2 including Caribou) were markedly higher than those of semi-dormancy type (FD rating 4-6) and non-dormancy type (FD rating 7-8) varieties, suggesting that dormancy type varieties have stronger cold tolerance. It is likely that the accumulation of raffinose and amino acid (Threonine, Histidine, Proline and Glycine) contributes for enhancing cold tolerance in dormancy type varieties (Liu et al., 2019). This positive association between the WSR and FD has been reported in previous researches (Li et al., 2015; Wang et al., 2021), which recommended that FD rating be used as an important index for selecting alfalfa varieties for winter hardiness (Barnes et al., 1979).
 
Crude protein content
 
Differences were examined among the different varieties in terms of CP contents (Table 2). The average CP contents over three years ranged from 18.43% to 19.29%, which is similar to those measured by others researchers (Avci et al., 2017; Albayrak et al., 2018; Wang et al., 2021). Gongnong No. 2 and Zhaodong (FD rating 1) showed greater CP contents than the other varieties. Their superiority, in terms of CP content, suggests that higher CP contents were related to their FD categories. As the FD of varieties has a major effect on the CP content of dry matter, the very dormancy varieties almost always had greater CP contents (Strbanovic et al., 2017). Some studies have also shown that the decrease in LSR is one of the main factors causing alfalfa quality depletion due to a decline in crude protein and an increase of fibrous constituents (Rimi et al., 2012; Avci et al., 2017; Wang et al., 2021).
 
Leaf stem ratio
 
The variance analysis of the LSR of the alfalfa varieties is shown in Table 2. The greatest LSR was observed in the Gongnong No. 2 (FD rating 1). Alfalfa leaves are the most valuable (i.e., nutritive and CP content) parts of alfalfa plants and varieties with greater LSRs are characterized by better quality (Strbanovic et al., 2017). According to the correlation analysis, there was a positive correlation between the LSR and the CP contents (Table 1), suggesting that the LSR is the most important determinant of quality for alfalfa among the dormancy varieties included in our correlation analysis. Leaves account for 55% to 65% (LSR=1.22-1.85) of high-quality alfalfa plants and 35% to 45% (LSR=0.53-0.82) of low-quality alfalfa plants (Putnam et al., 2008). According to this assessment, while the Gold Empress, Adrenalin, Sanditi, Sardi and WL525 variety (LSR=0.74-0.82) could be defined as low-quality alfalfa, the quality of Gongnong No. 2, Zhaodong and Caribou (LSR=0.83-0.92) falls between the low- and high-quality classes. Rotili et al. (1999) also found that the quality of alfalfa is mainly influenced by LSR and that plants with an LSR from 0.85 to 1.0 are defined as having high plant quality. When considered from this perspective, Zhaodong and Gongnong No. 2 are high-quality alfalfa varieties (Table 2).
 
Neutral detergent fiber and acid detergent fiber
 
The means of the NDF and ADF contents for the different alfalfa varieties are shown in Table 2. The NDF contents of alfalfa varieties ranged from 47.76% to 49.88%, over three years. The lowest NDF content was recorded for Zhaodong. Previously published reports found that the NDF contents of alfalfa depended on the variety (Albayrak et al., 2018; Avci et al., 2017; Spandel and Hesterman, 1997; Wang et al., 2021). There was a negative correlation between CP and NDF contents (Table 1), which was also reported by Avci et al., (2017). When indicated from this perspective, the Zhaodong is a good quality variety. The FD rating 1 varieties had greater nutritive value than less dormant varieties and that within FD rating 1 Zhaodong had better quality than Gongnong No. 2.
       
The three-year average of the ADF contents ranged from 30.79% to 32.84%, which is consistent to that reported elsewhere for alfalfa (Spandel and Hesterman, 1997; Avci et al., 2017; Albayrak et al., 2018; Wang et al., 2021). The ADF contents of Gongnong No. 2 and Zhaodong (FD rating 1) were significantly lower (P<0.05) than those of the other varieties. The lower the ADF value in alfalfa, the lower the content of cellulose and hemicellulose in the forage, so the palatability of Gongnong No. 2 and Zhaodong was good. The ADF content has a negative effect on digestibility and intake (Mader et al., 1991), suggesting that the nutritional values of Zhaodong and Gongnong No. 2 were better than those of the foreign varieties (i.e., Caribou, Gold Empress, Adrenalin, Sanditi, Sardi and WL525). This may be related to ecological factors, such as the relatively high evaporation and low precipitation (May and June) in Qiqihar and it is possible that the foreign varieties were affected by the arid climate conditions in this region (Rimi et al., 2012). Other studies indicate that the nutrient composition of alfalfa mainly depends on the variety and ecological factors (Karayilanli and Ayhan, 2016; Albayrak et al., 2018).
 
Digestible dry matter and relative feed value
 
Differences were observed in the DDM content among the varieties (Table 2). The DDM of Zhaodong was the greatest in 2019 and 2020. In 2021, the DDM values of Gongnong No. 2 (63.93%) and Zhaodong (64.42%) were significantly higher (P<0.05) than those of the other varieties. The three-year average of the DDM values of the varieties ranged from 63.32% to 64.91%. Zhaodong and Adrenalin had the highest and the lowest DDM values, which was mainly due to these alfalfa varieties having the greatest and least LSRs, respectively. As already noted, alfalfa leaves have relatively higher nutritive value and intake than stems. Julier and Huyghe (1997) also found that the digestibility of alfalfa varieties showed differences depending on their LSRs. The DDM value measured for the varieties in our study was generally similar to that reported in a previous research by Avci et al., (2017).
 
Significant differences were found in the RFVs among the alfalfa varieties (Table 2). The three-year average RFVs of the varieties ranged from 118.33 to 126.43. The highest and the lowest RFVs were recorded for Zhaodong and Adrenalin. This result suggested that the RFVs were significantly different among the alfalfa varieties, which may be mainly due to the large amount of variation in the DDM and DMI (not shown here). RFV is derived from the DDM and DMI values of alfalfa (Avci et al., 2017). Correlation analysis showed that there was a significant negative correlation (P<0.01) between the RFV and the NDF, ADF contents (Table 1), indicating that the NDF and ADF contents have negative effect on quality and feed value of alfalfa. RFV of full bloom alfalfa variety has a value of 100 and the higher values indicate better quality relative to the base of 100 (Avci et al., 2017). Legume forages with RFVs between 150-125, 124-103, 102-87 and 86-75 are categorized as premium, good, fair and poor, respectively (Kiraz, 2011). According to aforementioned assessment, Zhaodong is classified as premium, while the other alfalfa varieties are classified as good. The RFV determined for the varieties in our research was similar to that found in a previous study by Wang et al., (2021).
 

The FD rating 1 had significant effect on alfalfa production performance, such as the DMY and nutrient composition and it was negatively correlated with the WSR. Gongnong No. 2 and Zhaodong making them more suitable for planting in the Qiqihar, Heilongjiang, China and may be suitable for similar upper-latitude semiarid environments in the world.

This work was supported by Natural Science Foundation of Heilongjiang (YQ2022C033); Science and Technology Project in Qiqihar (CNYGG-2021009).

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