Scientia Agricultura Sinica

Origin, Evolution and Spread of Crop Buckwheat

WEI YiMin, ZHOU MeiLiang, TANG Yu

Scientia Agricultura Sinica. 2025, 58(21): 4305-4316. doi:10.3864/j.issn.0578-1752.2025.21.001

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China is one of the four major centers of agricultural origin in the world, where two distinct agricultural systems were established: the rice-based system represented by the middle and lower reaches of the Yangtze River, and the dryland farming system represented by the Yellow River basin. Historical records and archaeological evidence indicate that as early as the Shang Dynasty, oracle bone inscriptions already mentioned crops such as millet (Setaria italica), broomcorn millet (Panicum miliaceum), wheat, rice, and soybeans. During the pre-Qin period, the concept of the ‘Five Grains’ was established, and in The Book of Songs (Shijing), the broader term ‘Hundred Grains’ also appeared as a general reference to food crops. However, it is noteworthy that buckwheat, a crop native to China, has long been absent from these documented grain systems. This omission is inconsistent with the fact that buckwheat is an indigenous Chinese crop with high genetic diversity, significant local variation, and a long history of cultivation and domestication in cold mountainous regions. This study conducts a systematic review of the literature related to the origin, evolution, and spread of buckwheat, integrating recent findings in archaeobotany and genetic diversity analysis. Following internationally accepted principles for identifying crop origin centers, and drawing on historical texts, biological characteristics, and distribution patterns, the study presents comprehensive evidence supporting the hypothesis that southwestern China-particularly Yunnan, Sichuan, Guizhou, and the southern fringe of the Qinghai-Tibet Plateau-is the center of origin, genetic diversity, and domestication for Fagopyrum species. There are 23 species of Fagopyrum identified in China, including three cultivated species-common buckwheat (F. esculentum), tartary buckwheat (F. tataricum), and golden buckwheat (F. cymosum)-and 20 wild species, the majority of which are concentrated in southwestern China. This region is not only the native habitat of the ancestral subspecies of common and tartary buckwheat (F. esculentum ssp. ancestrale and F. tataricum ssp. potanini), but also the area with the richest diversity of Fagopyrum, strongly indicating its status as the origin center. Furthermore, molecular markers and phylogenetic studies confirm close genetic relationships between wild and cultivated buckwheat species in this region, providing key evidence for reconstructing domestication pathways. With advancements in modern research, buckwheat has gained recognition not only for its short growth cycle, broad adaptability, and resilience to poor soils and cold climates, making it suitable for cultivation in remote and mountainous areas, but also for its grain's rich content of proteins, flavonoids, and functional sugar alcohols. As a highly promising functional coarse grain crop, buckwheat is particularly suited to the development of characteristic agriculture in central and western China. It holds significant potential for ecological sustainability, nutritional health, and high-value agricultural development, and is expected to play an important role in China’s national nutrition strategy and food diversity conservation. This study provides theoretical and empirical evidence to support the scientific designation of China as the center of origin and domestication of buckwheat, laying a solid foundation for future work in germplasm conservation, variety improvement, and industrial development.

Comprehensive Evaluation of Salt Tolerance at the Seedling Stage and Screening of Tolerant Germplasm in Adzuki Bean (Vigna angularis)

CHEN TianXiao, CAO Rong, SONG QianNan, HU LiangLiang, WANG SuHua, WANG LiXia, CHENG XuZhen, CHEN HongLin

Scientia Agricultura Sinica. 2025, 58(21): 4317-4332. doi:10.3864/j.issn.0578-1752.2025.21.002

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【Objective】Adzuki bean (Vigna angularis) is an important legume crop in China, yet, its production is severely constrained by soil salinity. This study aimed to systematically identify and evaluate the salt tolerance of a large-scale adzuki bean germplasm collection to provide elite genetic resources and a theoretical basis for the genetic improvement of salt-tolerant adzuki bean cultivars. 【Method】A total of 398 adzuki bean accessions were evaluated in a hydroponic system under 100 mmol·L^-1 NaCl stress a concentration determined as suitable for screening in preliminary experiments. The salt injury index and 10 root morphological traits of each accession were measured post-treatment. A comprehensive salt tolerance value (D-value) for each accession was calculated using a combination of principal component analysis (PCA) combined with the subordinate function method. Based on the D-value, all accessions were systematically evaluated and classified into different salt tolerance grades. Subsequently, differences in salt tolerance among three germplasm types (cultivars, landraces, and wild accessions) were compared. Finally, stepwise regression analysis was employed to identify key indicators for evaluating seedling salt tolerance and to construct a simplified evaluation model. 【Result】Salt stress significantly inhibited root growth in adzuki bean, but extensive genetic variation was observed among the accessions. PCA effectively reduced the 10 root traits to three independent principal components, accounting for a cumulative 88.76% of the total variation. According to the criteria of a comprehensive salt tolerance value (D-value) and salt tolerance grade, a group of highly tolerant accessions at the seedling stage, such as B552 and B533, were identified. Comparative analysis indicated that wild accessions and landraces exhibited stronger seedling stage salt tolerance potential than cultivars, with wild accessions showing particularly outstanding tolerance. Stepwise regression analysis identified the salt tolerance coefficients of five key traits, including root volume, root fresh weight, root dry weight, average root diameter, and number of root crossing as the key indicators. Based on these indicators, an optimal regression equation with a very high coefficient of determination was established. 【Conclusion】This study systematically evaluated the salt tolerance of a large-scale adzuki bean germplasm collection at the seedling stage. It not only identified a group of elite salt-tolerant accessions, but also established a simplified and efficient comprehensive evaluation system for seedling salt tolerance in adzuki bean based on five key root traits.

