Alfalfa Cultivars Research Articles

Review of Partial Hybrids between Herbaceous Medicago sativa and Woody Medicago arborea and Their Potential Role in Alfalfa Improvement

Medicago sativa (2n = 4x = 32) and M. arborea (2n = 4x = 32) were thought to be reproductively isolated until hybrids (Alborea) were produced by sexual reproduction for the first time in 2003 in Wisconsin. The hybrids were asymmetric, at or near 2n = 4x = 32, and with a predominance of the alfalfa genome. Only M. sativa seed parents with reproductive abnormalities, including unreduced eggs, have produced hybrids; where M. arborea has been used as the seed parent, no hybrids have resulted. Pedigree selection within derivatives of the two original M. sativa seed parents (MB and M8) has been successful in increasing the frequency of hybrids produced. While Alborea individuals more closely resemble M. sativa, a number of M. arborea-specific traits have been observed across different hybrid individuals. These include single-coil flat pods, large seeds, yellow flowers, indeterminate growth, a minimal crown, lodging, frost resistance, and anthracnose resistance. These M. arborea traits have the potential to restructure alfalfa to increase its versatility and utilisation. There is emerging evidence from North and South America and Australia that some Alborea selections have the capacity to complement adapted alfalfa cultivars for yield. Work is continuing to introgress M. arborea traits of value into alfalfa.

Germination and Invasion of Paraphoma radicina on Roots of a Susceptible and a Resistant Alfalfa Cultivar (Medicago sativa).

Alfalfa Paraphoma root rot (APRR) (Paraphoma radicina) is a recently described alfalfa disease widely distributed in China, first reported in 2020. So far, the resistance levels of 30 alfalfa cultivars to APRR have been characterized; however, the resistance mechanisms among these cultivars remain unknown. In the present study, the alfalfa resistance mechanisms against APRR were investigated by studying the difference of P. radicina infecting susceptible (Gibraltar) and resistant (Magnum II) alfalfa cultivars under the light microscope and scanning electronic microscope. The conidial germination and germ tube growth in the root exudates of different resistant cultivars were also compared. The results revealed that conidial germination, germ tube development, and P. radicina penetration into root tissues of resistant plants were delayed. In susceptible and resistant cultivars, P. radicina infected roots by penetrating epidermal cells and the intercellular space between epidermal cells. During the infection process, germ tubes penetrated the root surface directly or formed appressoria. However, the penetration percentage on the susceptible cultivar was significantly higher than on the resistant cultivar, irrespective of the infection route. Moreover, disintegrated conidia and germ tubes were observed on resistant cultivar roots at 48 h postinoculation. The conidial germination and germ tube growth in root exudates of susceptible cultivars were significantly higher than in resistant cultivars. The current findings implied that the alfalfa resistance mechanism might be related to root exudates. These findings could provide insights into the alfalfa resistance mechanism following P. radicina infection.

Productivity, nutritive value, and profitability of high-digestibility alfalfa cultivars harvested at two stages of development in eastern Canada

Alfalfa ( Medicago sativa L.) cultivars developed for improved digestibility by conventional breeding or genetic modification have not been evaluated in eastern Canada. This study compared yield, nutritive value, and profitability of two reduced-lignin genetically modified (GM) cultivars, three conventionally selected for low lignin or high pectin, and one population with improved stem degradability, with two commercially adapted cultivars as controls. Alfalfa was harvested at the early bud (intensive management) or early flower (extensive management) stage of development during the first post-seeding year at three sites and the second post-seeding year at one site. All cultivars/populations had similar annual dry matter (DM) yields, except for the lower DM yield (−20%) of the improved stem degradability population. Conventionally selected cultivars for improved digestibility did not differ from control cultivars for in vitro DM digestibility (IVTD) and neutral detergent fiber digestibility (NDFd). Reduced-lignin GM cultivars, however, had a greater IVTD and NDFd (+10%) and less lignin (−10%) than control cultivars at the same stage of development. Reduced-lignin GM cultivars under extensive management had similar NDFd and greater annual DM yield (+1 to 2 Mg DM ha−1), but reduced total digestible nutrients (−41 to 44 g kg−1 DM), in comparison to control cultivars under intensive management. Using partial budget analysis, the comparison of more digestible GM cultivars to the control cultivars resulted in an increase in annual farm net profits ranging from CAD$7.40 to $79.60 cow−1 year−1 depending on stage of development at harvest. Further investigations are needed to quantify the performance of dairy cows fed cultivars with improved digestibility.

