Zhenyi Li

Background: Alfalfa (Medicago sativa) is a high-quality legume forage. Phosphorus (P) is essential for plant productivity. Approximately half of global arable soils exhibit available-P deficiency, constraining plants growth and yield. This study aimed to explore how hormones mediate the responses of alfalfa to phosphate starvation. Methods: We studied the morphological and physiological changes alfalfa (Zhongmu No.3) seedlings treated with auxin, ethylene and gibberellin under conditions of phosphate deficiency. 20-day-old alfalfa seedlings were exposed to varying concentrations of hormones in Hoagland solution. Changes in root morphology, photosynthesis, physiological characters and the levels of phosphate starvation induced (PSI) genes expression were monitored. Result: IAA stimulated lateral-root growth (1.16-fold vs. control), up-regulated the organic phosphate transporter (PHT1-4) and purple acid phosphatase (PAP23) and inhibited primary-root elongation. Ethylene promoted the growth of root hairs and 10 μM aminocyclopropane-1-carboxylic acid (ACC) increased the elongation of roots and their surface area.1 μM ACC boosted shoot and root acid phosphatase activity and up-regulated 1-aminocyclopropane-1-carboxylate oxidase (ACO), whereas 5 μM gibberellin A3 (GA3) promoted stem and leaf growth, increasing stem diameter by 16.10% and leaf area by 36.90%, while elevating sulfoquinovosyl diacylglycerol (SQD2) content and DELLA expression. There was a strong negative correlation (-0.79) between the plant height and root-to-shoot ratio and a positive correlation (0.62) between the number of lateral roots and the activity of acid phosphatase (ACP) in the roots. The most effective concentrations of hormones was 1 μM ACC, which enhanced the plant height, stem diameter, lateral root formation, chlorophyll biosynthesis and nitrogen content and induced the levels of expression of PSI and key genes in hormone signaling.

Alfalfa (Medicago sativa L.) is perennial leguminous forage, which is cultivated throughout the world due to its high yield, high quality, satisfactory palatability, and wide adaptability. With the increase of planting area in China, root diseases caused by Fusarium spp., Sclerotium rolfsii, Phytophthora spp. (Yang et al. 2022), and new pathogens have been found that reduce the yield and quality of alfalfa and cause economic losses (Li at al. 2019). In 2021, an alfalfa disease occurred under conditions of high temperature and high humidity at the Jiaozhou Experimental Base of Qingdao Agricultural University (Jiaozhou Modern Agricultural Science and Technology Demonstration Park, 36.33°N 120.40°E, Qingdao, Shandong, China), and about 2 ha of alfalfa were infected. The disease affected up to 35% of the plants and caused grass spots. Infected plants developed black-brown lesions with irregular shapes on roots with yellowing of the foliage; the leaves of the whole plant turned yellow. In the late stage of the disease, defoliation occurred and the plants stopped growing, wilted and died. Ten infected plants with typical symptom were collected for isolation and identification of pathogen. The infected roots were cut into 3-5 mm2 sections and then soaked in 75% ethanol for 30 s, followed by a 3-minute immersion in 2% sodium hypochlorite for surface sterilization. Next, the tissues were rinsed in sterile water five times and then placed on potato dextrose agar (PDA) medium. After three subcultures and subsequent single spore isolation, one representative strain named as DC1 was isolated from the infected roots. Based on morphological observation, the colony of DC1 was flat, granular, and powdery in appearance. Four days after inoculation on PDA medium, the size of the colony were 2.1-2.6 cm. After 8 to 20 days, the colonies were initially white and gradually change a light pink to peach color. The conidia are two-celled (Hamid et al. 2014), elliptic to pear-shaped, colorless or translucent, smooth to slightly rough with thick walls. The size of conidia ranged from 11.3 to 23.5 μm long × 6.1 to 12.7 μm wide (n =30). For the identification, the rDNA--ITS gene of the fungus was amplified using the primers ITS1/ITS4 (White et al.1990), and the EF1α gene was amplified using primers EF1-983F/EF1-2218R (Rehner and Buckley 2005). Then the PCR amplicons were cloned into the pCE2 TA/Blunt-Zero vector. The results of the rDNA-ITS (OM049197.1, 515 bp) and EF1α (OM069381.1, 926 bp) sequences were deposited in GenBank. DNA analysis showed that the two sequences were 100% similar to the rDNA-ITS sequence (MN882763.1) and EF1α sequence (DQ676610.1) of Trichothecium roseum, respectively. A pathogenicity test was done by placing one piece (0.5 cm in diameter) of fungal culture (PDA plug) 1cm below the crown of 40-day-old healthy alfalfa (cv. Zhongmu No.3) plants, 3 replicates and 20 plants in each replicate. PDA plug without the pathogen were used for control. All plants were cultivated in a growth chamber at 25±1°C with a light cycle of 15 h (90% relative humidity). After 18 days, the roots of inoculated plants had dark brown lesions and the leaves of their plants turn yellow, while those control plants had no symptoms. To fulfilling Koch’s postulates, the same pathogen was re-isolated from necrotic root tissue of inoculated plants and confirmed by morphology and the rDNA-ITS and EF1-α sequences. Based on disease symptoms, morphological characteristics DNA sequences and pathogenicity, the pathogen of alfalfa disease in Jiaozhou Experimental Base of Qingdao Agricultural University was identified as T. roseum. To our knowledge, this is first report of T. roseum causing alfalfa root rot. The newly emerging disease may pose threat to alfalfa production of central and southern China in future.