Effects of Variety, Over-Winter Regulation, and Pest and Disease Control on Rapeseed Yield

YANG Long, ZHANG XueKun, CHEN AiWu, LI Mei, CHENG Tai, MA WuHui, XU BenBo, XU JinSong

Scientia Agricultura Sinica. 2025, 58(21): 4333-4345. doi:10.3864/j.issn.0578-1752.2025.21.003

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【Objective】Rapeseed (Brassica napus L.) is the first major oil-bearing crop in China. Enhancing rapeseed yield per unit area is the key to increase its effective supply and economic benefits. Under the condition of mechanical direct seeding, further clarifying the impacts of major technical factors, such as variety selection, overwintering regulation, and disease and pest control, on the yield per unit area could provide a theoretical basis for the improvement of rapeseed yield per unit area in China. 【Method】Nine high-yield and high-quality rapeseed varieties were selected, and mechanical direct-seeding experiments were conducted at 22 experimental sites (major rapeseed-producing counties) over 2 years from 2021 to 2023. A systematic study was carried out to investigate the impacts of factors such as rapeseed variety, seed treatment, overwintering regulation, and Sclerotinia sclerotiorum control on the yield per unit area under the condition of reduced topdressing application of nitrogen fertilizer. 【Result】The application of three combined treatment techniques, namely seed treatment (Maishuping coating for pest prevention, D1), overwintering regulation (D2), and novel Sclerotinia sclerotiorum control (fluxapyroxad, D3), all significantly increased the yield per unit area of rapeseed under reduced nitrogen topdressing (urea 45 kg·hm^-2). Compared with the traditional loss-reduction technology mode (uncoated seeds+no overwintering regulation and prochloraz for Sclerotinia sclerotiorum control+urea topdressing, CK), with a yield of 112.5 kg·hm^-2, the yields under D1, D2 and D3 treatments increased significantly by 6.2%, 6.4%, and 10.9%, respectively, while the corresponding yields reached 2 594.2, 2 600.4 and 2 708.9 kg·hm^-2, respectively. However, only the incidence of Sclerotinia sclerotiorum was extremely significantly reduced by 49.1% under the D3 treatment. Different technical combinations exhibited significant interaction effects on yield per unit area improvement. When seed treatment and overwintering regulation were combined with the novel Sclerotinia sclerotiorum control technology respectively, the yields were significantly 15.7% and 16.1% higher than those under the traditional technology, respectively, reaching 2 820.0 and 2 834.2 kg·hm^-2, respectively, while the incidence of Sclerotinia sclerotiorum decreased by 55.6% and 55.3%, respectively. Additionally, there was a significant interaction effect between rapeseed variety genotypes and technology. The application of new technologies could exceed the national regional trial yields of the corresponding varieties. For instance, the regional trial yields of Yangguang 131, Dadi 199 and Zhongyouza 39 were 2 341.2, 3 085.5, and 2 982.0 kg·hm^-2, respectively. Under optimized combinations of regulatory technology, the yields were 11.7%, 5.0% and 3.5% higher than their respective regional trial yields, respectively. A multiple stepwise regression analysis was conducted between the experimental yield and factors including the regional trial yield, disease resistance of rapeseed varieties, as well as different technological combinations, and the results revealed that the critical factors determining yield levels were the 1000-seed weight in regional trials, regional trial yield of varieties, novel Sclerotinia sclerotiorum control, and reduced winter urea topdressing rate. Canonical correlation analysis further confirmed that the regional trial yield, regional trial 1000-seed weight, and regional trial silique number of varieties determined the yield levels. Meanwhile, fluxapyroxad and Maishuping played a very important role in enhancing yield increase rates. Additionally, the novel Sclerotinia sclerotiorum control technology and overwintering stress resistance regulation were related to reducing the incidence of Sclerotinia sclerotiorum. 【Conclusion】In the mechanical production of rapeseed, the regional trial yield of varieties was a key factor affecting the rapeseed yield levels. The use of fluxapyroxad to control Sclerotinia sclerotiorum was an effective factor for reducing the yield loss caused by Sclerotinia sclerotiorum at present. The combination of seed treatment, overwintering regulation and the novel Sclerotinia sclerotiorum control technology could further exert the yield potential of rapeseed varieties. It was suggested that in practical production, high-yield and high-quality varieties should be selected, and loss-reduction technologies, such as supporting seed treatment, overwintering regulation and fluxapyroxad, should be matched to achieve a significant improvement in rapeseed yield per unit area.

Effects of Nitrogen Fertilization on Photosynthetic Carbon Allocation in Pasture Based on ¹³C Pulse-Labeling Experiments

XU XiuYuan, ZHANG HongZhi, XU LiJun, XUE Wei, NIE YingYing, GUO MingYing, LI JinXia, ZHAO YaRu, SHI MingJiang

Scientia Agricultura Sinica. 2025, 58(21): 4346-4356. doi:10.3864/j.issn.0578-1752.2025.21.004

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【Objective】Field experiments were conducted to examine the effects of nitrogen fertilization on the biomass yields of alfalfa and oat by measuring the plant’s ¹³C fixation and the ¹³C abundance in different plant organs, aiming to shed light on the transport and distribution of photosynthetic carbon in the plant-soil system and to explore effective means of soil carbon sequestration and soil quality improvement. 【Method】The ¹³C pulse labeling method was adopted in the field experiment. Two treatments were incorporated for alfalfa and oat crops, including nitrogen fertilization at 75 kg·hm^-2 in addition to a control experiment (no nitrogen). Plant aboveground and belowground biomass were measured. ¹³C abundances in different plant organs were also measured. 【Result】Nitrogen fertilization increased soil organic carbon (SOC) content in oat plots, while no significant SOC effects were observed in alfalfa plots. However, nitrogen fertilization increased the leaf, stem, and root biomass in alfalfa by 117.5% for leaf and stem and 97.8% for roots. The biomass increases in oat under nitrogen fertilization were smaller than that in alfalfa. The aboveground and belowground biomass under nitrogen fertilization in oat was increased by 19.1% and 9.6%, respectively, compared with the control experiment. Moreover, nitrogen fertilization also increased ¹³C abundance in alfalfa and oat by 54.36‰ and 28.6‰, respectively. Additionally, more carbon was allocated to roots in alfalfa under nitrogen fertilization than the control experiment (11.5% versus 5%). No significant differences in carbon allocation were observed in oat, despite slight increases in root carbon allocation in oat. 【Conclusion】Nitrogen fertilization increased soil organic carbon content of alfalfa and oat, increased alfalfa biomass yield, and stimulated carbon allocation to roots in alfalfa. Nevertheless, nitrogen fertilization showed no significant effects on both carbon fixation and allocation in oat.