Simulation of alfalfa yield with AquaCrop

The AquaCrop simulation model, originally designed for annual crops, is expanded here to simulate alfalfa, a perennial forage crop. A new routine was added to the model to mimic the assimilate partitioning between above and below-ground plant parts to account for the utilization of reserves in Spring and for their storage in the Fall. The simulation of the transfer of assimilates requires only three extra crop parameters which makes the model also easy to calibrate. To evaluate the model, yield data collected in Louvain-La-Neuve (Belgium), Isparta (Turkey), and Ottawa (Canada) for different alfalfa cultivars, various years and field and irrigation management strategies were used. To assess the accuracy and robustness of the simple assimilate remobilization process, simulations were run for the three different environments with a common set of crop parameters which were parameterized. The dispersion between the observed and simulated cumulative dry above-ground biomass during the growing cycle was small (r² = 0.97; nRMSE = 11%; Nash-Sutcliffe model EF = 0.97), and a systematic over- or underestimation by the model was not observed (Willmott’s index of agreement, d = 0.99). When evaluating the goodness of fit of the 81 individual harvest events, the results were still very satisfactory although the nRMSE doubled. The simulations indicated that the AquaCrop model adapted to perennial crops and with a novel storage-remobilization routine, could predict well alfalfa yields in various climates and environments, with and without water and fertility stress, and for three different alfalfa cultivars.

Sandy silt loam soil may hamper the inoculation effect on lucerne (Medicago sativa L.) growth

ABSTRACT Despite newly approved lucerne cultivars, this has not led to increased use of this legume in high-latitude agriculture. Challenges with inoculation by Rhizobium meliloti have been identified as a bottleneck to adaptation. Here we tested inoculation sources (ISs) with soil types and cultivars in pot and field experiments. During a one-year outdoor pot experiment, we tested the impact of IS (wet peat slurry and Nitragin Gold dry inoculation) and three soil types (sand, sandy silt and /peat soil) on nodule development, shoot and root growth and winter survival of one hybrid lucerne cultivar (‘Ludvig’). The pot experiment revealed that dry inoculation led to significant better plant growth, flower and nodule development as well as plant regrowth after winter survival. Peat soil appeared as the best growth medium and silty soil limited inoculation efficiencies. In field trials at two locations differing in soil characteristics using similar ISs, and three hybrid lucerne cultivars (‘Lavo’, ‘Live’ and ‘Lotte’) biomass yield during two ley years showed site as well as cultivar differences. Such environmental interactions in the field trials justify the use of adapted cultivars, and dry inoculation should be recommended for practical use replacing peat slurry inoculation.

Five flavonoids from Lucerne (Medicago sativa L.) varieties

Alfalfa (Medicago sativa L., Fabaceae) is knowed as perennial herbaceous leguminous plant species that originated in southwestern Asia and used as folk medicine for the treatment of various ailments. The upperground part of Lucerne contain phenolic compound such as flavonoids etc., that contributes to its biological activities. The aim of this study was to determine five widely knowing flavonoids in extracts 20 alfalfa varieties herb at the Ukrainian steppe growing. We selected 50 seeds of the same size from twenty alfalfa cultivars from different countries, were cultivated under controlled areas of southern part of the Ukrainian left-bank at border of forest-steppe and steppe zones (Zaporizhzhya, Ukraine) from April to June, with 15 °C/ 07 °C (day/night), 14 h/10 h (light/dark) and 60–65% relative humidity. The content of flavonoids were found unequable in ethanol extracts. The chemical compositions and their content were assessed by ultrahigh-performance liquid chromatography. The content of five flavonoids were different in the 20 alfalfa varieties raw materials. Umbelliferone were found high in ethanol extract of Mongolian colorful hybrid (Mongolia, 0,23 mg|g). Four sorts have not contained umbelliferone: Кisvardai (Hungary), Nizona (Cuba), Тanhuato (Mexico) and Mesopotamian (Iraq). The leader from cinaroside content was sort Commercial 2-52-75 of UK origin. Routine has been found in the highest quantities in WL 50 from USA. Ferganska 700 from Uzbekistan was leader in luteolin content and Кisvardai, Hungary was leader in average of kaempferol content (0,030 mg|g). The present article comprises the hierarchical cluster analyses from the data flavonoid assay.