To understand how light intensity influences plant morphology and photosynthesis in the forage crop alfalfa (Medicago sativa L. cv. Zhongmu 1), we investigated changes in leaf angle orientation, chlorophyll fluorescence, parameters of photosynthesis and expression of genes related to enzymes involved in photosynthesis, the Calvin cycle and carbon metabolism in alfalfa seedlings exposed to five light intensities (100, 200, 300, 400 and 500 μmol m⁻² s⁻¹) under hydroponic conditions. Seedlings grown under low light intensities had significantly increased plant height, leaf hyponasty, specific leaf area, photosynthetic pigments, leaf nitrogen content and maximal PSII quantum yield, but the increased light-capturing capacity generated a carbon resource cost (e.g., decreased carbohydrates and biomass accumulation). Increased light intensity significantly improved leaf orientation toward the sun and upregulated the genes for Calvin cycle enzymes, thereby increasing photosynthetic capacity. Furthermore, high light (400 and 500 μmol m⁻² s⁻¹) significantly enhanced carbohydrate accumulation, accompanied by gene upregulation and increased activity of sucrose and starch-synthesis-related enzymes and those involved in carbon metabolism. Together, these results advance our understanding of morphological and physiological regulation in shade avoidance in alfalfa, which would guide the identification of suitable spatial planting patterns in the agricultural system.

Alfalfa (Medicago sativa L.) is a perennial leguminous forage cultivated globally. Fusarium spp.-induced root rot is a chronic and devastating disease affecting alfalfa that occurs in most production fields. Studying the disease resistance regulatory network and investigating the key genes involved in plant–pathogen resistance can provide vital information for breeding alfalfa that are resistant to Fusarium spp. In this study, a resistant and susceptible clonal line of alfalfa was inoculated with Fusarium proliferatum L1 and sampled at 24 h, 48 h, 72 h, and 7 d post-inoculation for RNA-seq analysis. Among the differentially expressed genes (DEGs) detected between the two clonal lines at the four time points after inoculation, approximately 81.8% were detected at 24 h and 7 d after inoculation. Many DEGs in the two inoculated clonal lines participated in PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) mechanisms. In addition, transcription factor families such as bHLH, SBP, AP2, WRKY, and MYB were detected in response to infection. These results are an important supplement to the few existing studies on the resistance regulatory network of alfalfa against Fusarium root rot and will help to understand the evolution of host–pathogen interactions.

Alfalfa, an important forage legume, is an ideal crop for sustainable agriculture and a potential crop for bioenergy resources. Drought, one of the most common environmental stresses, substantially affects plant growth, development, and productivity. MicroRNAs (miRNAs) are newly discovered gene expression regulators that have been linked to several plant stress responses. To elucidate the role of miRNAs in drought stress regulation of alfalfa, a high-throughput sequencing approach was used to analyze 12 small RNA libraries comprising of four samples, each with three biological replicates. From the 12 libraries, we identified 348 known miRNAs belonging to 80 miRNA families, and 281 novel miRNAs, using Mireap software. Eighteen known miRNAs in roots and 12 known miRNAs in leaves were screened as drought-responsive miRNAs. With the exception of miR319d and miR157a which were upregulated under drought stress, the expression pattern of drought-responsive miRNAs was different between roots and leaves in alfalfa. This is the first study that has identified miR3512, miR3630, miR5213, miR5294, miR5368 and miR6173 as drought-responsive miRNAs. Target transcripts of drought-responsive miRNAs were computationally predicted. All 447 target genes for the known miRNAs were predicted using an online tool. This study provides a significant insight on understanding drought-responsive mechanisms of alfalfa.

Alfalfa, an important legume forage, is an ideal crop for sustainable agriculture and a potential bioenergy plant. Drought, one of the most common environmental stresses, substantially affects plants’ growth, development and productivity. MicroRNAs (miRNAs) are newly discovered gene expression regulators that have been linked to several plant stress responses. To elucidate the role of miRNAs in drought stress regulation of alfalfa, a high-throughput sequencing approach was used to analyze 12 small RNA libraries comprising of 4 samples, each with 3 biological replicates. We identified 348 known miRNAs, belonging to 80 miRNA families, from the 12 libraries and 281 novel miRNAs using Mireap software. 18 known miRNAs in roots and 12 known miRNAs in leaves were screened out as drought-responsive miRNAs. Except for miR319d and miR157a which were upregulated under drought stress, the expression pattern of drought-responsive miRNAs were different between roots and leaves in alfalfa. This is the first study discovering miR157a, miR1507, miR3512, miR3630, miR5213, miR5294, miR5368 and miR6173 are drought-responsive miRNAs. Target transcripts of drought-responsive miRNAs were computationally predicted. All 447 target genes for the known miRNAs were predicted using an online tool. This study provides a significant insight on understanding drought-responsive mechanisms of alfalfa.