Green Control of Major Cowpea Pests and Fusarium Wilt: Research and Practical Innovations

WU ShengYong, XIE Wen, SHI CaiHua, PAN HuiPeng, CHEN JunYu, KONG XiangYi

Scientia Agricultura Sinica. 2025, 58(21): 4357-4360. doi:10.3864/j.issn.0578-1752.2025.21.005

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GENETIC DIVERSITY OF MTCO1 IN DIFFERENT GEOGRAPHICAL POPULATIONS OF MEGALUROTHRIPS USITATUS

LIU XiaoXu, ZHONG ZeXin, QIU JiaRen, YANG ChunXiao, ZHANG YongJun, XIE Wen, ZHANG YouJun, PAN HuiPeng

Scientia Agricultura Sinica. 2025, 58(21): 4361-4371. doi:10.3864/j.issn.0578-1752.2025.21.006

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【Objective】Megalurothrips usitatus is a significant vegetable pest in China, causing substantial damage to legume crops as economic plants. The purpose of this study is to compare the genetic diversity of M. usitatus in different geographical populations and ascertain the genetic differentiation of M. usitatus, and to provide a theoretical basis for the precise control of M. usitatus. 【Method】A total of 18 geographical populations of M. usitatus were collected from 6 prefecture-level cities in Guangdong Province and 9 prefecture-level cities outside Guangdong Province. The mitochondrial DNA (mtDNA) cytochrome c oxidase subunit 1 (CO1) gene sequence was obtained based on PCR. The haplotype diversity (Hd), nucleotide diversity (π) of different geographical populations were analyzed, and Tajima’s D neutrality test based on the mtCO1 was performed to evaluate the genetic structure of M. usitatus populations across different geographical regions. The fixation index (F_ST) and gene flow (Nm) were calculated to evaluate the degree of genetic differentiation among populations. 【Result】A total of 111 mtCO1 gene sequences were detected in 18 geographical populations of M. usitatus. A total of 617 conserved sites and 24 variable sites were detected, accounting for 3.68% of the total sequence length. A total of 20 haplotypes (H1-H20) were detected, of which H4 was the dominant haplotype, accounting for 48.6% of the total sequence and distributed in 17 populations. The overall population of M. usitatus exhibited relatively high haplotype diversity (Hd=0.677) and nucleotide diversity (π=0.00425), but nucleotide sequence divergence among different genetic types was not pronounced. The nonsignificant Tajima’s D neutrality test indicated stable population size without marked expansion. Furthermore, the population fixation coefficient (F_ST=0.04973<0.05) and gene flow (Nm=9.55>1) suggested sufficient genetic exchange and low genetic differentiation among geographically distinct populations. The results of molecular variance analysis showed that the main factor causing the overall population variation was intra-population variation. 【Conclusion】The geographically distinct populations of M. usitatus exhibited relatively high genetic diversity, with frequent gene flow and minimal genetic differentiation among populations, suggesting a stable overall population size. These findings provide a theoretical basis for the integrated management of M. usitatus field populations in diverse regions.

Effects of Acoustic Interference on the Courtship Behavior of Liriomyza sativae

XING GuangTao, LÜ BaoQian, WU ShengYong, WU JianTao, ZHOU Ying, GE Jin, ZHANG QiKai

Scientia Agricultura Sinica. 2025, 58(21): 4372-4381. doi:10.3864/j.issn.0578-1752.2025.21.007

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【Objective】The objective of this study is to investigate the disruptive effect of acoustic stimuli on the courtship behavior of Liriomyza sativae and its application potential in green pest control. 【Method】Acoustic signals of varying frequencies were emitted by directional loudspeakers. Laser vibrometry coupled with behavioral video recording was employed to measure vibrational responses of cowpea leaves and courtship and mating parameters of L. sativae (latency, frequency, duration, mating success rate). The laboratory population control trial of L. sativae larvae was carried out by using 200 Hz acoustic frequency, and the population density change of L. sativae and effect on cowpea quality (soluble sugar, soluble protein and vitamin C content) were analyzed by setting sound source distance gradient with spatiotemporal dynamic monitoring in the field. 【Result】External acoustic stimulation induced differential leaf vibrational responses, disrupting L. sativae courtship. Cowpea leaves exhibited the strongest vibrational response within 200-300 Hz with peak vibration velocity of ±4 mm·s^-1. The 200 Hz frequency significantly prolonged male courtship latency ((15.92±3.99) min vs. control (5.24±1.63) min, P<0.05) and duration ((44.50±2.68) s vs. control (20.22±1.97) s, P<0.05), increased male courtship attempts ((13.20±2.58) times vs. control (5.40±1.21) times, P<0.05), and reduced mating success rate to 20% (control: 60%). Markov chain analysis confirmed greatest behavioral disruption in the mid-frequency band (200-300 Hz), specifically blocking the critical “male courtship→female response” transition pathway. Laboratory trials demonstrated 42.14% larval number reduction under 200 Hz acoustic treatment ((57.67±3.18) individuals vs. control (99.67±9.61) individuals, P<0.05). Field data indicated that with the increase of audio playback time, the mining density in near-source zones (0-25 m) decreased significantly, though efficacy diminished in far-source areas (25-70 m). No significant adverse effects occurred on cowpea quality parameters (soluble sugar, soluble protein and vitamin C content). 【Conclusion】The 200 Hz acoustic frequency disrupts L. sativae courtship signaling by maximizing cowpea leaf vibrations, effectively suppressing mating behavior and population growth without compromising crop quality. This technique provides an innovative perspective for developing environmentally-friendly pest control strategies.

Development and Application of Novel Physical Control Technologies Based on the Correlation Between Megalurothrips usitatus Outbreaks and Cowpea Flower Development Dynamics

SHI CaiHua, JIN Jie, HUA DengKe, HU JingRong, ZHANG YouJun, HUANG ShengLin, WU MingYue, KONG XiangYi, XIE Wen

Scientia Agricultura Sinica. 2025, 58(21): 4382-4392. doi:10.3864/j.issn.0578-1752.2025.21.008