Evaluation of genetic diversity among Saudi Arabian and Egyptian cultivars of alfalfa (Medicago sativa L.) using ISSR and SCoT markers

Medicago sativa (Alfalfa) is valued for its high nutrient content as livestock feed for the production of dairy and meat products globally. The genetic improvement of alfalfa is limited because it is an allogamous autotetraploid. In this study, Inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) markers were used to assess the extent of genetic divergence between sixteen cultivars: twelve from Saudi Arabia and four from Egypt. The genetic diversity was assessed using eleven ISSR and eight SCoT primers. The ISSR primers produce 163 amplified bands with an average of 14.82 bands per primer and 60% polymorphism. The SCoT primers reveal 150 bands, with an average of 18.75 bands per primer and 77% polymorphism. The dendrogram clustered the Saudi and Egyptian cultivars into two distinct populations. The information revealed here will help in the breeding program of alfalfa, which will produce more drought tolerant and high-yielding varieties.

Multiplex CRISPR/Cas9-mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality.

Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.

Genetic evaluation and germplasm identification analysis on ITS2, trnL-F, and psbA-trnH of alfalfa varieties germplasm resources.

In this study, genetic diversity and germplasm identification of 28 alfalfa germplasm cultivars materials were evaluated by analyzing their internal transcribed spacer 2 (ITS2), trnL-F, and psbA-trnH sequences to provide the innovative reference of alfalfa varieties genetic diversity and identify research. The results showed that the fragment average length of ITS2, trnL-F, and psbA-trnH sorting sequences were 455.7 bp, 230.3 bp, and 345.6 bp, respectively. The ITS2 sequence was too conservative to reflect the individual differences between intercultivars and intracultivars in the preliminary experiment. Furthermore, trnL-F and psbA-trnH sequence differences were relatively small between intercultivars but significant between intracultivars. Alfalfa cultivars were divided into four groups by sequence similarity clustering. Alfalfa cultivars trnL-F and psbA-trnH sequences have apparent differences, showing that chloroplast conservative sequences were independent evolution. Compared with trnL-F and psbA-trnH sequences of alfalfa cultivars, psbA-trnH sequence has abundant variation sites and can better reflect the differences between cultivars than the trnL-F sequence. Therefore, the psbA-trnH sequence can identify different alfalfa cultivars and establish the DNA sequence fingerprint.

Evaluation of Rumen Degradation Kinetics of Low-Lignin Alfalfa 'Hi-Gest® 360' in Saskatchewan Canada.

The objective of this study was to determine rumen degradation kinetics of new low-lignin alfalfa (Medicago sativa L.) cv. Hi-Gest®360 (HiGest) in comparison with conventional alfalfa cv. AC Grazeland (Grazeland) in monoculture and binary mixtures at different maturity stages. Two cultivars of alfalfa (HiGest, and AC Grazeland) and their binary mixtures with hybrid bromegrass (HBG; cv. AC Success), grown in 2019 at two locations (Saskatoon and Lanigan), were cut at three maturity stages of alfalfa (1 = 10% bloom; 2 = 40% bloom; and 3 = 100% bloom). Rumen degradation characteristics, including rapidly degradable fraction (S), potentially degradable fraction (D), undegradable fraction (U), degradation rate (Kd), lag time (T0), and effective degradability (ED) of each component were determined using in situ technique and were analyzed by a first-order kinetic equation described by Ørskov and McDonald with lag time. Generally, in alfalfa monoculture, S or D were decreased and U was increased without affecting Kd and T0, resulting in decreased ED fraction with increasing stage of maturity. In binary mixtures, plant maturity stages have negligible effects on rumen degradation characteristics of CP. HiGest had higher effective degradability of DM (EDDM) as well as of NDF (EDNDF) than Grazeland. In conclusion, HiGest had greater DM and NDF rumen degradation potential relative to Grazeland. HiGest and Grazeland were different in DM and CP degradation patterns, with HiGest having higher EDDM and EDCP than Grazeland.