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【Objective】Megalurothrips usitatus is a major biotic disaster for leguminous crops, particularly prone to damaging cowpeas. The study aims to clarify the relationship between field population dynamics of M. usitatus and cowpea flowers, and to develop new green control technologies. 【Method】The population dynamics of M. usitatus across different growth stages and plant organs of cowpeas were investigated, along with the influence of flower opening and shedding times on their activity patterns. The optimal timing for pesticide application was determined, and the abscission efficiency of five abscission methods (natural abscission, fan-induced abscission, lever-induced abscission, climbing net-induced abscission, and a combined method using both fan and lever) was compared. Additionally, the effectiveness of various flower-absorbing carriers (silver-gray plastic film, shading net, weed-proof fabric, hundred-knot net, and bare soil) was evaluated. Based on these findings, a novel integrated green control strategy, termed the “trinity, internal and external killing” approach, was developed and assessed for its efficacy in controlling M. usitatus, as well as its impact on cowpea quality and yield. 【Result】The population density of M. usitatus exhibited a trend of initial increase followed by a decline from the seedling stage to the end of flowering. The lowest population density was observed during the seedling stage, with a significant increase noted during the early flowering stage, peaking at full flowering stage. M. usitatus adults predominantly reside within flowers, while nymphs preferentially inhabit pods, followed by flowers and tender shoots. As flower buds open, the number of M. usitatus adults within flowers increases progressively. Flower closure typically occurs after 10: 00, encapsulating the insects within the floral structures. These closed flowers generally fall off gradually the following day; however, substantial numbers of M. usitatus remain concealed within the fallen flowers. Notably, flowers with receptacles harbor significantly higher numbers of M. usitatus compared to those without. A mass exodus of M. usitatus from the floral structures typically begins after 6: 00, at which point they migrate to new plant organs. For effective chemical control, application during the evening or early morning—when flowers are open—is most effective. Among the tested abscission methods, the combined use of a fan and lever demonstrated the highest efficiency in removing flowers. Additionally, the hundred-knot net significantly enhanced the effectiveness of flower collection. The implementation of the “trinity, internal and external killing” integrated green control strategy resulted in a 93.71% reduction in black head and black tail symptoms, a 96.87% reduction in wrapper tail, and a 91.55% reduction in overall malformation in cowpeas, accompanied by a 20.40% increase in yield. 【Conclusion】The “trinity, internal and external killing” technique not only challenges the conventional reliance on chemical pesticides, but also effectively suppresses M. usitatus populations, enhances cowpea quality and yield, and provides a valuable theoretical and practical framework for the development of green pest control strategies in other crops.

Control Effect of New Insect-Repellent Screen on Megalurothrips usitatus and Its Impact on the Microclimate in the Field

ZHAO HanYang, LI YiHong, XU ShuGuang, WU YueMin, WU ShengYong

Scientia Agricultura Sinica. 2025, 58(21): 4393-4404. doi:10.3864/j.issn.0578-1752.2025.21.009

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【Objective】The objective of this study is to evaluate the control effect of a new type of insect-repellent screen on Megalurothrips usitatus, and to clarify its effect on the microclimate environment in the field, so as to provide new technical support for the control of cowpea thrips. 【Method】The barrier effects of a new type of insect-repellent screen and conventional insect-proof screen on M. usitatus were investigated under laboratory and field conditions. Light intensity, ventilation, temperature, and humidity were measured using the illuminometer, anemometer, and thermo-hygrometer, respectively, under three treatments of insect-repellent screen, conventional insect-proof screen and open field. The effect of insect-repellent screen on the field microclimate was further analyzed based on the observed data. Gas chromatography-mass spectrometry (GS-MS) was employed to detect the types, contents of insect-repellent volatile compounds in the screen after two months of field application. 【Result】The laboratory results showed that the barrier rate of the new insect-repellent screen cage against M. usitatus adults generally exhibited a slowly declining trend within one month. The barrier rate of the insect-repellent screen was 52.81%-67.11%, significantly higher than that of the conventional insect-proof screen (46.32%-51.23%). Field results indicated that within one month of investigation during the flowering and pod-setting stage of cowpeas, the adult populations of M. usitatus in the insect-repellent screen, conventional insect-proof screen (both with a porosity of 69.40%), and open field were 4.3-7.4, 6.2-11.1 and 7.8-14.2 individuals/flower, respectively. The overall trend of population in the insect-repellent screen remained at a lower level, significantly lower than that in the conventional insect-proof screen and open-field cowpeas. No significant differences were observed among the three treatments (insect-repellent screen, conventional insect-proof screen, and open field) in terms of light intensity (11 900-73 800, 11 400-73 100, and 12 000- 73 900 lx, respectively), wind speed (0.16-1.38, 0.12-1.39, and 0.20-1.47 m·s^-1, respectively), daily mean temperature (18.73-25.75, 19.50-25.62, and 19.51-26.00 ℃, respectively), and daily mean relative humidity (65.00%-72.15%, 66.32%-73.78%, and 62.10%-69.66%, respectively). During a single day at the full blooming stage of cowpea, the temperature and relative humidity under all three treatments exhibited an inverted “U”-shaped and a positive “U”-shaped trend, respectively. Specifically, the temperature reached the highest value in the afternoon (13: 00-16: 00), while the relative humidity dropped to the lowest value in the afternoon (14: 00-15: 00). GC-MS analysis revealed that the volatile components of the insect-repellent screen contained compounds with high similarity to menthol and limonene, with similarity scores of 79.50% and 80.00%, and the volatilization rates after two months of application were 98.47% and 92.86%, respectively. 【Conclusion】The barrier effect of the new insect-repellent screen against M. usitatus is better than conventional insect-proof screen, showing dual effects of physical barrier and biological repellence, and has negligible impact on the field microclimate environment. The development and application of the new insect-repellent screen provides a novel approach for integrated control of cowpea thrips.

Effects of Different Organic Fertilizers on the Control of Cowpea Wilt by Bacillus velezensis SD13

XIE HaiPeng, LIN JunXu, LIU Yong, MAI XianJun, LUO Feng, WANG XueWu, XIE Wen, LI ShaoKa, KONG XiangYi, WU XiaoYan

Scientia Agricultura Sinica. 2025, 58(21): 4405-4420. doi:10.3864/j.issn.0578-1752.2025.21.010

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【Objective】The objective of this study is to investigate the biological control effect of Bacillus velezensis SD13 against cowpea wilt when combined with different organic fertilizers, and to provide a theoretical basis for the application of biological control technology of cowpea wilt. 【Method】A pot experiment was conducted to evaluate the effects of applying 10% sheep manure, 10% vermicompost, and a 5% sheep manure+5% vermicompost mixture as fertilizer carriers on the efficacy of B. velezensis SD13 in controlling cowpea wilt. The experiment included eight treatments: blank control (CK), pathogen control (F), pathogen+SD13 (FB), pathogen+10% vermicompost (FW), pathogen+SD13+10% vermicompost (FBW), pathogen+10% sheep manure (FS), pathogen+SD13+10% sheep manure (FBS), and pathogen+SD13+5% vermicompost+5% sheep manure (FBWS). Each treatment was replicated three times, with three pots per replicate. After 45 d of cultivation in a greenhouse, the incidence rate of cowpea wilt, plant biomass (above-ground and below-ground dry weight), and defense enzyme activity (catalase) were measured for each treatment. Rhizosphere soil samples were collected, and the structure, diversity, and key microbial taxa of the soil bacterial community were analyzed using 16S rRNA high-throughput sequencing. 【Result】The control effect of sheep manure, vermicompost and mixed organic fertilizer combined with B. velezensis SD13 on cowpea wilt was more than 75%. The combination of sheep manure and SD13 significantly increased soil total nitrogen by 30%, with the dominant soil bacteria being Firmicutes, which showed a negative correlation with other bacterial communities. The combination of vermicompost and SD13 significantly increased the above-ground dry weight of diseased cowpea plants by 29% and the below-ground dry weight by 30%, enhanced the activity of catalase in cowpea by 10%, and significantly increased the richness and diversity of soil bacteria, restoring the community structure to a healthy level. The combination of mixed organic fertilizers and SD13 significantly increased the below-ground dry weight of diseased cowpea plants by 155%, with the soil community structure being influenced by the microbial communities of both vermicompost and sheep manure. 【Conclusion】The type of organic fertilizer affects the biocontrol effect of B. velezensis SD13 against cowpea wilt in the soil. Vermicompost is more suitable for combination with SD13 than sheep manure. This combination not only ensures high biocontrol effect, but also further enhances cowpea biomass, catalase activity, and soil bacterial richness and diversity. It increases the abundance of beneficial bacterial communities and restores the soil bacterial community structure and composition to levels associated with healthy cowpea soil, making it a suitable organic fertilizer for the application of SD13 in controlling cowpea wilt in soil.

Comparison of the Predation Ability of Four Species of Orius Against Megalurothrips usitatus, Tetranychus urticae and Aphis craccivora

DAI XiaoYan, ZHAO JinFeng, WANG RuiJuan, SU Long, WANG Yu, CHENG WenHua, XU QianQian, ZHAO Shan, ZHENG Li, LIU Yan, ZHAI YiFan

Scientia Agricultura Sinica. 2025, 58(21): 4421-4428. doi:10.3864/j.issn.0578-1752.2025.21.011

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【Objective】The purpose of this study is to identify the dominant species of commercial Orius species (O. nagaii, O. sauteri, O. minutus and O. strigicollis) against the main pest of cowpea Megalurothrips usitatus, Tetranychus urticae and Aphis craccivora, and to provide a scientific basis for field demonstration and commercial production of Orius species. 【Method】The single-headed female adult of a single species of Orius was used to test the predation, functional responses and search effects of different densities of M. usitatus (10, 20, 30, 40 /tube), T. urticae (5, 10, 15, 20 /dish) and A. craccivora young nymphs (5, 10, 15, 20 /dish) under indoor conditions. Holling II equation was used to analyze the predation ability and biological control potential of four Orius species on three pests. 【Result】The daily predation of four Orius species increased with the increase of the density of pests (M. usitatus, T. urticae and A. craccivora). The predatory functional response of the four Orius species conformed to the Holling II model equation, and the search effect of the four Orius species gradually decreased with the increase of prey density. While hunting M. usitatus, the transient attack rate (a′) (0.861), predation ability (a′/Th) (661.932), maximum daily predation amount (1/Th) (769.231) and processing time of single prey (Th) (0.001 d) of O. nagaii were higher than those of the other three species. While hunting T. urticae, the processing time of single prey (0.004 d), predation ability (260.617) and maximum daily predation amount (285.714) of O. nagaii were also higher than those of the other three species. There was no significant difference in the predation ability of the four Orius species on A. craccivora. 【Conclusion】At room temperature, compared with the other three Orius species, O. nagaii has better predation ability on M. usitatus and T. urticae, but the four species have little difference in predation on A. craccivora.

Effects of Different Host Plants on Digestive Enzyme Activity and Nutrient Content in Megalurothrips usitatus

WANG HuanTing, HUANG LiFei, CAO XueMei, GONG Rui, SU GuoLian, ZHENG XiaLin, WU MingYue, YANG Lang

Scientia Agricultura Sinica. 2025, 58(21): 4429-4438. doi:10.3864/j.issn.0578-1752.2025.21.012

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【Background】Megalurothrips usitatus can successfully develop and reproduce on host plants such as cowpea (Vigna unguiculata), green bean (Phaseolus vulgaris), and white bean (Vigna cylindrica), with cowpea being the most suitable host. In contrast, its development and reproductive capacity are significantly limited on wax gourd (Benincasa hispida). 【Objective】The objective of this study is to determine the digestive enzyme activity and nutrient content in M. usitatus after feeding on different hosts, and to explore the adaptation mechanism of M. usitatus to different host plants. 【Method】F₁ adults of M. usitatus reared on cowpeas for 3 d were transferred to petri dishes containing cowpea, green bean, white bean, or wax gourd, with four replicates per treatment. Sampling was conducted at 0, 3, 6, 12, 24, 48, and 72 h after feeding. The dynamic changes in the activities of key digestive enzymes (including α-amylase, trehalase, trypsin, and chymotrypsin) were systematically measured. Concurrently, changes in the content of major nutrients, such as glycogen, soluble protein, and fat, were analyzed. 【Result】Significant changes were observed in the activities of digestive enzymes and the content of nutrients in M. usitatus after feeding on different host plants. The activities of various digestive enzymes in M. usitatus fed on cowpea, green bean and white bean were generally higher than those fed on wax gourd. Specifically, the α-amylase activity of M. usitatus fed on cowpea was the highest (1.33 mg·min^-1·mg^-1 protein), along with high trehalase activity (469.80 nmol·min^-1·mg^-1 protein). Trypsin and chymotrypsin activities reached 20.42 and 24.86 U·mg^-1 protein, respectively. M. usitatus fed on green bean had the highest α-amylase activity (1.49 mg·min^-1·mg^-1 protein), but the activities of trehalase (304.81 nmol·min^-1·mg^-1 protein) and two proteases were slightly lower than those of cowpea group. Trehalase activity in white bean group was significantly decreased (175.61 nmol·min^-1·mg^-1 protein), while trypsin activity was increased (21.15 U·mg^-1 protein). In contrast, all enzyme activities of M. usitatus fed on wax gourd were at the lowest level, the trehalase activity was only 152.89 nmol·min^-1·mg^-1 protein. Regarding nutrient content, glycogen content (1.61 mg·mg^-1 protein) and fat content (5.54 μg per individual) were highest in M. usitatus fed on cowpea, while the lowest values were observed in the wax gourd group (glycogen content 0.79 mg·mg^-1 protein; fat content 3.37 μg per individual). 【Conclusion】Different host plants significantly influenced the digestive enzyme activities and nutrient contents of M. usitatus. M. usitatus rapidly adjusted these physiological parameters after feeding on cowpea, green bean, and white bean, but exhibited slower adaptation on wax gourd. These findings indicate that M. usitatus has higher adaptability to cowpea, green bean, and white bean, whereas its adaptability to wax gourd is comparatively poorer.