Estimation of alfalfa fall dormancy using spaced plant and sward trials across multiple environments

AbstractBackgroundAlfalfa is a globally important forage crop. Cultivars are characterized by fall dormancy (FD). FD affects biomass yield and winter survival and is used to identify appropriate growing regions of cultivars. It has historically been assessed by measuring the natural height of regrowth in autumn of spaced plants in the field. Because commercial alfalfa is normally grown as a solid planted sward, FD could be different if plants were grown under real production conditions. The objective of this experiment was to assess whether FD ratings obtained from swards were similar to those obtained on spaced plants.MethodsWe evaluated 20 alfalfa cultivars in field trials established in 2015 at five locations in the United States. We harvested the trials in early autumn and measured regrowth plant height approximately 3 weeks later in 2015 and 2016.ResultsAutumn plant height responded as expected for the established check cultivars in all locations. Fall dormancy ratings for cultivars under both management systems were highly correlated.ConclusionsEstimating FD from spaced plant height measurements in autumn is very robust, and using height data from sward plots gives equivalent results to that measured in spaced plant nurseries. This finding has many practical advantages.

Results of symbiotic breeding of alfalfa

The results of the studies on the effectiveness of alfalfa symbiosis varieties created by different methods are presented. Some peculiarities of plant-rhizobial interactions are identified. When alfalfa was grown in low- and near-neutral soils (pH 5.1-6.0), the highest increase in dry matter yield was achieved by pre-sowing inoculation with the CXM 412b strain, and on strongly and moderately acidic soils (pH 4.1-5.0) by the CXM 404b strain. In the year of sowing, the efficiency of symbiosis was at least 25%, and the yield reduction to the control level occurred during 3-4 years of use. Strain СХМ 404b has the unique ability to increase the seed productivity of different varieties under different growing conditions by 23-56%, other strains - by 3-32%. It was found that the productivity of variety microbial systems is mainly determined by the strain of nodule bacteria (the effect of inoculation was 60-62%). No relationship between the yield of the aboveground biomass and its crude protein content was found. The correlation coefficients obtained in the different field experiments were -0.41 ± 0.34 to 0.26 ± 0.39. Pre-sowing inoculation with highly active rhizobia strains of alfalfa variegated seeds of Selena, Agnia, Taisia, created using the methods of conjugate symbiotic breeding, increased the yield in the year of sowing by 26-35% in dry matter and by 44-56% in seeds. The yield of Vega 87, Lugovaya 67, Pastbishnaya 88, and Nakhodka varieties created by traditional methods increased by 3-20% in dry matter and by 23-29% in seeds. Pre-sowing inoculation with active rhizobia strains of alfalfa cultivars created by symbiotic breeding methods increased the yield in the first year of use by 46-128% in plots where alfalfa was not grown before and by 32-35% in the crop rotation with high alfalfa crop saturation.

Screening of Alfalfa Varieties Resistant to Phytophthora cactorum and Related Resistance Mechanism

Alfalfa is one of the most important legume forages in the world. Root rot caused by soil-borne pathogens severely restricts the production of alfalfa. The knowledge of the interaction between alfalfa and root rot-pathogens is still lacking in China. Phytophthora cactorum was isolated from symptomatic seedlings of an alfalfa field in Nanjing with high levels of damping-off. We observed the different infection stages of P. cactorum on alfalfa, and found that the purified P. cactorum strain was aggressive in causing alfalfa seed and root rot. The infecting hyphae penetrated the epidermal cells and wrapped around the alfalfa roots within 48 h. By evaluating the resistance of 37 alfalfa cultivars from different countries to P. cactorum, we found Weston is a resistant variety, while Longdong is a susceptible variety. We further compared the activities of various enzymes in the plant antioxidant enzyme system between Weston and Longdong during P. cactorum infection, as well as gene expression associated with plant hormone biosynthesis and response pathways. The results showed that the disease-resistant variety Weston has stronger antioxidant enzyme activity and high levels of SA-responsive PR genes, when compared to the susceptible variety Longdong. These findings highlighted the process of interaction between P. cactorum and alfalfa, as well as the mechanism of alfalfa resistance to P. cactorum, which provides an important foundation for breeding resistant alfalfa varieties, as well as managing Phytophthora-caused alfalfa root rot.