Analysis of Annual Population Dynamics of Megalurothrips usitatus on Cowpea in Hainan

HUANG ShengLin, XU YiBo, KONG XiangYi, SHI CaiHua, JIAO XiaoGuo, ZHANG YouJun, WU MingYue, XIE Wen

Scientia Agricultura Sinica. 2025, 58(21): 4439-4450. doi:10.3864/j.issn.0578-1752.2025.21.013

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【Objective】Megalurothrips usitatus is a persistent pest in major cowpea production areas in southern China such as Hainan. Its continuous infestation has led to a reduction in cowpea yields and quality, which has become a key biotic factor constraining the sustainable development of the industry. This study aims to provide theoretical support for cowpea thrips disaster early warning and control decision-making in tropical and subtropical cowpea-growing regions of China by systematically monitoring the annual pattern of M. usitatus in the main cowpea production area of Sanya, Hainan. 【Method】The population size of M. usitatus on host plants including Fabaceae, Cucurbitaceae, and Poaceae in Yazhou District, Sanya City, Hainan Province was investigated using the sticky trap method: August 2021 to July 2022 (the first year) and October 2023 to October 2024 (the second year). Based on the survey data, population dynamics of M. usitatus were analyzed through graphical visualization. Furthermore, by comparing population dynamic changes of M. usitatus across different years and seasons, the occurrence patterns and host transition behavior of M. usitatus on cowpea and other host plants were further analyzed. 【Result】(1) M. usitatus could cause damage on cowpeas throughout the year, and at all stages of growth. In the first year, the occurrence peaks of M. usitatus on cowpea and non-cowpea host plants occurred in winter and spring (December and January of the following year), which coincided with the peak planting period of cowpeas. In the second year, the occurrence peaks of M. usitatus on cowpeas occurred in summer and autumn (June and September), corresponding to the sporadic planting period of cowpeas. Its occurrence peaks on non-cowpea host plants occurred from early March to late April, coinciding with the large-scale crop termination period of cowpeas (pulling up seedlings after harvest). (2) M. usitatus exhibited a wide range of hosts, with significantly higher population densities on leguminous host plants (such as cowpeas, green beans, peanuts, and sesbania) than on non-leguminous host plants. (3) Studies on thrips host transfer patterns found that M. usitatus primarily infested cowpeas during the concentrated planting period of cowpea. Once cowpeas undergo large-scale crop termination (pulling up seedlings after harvest), it was likely to be transferred to the surrounding host plant plots such as eggplant, sesbania to conceal its infestation. 【Conclusion】In the Sanya region, the population density of M. usitatus on Fabaceae host plants was significantly higher than that on non-Fabaceae host plants. This pest can occur year-round in fields of cowpea, eggplant, and weeds, and exhibits the characteristic of shifting hosts to cause damage. The population dynamics of M. usitatus on cowpea and other plants show inter-annual and seasonal variations. Based on the annual population dynamic patterns of thrips in cowpea fields, it is recommended to conduct field sanitation before cowpea planting, including the removal of surrounding Fabaceae crops and weeds.

Investigation on Species Composition and Damage Dynamics of Cowpea Pests in Hainan Winter-Spring Cropping System (2020-2025)

WU MingYue, FU QiuMei, HUANG ShengLin, LUO Feng, LIU Yong, SHI CaiHua, ZHANG YouJun, KONG XiangYi, XIE Wen

Scientia Agricultura Sinica. 2025, 58(21): 4451-4466. doi:10.3864/j.issn.0578-1752.2025.21.014

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【Objective】This study aimed to identify the species composition and population dynamics of major pests in the main cowpea-producing areas of Hainan, and to evaluate the effects of different planting patterns and cultivation managements on the pest community structure, thus providing a theoretical basis for developing precise control strategies. 【Method】Systematic investigations were conducted on the pest species and dominant pests on cowpeas during the winter-spring seasons from 2020 to 2025 in Hainan cowpea planting areas, using plant visual inspection and colored sticky trap methods. The population dynamics of pests were compared under different management practices (non-pesticide-treated and conventionally pesticide-treated) and different cultivation modes (open-field, semi-enclosed, and fully-enclosed). 【Result】(1) A total of 16 pest species infesting cowpea were identified in Hainan Province, which belonged to 6 orders, 9 families. In non-pesticide-treated cowpea fields, a higher diversity of pests was observed (6 orders, 9 families, 16 species), with the dominant species being Megalurothrips usitatus, Frankliniella intonsa, Liriomyza trifolii, Spodoptera exigua, and Aphis craccivora. In contrast, conventional pesticide-treated fields exhibited reduced pest diversity (4 orders, 5 families, 10 species), with thrips and leafminers remaining as the predominant pests. (2) Regardless of pesticide application, thrips and leafminers were present throughout the entire cowpea growth cycle. Thrips populations were significantly higher, showing low numbers at the seedling stage, a continuous increase during vine elongation, and multiple population peaks during the flowering stage coinciding with bloom. Leafminers were abundant from January to March in 2021, but caused only sporadic damage during 2024-2025. (3) Under both open-field and semi-enclosed planting modes, the pest species composition was similar, primarily consisting of thrips and leafminers, with M. usitatus as the dominant thrips species and L. trifolii as the dominant leafminer species. Thrips outbreaks generally occurred around January of the following year, while spider mite outbreaks were occasionally observed in semi-enclosed planting mode in certain years. Compared with open-field and semi-enclosed modes, the species of pests in the fully-enclosed planting mode were relatively few, and only thrips accounted for approximately 66% of the total pest population. During the later growth stages under higher temperatures (i.e., January to March of the following year), populations of Bemisia tabaci and Tetranychus urticae increased significantly. In conclusion, thrips caused the most severe damage throughout the cowpea growth cycle, significantly affecting both yield and quality. In some years, leafminers and T. urticae also inflicted considerable damage, while other pests were less detrimental. 【Conclusion】There are many kinds of pests in cowpea in Hainan, among which thrips are the most serious. The occurrence of pests in open-field planting mode is more serious, and the damage of pests in net-enclosed field is relatively light. The net-enclosed mode can effectively block some pests, but the fully-enclosed planting mode is affected by the micro-environment such as high temperature and high humidity, and the probability of outbreak of B. tabaci, T. urticae and other pests in the late growth stage of cowpea is large.