Genetic diversity and local adaption of alfalfa populations (Medicago sativa L.) under long-term grazing

Genomic information on alfalfa adaptation to long-term grazing is useful for alfalfa genetic improvement. In this study, 14 alfalfa populations were collected from long-term grazing sites (> 25 years) across four soil zones in western Canada. Alfalfa cultivars released between 1926 and 1980 were used to compare degree of genetic variation of the 14 populations. Six agro-morphological and three nutritive value traits were evaluated from 2018 to 2020. The genotyping-by-sequencing (GBS) data of the alfalfa populations and environmental data were used for genotype-environment association (GEA). Both STRUCTURE and UPGMA based on 19,853 SNPs showed that the 14 alfalfa populations from long-term grazing sites had varying levels of parentages from alfalfa sub-species Medicago sativa and M. falcata. The linear regression of STRUCTURE membership probability on phenotypic data indicated genetic variations of forage dry matter yield, spring vigor and plant height were low, but genetic variations of regrowth, fall plant height, days to flower and crude protein were still high for the 14 alfalfa populations from long-term grazing sites. The GEA identified 31 SNPs associated with 13 candidate genes that were mainly associated with six environmental factors of. Candidate genes underlying environmental factors were associated with a variety of proteins, which were involved in plant responses to abiotic stresses, i.e., drought, cold and salinity-alkali stresses.

Diversity of Agro-Biological Traits and Development of Diseases in Alfalfa Cultivars during the Contrasting Vegetation Seasons

Alfalfa exhibits high adaptability to a range of environmental conditions. The aim of this study was to evaluate the agro-biological traits of alfalfa and select its most promising cultivars under different contrasting vegetation seasons. The field experiment was carried out at the Institute of Agriculture of Lithuanian Research Centre for Agriculture and Forestry. In 2016 and 2018, eleven alfalfa cultivars of different origin were established in the Endocalcari Epigleyic Cambisol. In the seasons of 2017–2018 and 2019–2020, the agro-biological traits of alfalfa cultivars were evaluated during winter; at the height of spring regrowth; before flowering at the three cuts; and during the fresh and dry matter yields; we also examined the development of diseases at the three cuts. The cultivar Birutė from Lithuania was distinguished by its wintering, its plant height at spring regrowth, its height before flowering, and its fresh and dry matter yields. The cultivar Timbale from France was distinguished by its wintering, spring regrowth, and its height before flowering. The cultivar Magnat from Romania was distinguished by its height before flowering and the fact that it was less damaged by downy mildew. The cultivars Jõgeva 118, Juurlu, and Karlu from Estonia were distinguished by their wintering and the fact that they were less damaged by diseases. The cultivar Eugenia from Italy was distinguished by the fresh and dry matter yields and the height before flowering.

In vitro Evaluation of Seed Germination in Twelve Alfalfa Cultivars under Salt Stress

Alfalfa (Medicago sativa L.), when exposed to abiotic stress such as salinity, suffers significant losses in yield and productivity. The present study evaluated the salinity tolerance of 12 alfalfa cultivars in vitro using five concentrations of sodium chloride (NaCl), ranging from 0 to 250 mmol L−1 . The results obtained in the current study revealed that the Saudi cultivars, Kasimi and Hassawi, and the German cultivar (Berlin) had the highest salinity tolerance in terms of germination percentage (GP), corrected germination rate index (CGRI), days to reach 50% germination (GT50), and ability to form cotyledonary and true leaves. Under mmol L−1 NaCl, the Saudi cultivar Kasimi cultivar showed GP, CGRI, and GT50 of 55.20%, 123.15, and 3.77 days, respectively. Similarly, the German cultivar (Berlin) showed GP, CGRI, and GT50 of 50.06%, 86.61, and 5.17 days, respectively. These findings might reveal a pivotal aspect in salt tolerance in alfalfa. Our results will help to select salt-tolerant alfalfa cultivars that could thrive in arid and semi-arid areas with salinity problems.

The Efficacy of Biological Control for the Suppression of the Pea Aphid (Acyrthosiphon Pisum): Does the Resistance of Alfalfa Cultivars Matter?