Exploration of Salt-Alkali and Drought Resistant Genes for Alfalfa Breeding

LI MingNa, LONG RuiCai, YANY QingChuan

Scientia Agricultura Sinica. 2025, 58(21): 4467-4470. doi:10.3864/j.issn.0578-1752.2025.21.015

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The Breeding History, Current Status and Prospects of Alfalfa

ZHANG Fan, YANG QingChuan

Scientia Agricultura Sinica. 2025, 58(21): 4471-4481. doi:10.3864/j.issn.0578-1752.2025.21.016

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Alfalfa (Medicago sativa subsp. sativa L.), as the world's most important leguminous forage, plays an indispensable role in modern livestock farming and ecological systems due to its high yield, superior quality, and excellent adaptability. However, China's alfalfa industry has long faced a "bottleneck" in its seed industry, primarily characterized by the insufficient innovation capacity in germplasm resources and a low supply rate of elite proprietary varieties. The root of this challenge lies not only in the relatively late start and underdeveloped technical framework of alfalfa breeding in China, but also in the complex genetic characters obstacles inherent to alfalfa itself—namely, its autotetraploid nature with high heterozygosity and a self-incompatibility mechanism. Therefore, a systematic review of alfalfa's breeding history, current research status, and a forward-looking perspective on biotechnological breeding is of significant referential value for accelerating genetic improvement in China. The history of alfalfa domestication and spread was extensive. Originating from the Transcaucasus region, it was introduced to China via the "Silk Road" and subsequently spread worldwide. The vast enrichment of its genetic diversity benefited historically from the introduction of germplasm from different geographical origins and inter/intraspecific hybridization, particularly through genetic introgression from the stress-tolerant subspecies, M. sativa subsp. falcata. This laid a solid genetic foundation for breeding varieties adapted to diverse ecological niches. Developed countries, such as the United States, have established a comprehensive system for alfalfa breeding that covered germplasm collection, evaluation, and new variety development, with a history spanning over a century. Nevertheless, even in the United States, yield improvement plateaued after 1990, revealing the potential limitations of conventional breeding strategies. Although China has made considerable progress in alfalfa breeding, successfully developing adapted varieties, such as the "Zhongmu" and "Gannong" series, the overall effort still faced significant challenges. Firstly, the number of registered varieties is relatively less (128 as of 2024, far fewer than the 1 738 in the U.S.), which severely limits the ability to select optimal varieties for China's diverse ecological environments. Secondly, conventional breeding strategies themselves have constrained the effective utilization of heterosis. Historically, breeders often employed a strategy of population mixing and recurrent selection with varieties from different geographical origins. However, this approach aggregated favorable genes to some extent, and it also led to a homogenization of the genetic backgrounds among different breeding populations, thereby diminishing the potential for generating strong heterosis through subsequent hybridization. Consequently, breaking through the limitations of traditional methods by introducing efficient modern biotechnologies has become an inevitable choice for contemporary alfalfa breeding. In recent years, modern biotechnology centered on genomics has presented unprecedented opportunities to overcome the challenges in alfalfa breeding. China has achieved notable success in foundational genomics research, completing tasks ranging from genetic analysis of germplasm resources and genetic mapping to the key molecular markers and the development of elite germplasm. These genomic resources have made it possible to dissect the genetic regulatory networks of complex traits, such as yield, quality, and stress resistance at the molecular level. Looking ahead, alfalfa breeding in China should adopt a strategy that integrates conventional breeding with modern biotechnology, transitioning from source innovation to the "Breeding 4.0" era of precision design. The integration of modern biotechnologies—including genomics, marker-assisted selection, and gene editing—to conduct precise and efficient molecular design breeding is the core pathway to resolving the "bottleneck" in China's alfalfa seed industry and achieving fast development.

Physiological Effects of 5-AzaC on Alleviating Salt‑Alkali Stress in Alfalfa and Its Impact on the Expression of DNA Methylation Enzyme Genes

GAO Rong, LI HengYu, CHEN LiJuan, MA HuiLing

Scientia Agricultura Sinica. 2025, 58(21): 4482-4496. doi:10.3864/j.issn.0578-1752.2025.21.017

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【Background】Soil salinization is one of the major ecological challenges threatening global agricultural production, severely restricting crop growth, yield formation, and quality improvement. Alfalfa (Medicago sativa), as an important perennial legume forage, is particularly constrained by salt-alkali stress, while the epigenetic regulatory mechanisms underlying its response remain largely unknown. DNA methylation, as a key epigenetic modification, plays an essential role in plant adaptation to abiotic stresses. 【Objective】This study aimed to systematically identify DNA methylation-related gene families in alfalfa, characterize their expression patterns under salt-alkali stress, and further explore the role of DNA methylation in salt-alkali tolerance by applying the DNA methylation inhibitor 5-azacytidine (5-AzaC), thereby providing the theoretical insights for the genetic improvement of salt-alkali-tolerant alfalfa. 【Method】Based on the reference genome of alfalfa, DNA methyltransferase and demethylase genes were identified genome-wide, and their functions were inferred through phylogenetic analysis and conserved domain annotation. RT-qPCR was employed to analyze the expression patterns of these genes under salt-alkali stress. Using the cultivar Gannong No. 3 as plant material, a hydroponic salt-alkali stress system was established. Different concentrations of 5-AzaC were applied as pretreatments, and the optimal concentration was selected for subsequent assays. Plant growth, physiological, and photosynthetic parameters were then measured to evaluate the regulatory role of 5-AzaC in alfalfa salt-alkali tolerance. 【Result】A total of 13 DNA methyltransferase genes and 4 DNA demethylase genes were identified in alfalfa, all of which were localized to the nucleus with complete conserved domains. Expression analysis revealed that MsCMT4, MsCMT6, MsCMT8, and MsDML2 were significantly upregulated under salt-alkali stress, indicating the involvement of both methylation and demethylation processes in stress responses. Physiological analyses showed that 100 μmol·L^-1 5-AzaC significantly alleviated growth inhibition caused by salt-alkali stress, so plant height, fresh weight, and dry weight increased by 12.62%, 23.50%, and 18.67%, respectively, compared with the control. Regarding chlorophyll metabolism, 5-AzaC suppressed the expression of chlorophyll degradation-related genes (PAO, CAO, NYC), thereby delaying pigment degradation. Photosynthetic analysis indicated that 5-AzaC treatment markedly increased net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr), as well as the quantum efficiency of photosystem II (YII) and photochemical quenching (qP), suggesting enhanced stability and efficiency of the photosynthetic system. In terms of osmotic adjustment, 5-AzaC promoted soluble sugar accumulation (+37.21%) but had no significant effect on soluble protein. Reactive oxygen species (ROS) measurements showed that 5-AzaC reduced H₂O₂ and O₂^-·levels by 22.8% and 35.8%, respectively, while superoxide dismutase (SOD) and catalase (CAT) activities increased by 13.58% and 21.82%, respectively, indicating that 5-AzaC enhanced antioxidant capacity and alleviated oxidative damage. 【Conclusion】This study systematically characterized DNA methylation-related gene families in alfalfa and their responses to salt-alkali stress, revealing the pivotal role of DNA methylation in shaping salt-alkali tolerance. Exogenous application of 5-AzaC improved alfalfa tolerance by maintaining photosystem stability, enhancing photosynthetic efficiency, promoting osmolyte accumulation, and strengthening ROS scavenging capacity. These findings provided the new experimental evidence for understanding the epigenetic mechanisms of forage adaptation to abiotic stress and offer theoretical guidance for the improvement and utilization of salt-alkali-tolerant alfalfa germplasm.