The pea aphid, Acyrthosiphon pisum Harris, is a major pest of alfalfa in northwestern China. However, the roles of different groups of natural enemies in combination with aphid-resistant cultivars in the suppression of the pea aphid have not been clarified under field conditions. In this study, we used experimental cages to better understand the top-down (natural enemies) and bottom-up (nine alfalfa cultivars) biological processes, as well as the individual roles of the two processes, in the control of the pea aphid. There was a significant difference in resistance among cultivar classes revealed when natural enemies were excluded. The functional contribution of top-down suppression was higher than the bottom-up process, with natural enemies significantly suppressing aphid populations, regardless of the resistance level of different alfalfa cultivars. The mean biological efficacies of predators, parasitoids, and mixed populations of natural enemies were 85%, 42%, and 88%, respectively. Overall, our study indicated that natural enemies play a critical role in suppressing aphid populations, especially in the summer, whereas cultivar resistance did not combine effectively with natural enemies to inhibit the growth of aphids. Conservation biological control (CBC) can be implemented in the alfalfa production regions in northwestern China to reduce the overreliance on insecticides for the control of pests and mitigate their harmful effects on humans, ecosystems, and biodiversity.

First report of Alfalfa root rot caused by Fusarium commune in China.

Alfalfa (Medicago sativa) is the most important perennial forage legume worldwide, with high yields and nutrient quality. Diseases are significant threats to alfalfa and cause substantial yield losses (Fang et al. 2021). In March 2022, a disease with symptoms similar to fusarium wilt was observed in an alfalfa (cultivar Aurora) field in Baima teaching and research base (119°18'07″E, 31°61'47″N) in Nanjing, China. Reddish-brown discoloration of the roots, stele, basal stems and the withering symptoms on the aerial portions are the specific symptoms of fusarium wilt. The disease incidence varied from 3 to 6% in around 0.3 hectares of alfalfa fields surveyed. Fresh tissues from symptomatic alfalfa were cut into pieces (3-5 mm) and surface sterilized with 75% ethanol for 30 seconds, followed by 1% sodium hypochlorite (NaClO) for 120 seconds, and three rinses with sterile distilled water. Tissue pieces were placed on selective potato dextrose agar (PDA) containing 50 mg/L rifampicin and ampicillin. Plates were sealed and incubated at 25°C for 48 to 72 hours. Over 70 tissue fragments plated, 20 isolates from different fragments showed similar colonies. These isolates were purified by single spore isolation and grown on PDA and mung bean medium for morphology identification, molecular identification and pathogenicity test. The colony showed white to pink, abundant, densely aerial mycelium, while its backside was light violet. Macroconidia were hyaline, falcate with septa, and ranged in size from 25.5 to 61.5 × 3.8 to 6.2 μm (n=50). Microconidia were non-septate, hyaline, oval, straight to slightly curved, and were 5.6 to 10.7 × 2.4 to 3.2 μm (n=50). Spherical chlamydospores were 10.6 ± 1.3 μm in size. The rDNA internal transcribed spacers (ITS), RNA polymerase II subunit 2 (RPB2) and translation elongation factor 1-α (TEF) genes were amplified and sequenced (White et al. 1990; Carbone et al. 1999; Wang et al. 2019). Blast analysis showed 99.41% to 100% identity to F. commune sequences in the GenBank database (KU341324.1, MN892350.1, MH341219.1). The pathogenicity test was conducted using the dipping method in roots. Fresh F. commune hyphae were cut into 3 x 3mm agar plugs from a 7 cm PDA plate and inoculated in 200 mL mung bean medium on a shaker at 160 rpm, 25°C for 5 days. Spores were filtered through a cheese cloth, adjusted to 1 × 106 spores/mL with distilled water and used immediately. Roots in two-week-old alfalfa seedlings were soaked in spore suspension for 30 mins before being transplanted to sterile vermiculate with four repeats, while those in sterile water were used as non-inoculated control. The plants were placed in the green house at 25°C with 16 h of light and 8 h of darkness. The symptoms were observed two weeks following the inoculation. 30% of the seedlings withered, and reddish-brown discoloration symptoms were visible on the roots and stem of the unwithered plant. F. commune was isolated from these lesions again, and no disease symptoms were observed in the control plant. F. commune has been reported to cause root and stem rot in many plants worldwide, including tobacco (Zhong et al. 2021), maize (Mezzalama et al. 2021; Xi et al. 2019), soybean (Detranaltes et al. 2022) and sugarcane (Wang et al. 2018). Our results show that F. commune was the causative agent of alfalfa root rot. To our knowledge, this is the first report of F. commune causing root rot in alfalfa. The finding provides insights for disease diagnosis of alfalfa root rot disease and management in the region.

Embryogenic Callus Differentiation in Short-Term Callus Derived from Leaf Explants of Alfalfa Cultivars

Alfalfa is a forage crop that accounts for one of the best sources of protein and is commonly grown all over the world. In vitro callus and embryogenic induction of alfalfa have been investigated previously; however, most of the investigations were almost limited with callus formation. In this study, leaves of 5 Medicago sativa L. cultivars (Alsancak, Sazova, Plato Iside, and Bilensoy) have been used. Influences of culture medium contents and hormones applications on callus and embryogenic callus induction were determined to optimize in vitro culture mediums of alfalfa (0.0125 g kinetin 2,4 D 1 mg mL-1; 1 g kinetin 2,4 D 1 mg mL-1 ; 0.25 g kinetin 2,4 D 2 mg mL-1; 0.5 g kinetin 2,4 D 2 mg mL-1). Callus formation was detected at a rate of 74% in 5 different cultivars used in the experiment. The five alfalfa cultivars were classified into four categories in terms of embryogenic differentiation capacity. The tested alfalfa cultivars varied in their callus formation and embryogenic callus differentiation. Sazova, Alsancak, and Bilensoy were detected for better callus formation; similarly, the same cultivars responded with better embryogenic callus formation in the culture mediums including various hormone concentrations. The present study shows that our methods have beneficial impacts on the somatic embryo induced by alfalfa. However, it depends strongly on genotype, hormone concentrations and the other medium components.

Characteristics of Carbon, Nitrogen and Phosphorus Stoichiometry and Nutrient Reabsorption in Alfalfa Leaves with Different Fall-Dormancy Levels in Northern Xinjiang, China

Alfalfa productivity and cold resistance in different regions are influenced by the fall-dormancy level of alfalfa. However, it is unclear whether the stoichiometric characteristics and nutrient resorption efficiency in alfalfa leaves also vary with the fall-dormancy level. In order to further understand the differences in nutrient absorption and requirements of different fall-dormant alfalfa, we conducted field trials on 30 different fall-dormancy alfalfa cultivars for 2 consecutive years in 2020 and 2021. We investigated the concentrations of carbon, nitrogen, and phosphorus in mature and senescent alfalfa leaves; nutrient stoichiometry ratios; and the coupling relationship between nutrient reabsorption efficiency and dry matter yield. The differences in nutrient reabsorption, fall dormancy, and dry matter yield of different fall-dormant alfalfa, and the correlation between indicators were utilized to further analyze the regulatory mechanisms of nutrient reabsorption in different fall-dormancy alfalfa varieties. The results demonstrated that the nitrogen reabsorption efficiency (NRE) and phosphorus reabsorption efficiency (PRE) of leaves increased first and then decreased with the increase in fall dormancy, whereas the carbon reabsorption efficiency (CRE) showed the reverse tendency. Different fall-dormancy alfalfa varieties significantly affected the dry matter yield and nutrient absorption in the first cut, while the last cut had the lowest variable coefficient and impact. There was a significant decrease in the over-winter survival rate of alfalfa as the fall-dormancy level increased, whereas the over-summer survival rate was less affected by the fall-dormancy level. As the growth year increased, there was a significant decrease in the over-winter survival rate. Among mature leaves, the NRE and PRE showed a significant positive correlation with the C concentration, while they showed a significant negative association with the N and P concentrations. In the same cut, the dry matter yield decreased with the increase in CRE but increased with the increases in NRE and PRE, while there was no significant trend in dry matter yield and nutrient resorption efficiency (NuRE) between different cuts. Taken together, the alfalfa survival rate and dry matter yield were relatively better in the moderate fall-dormancy (fall-dormancy level, FD = 4, 5) types and fall-dormancy (FD = 3) type, with a corresponding increase in the reabsorption requirements for nitrogen and phosphorus.