Cloning and Salt Tolerance Function Analysis of MsKTI3 Gene in Alfalfa

LÜ HuanHuan, LI RuYue, LIU QingSong, XU Lei, XU YanRan, YU HaoJie, GUO ChangHong, LONG RuiCai

Scientia Agricultura Sinica. 2025, 58(21): 4497-4511. doi:10.3864/j.issn.0578-1752.2025.21.018

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【Objective】Salt stress can severely damage plant cells, inhibit plant growth and development, and consequently lead to a substantial reduction in yield. Kunitz trypsin inhibitor (KTI), a representative type of serine protease inhibitors, is primarily involved in regulating physiological processes in plants, such as growth and development, pest and disease resistance, and responses to abiotic stress. Exploring and analyzing the molecular mechanisms underlying the regulation of salt stress by the alfalfa KTI would contribute to the provision of novel candidate genes for molecular breeding of salt-tolerant alfalfa. 【Method】In this study, a salt stress induced KTI was cloned from Medicago sativa ‘Zhongmu No. 4’, which was named MsKTI3. Through bioinformatics methods, the structural characteristics of the MsKTI3 and its encoded protein were deeply analyzed. Sequence alignment and evolutionary analysis were carried out with homologous genes of other species. The real-time fluorescence quantitative PCR (RT-qPCR) method was used to analyze the expression patterns of the MsKTI3 in different tissues and under different stress conditions. With the aid of the tobacco transient expression system, the subcellular localization of the MsKTI3 protein was analyzed. An MsKTI3 overexpression vector was constructed, and the MsKTI3 overexpressing lines of Arabidopsis thaliana were successfully obtained by the Agrobacterium tumefaciens-mediated method. Meanwhile, the Medicago sativa plants with MsKTI3 overexpression in roots were obtained by the Agrobacterium rhizogenes-mediated method. Phenotypic analysis and physiological index determination of the related lines were carried out under salt stress conditions. 【Result】Bioinformatics analysis indicated that the coding sequence (CDS) of the MsKTI3 gene was 627 bp, encoding 208 amino acids. The relative molecular weight was 23 220.81 Da, and the theoretical isoelectric point was 8.57. Phylogenetic tree analysis revealed that the amino acid sequence of MsKTI3 shared a high homology with that of MtKTI3 of Medicago truncatula, reaching 97%. RT-qPCR was employed to analyze the expression pattern of the MsKTI3. The results demonstrated that the expression level of the MsKTI3 was the highest in roots. Moreover, during the initial stage of NaCl (200 mmol·L^-1) and ABA (150 μmol·L^-1) stress, the expression level generally exhibited an up-regulation trend. Subcellular localization results showed that the MsKTI3 protein was located in the plasma membrane. Twelve overexpressing Arabidopsis lines were generated via the Agrobacterium-mediated method. Under salt-stress conditions, the germination rate of the Arabidopsis lines overexpressing the MsKTI3 was higher than that of the wild type, and the damage degree of the overexpressing seedlings was lower than that of the wild type. The relative electrolyte leakage (IEL) and malondialdehyde (MDA) content of the overexpressing plants were significantly lower than those of the wild type (P<0.05), while the chlorophyll (Chl) content and catalase (CAT) activity were significantly higher than those of the wild type (P<0.05). MsKTI3 was transferred into the roots of alfalfa using Agrobacterium rhizogenes. Phenotypic analysis indicated that overexpression of MsKTI3 in roots enhanced the salt tolerance of alfalfa. Additionally, the CAT activity of the overexpressing plant roots was higher than that of the control plants. 【Conclusion】The MsKTI3 gene played a positive regulatory role in responding to salt stress, and overexpression of MsKTI3 could improve the salt tolerance of plant.

Differential Proteomic Analysis of Alfalfa Seedlings Under Salt- Alkaline Stress

YANG YongNian, ZENG XiangCui, LIU QingSong, LI RuYue, LONG RuiCai, CHEN Lin, WANG Xue, HE Fei, KANG JunMei, LI MingNa

Scientia Agricultura Sinica. 2025, 58(21): 4512-4527. doi:10.3864/j.issn.0578-1752.2025.21.019

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【Objective】Soil salinization and alkalization are significant limiting factors for the growth and yield of alfalfa (Medicago sativa L.). To elucidate the molecular mechanisms underlying the response of alfalfa seedlings to salt or alkali stress, this study employed proteomic analysis to identify key differentially expressed proteins and their associated metabolic pathways under saline-alkali stress, so as to provide a theoretical foundation for elucidating the mechanisms of saline-alkali tolerance. 【Method】This study used Zhongmu No. 4 alfalfa as the experimental material. Seeds were subjected to salt stress (30 mmol·L^-1 NaCl and 30 mmol·L^-1 Na₂SO₄) and alkali stress (10 mmol·L^-1 Na₂CO₃ and 10 mmol·L^-1 NaHCO₃). TMT labeling combined with liquid chromatography- mass spectrometry (LC-MS/MS) was employed to analyze the proteomic changes in alfalfa seedlings after stress treatments. 【Result】In total, 6 829 proteins were identified, including 489 differentially expressed proteins (DEPs) (274 up-regulated and 215 down-regulated) under alkali stress and 376 DEPs (218 up-regulated and 158 down-regulated) under salt stress. GO annotation analysis revealed that the DEPs were primarily associated with biological processes, such as cellular metabolism, organic metabolism, and stress response, as well as cellular components including intracellular organelles, cytoplasm, and membranes. KEGG pathway enrichment analysis indicated that under alkali stress, DEPs were significantly enriched in pathways, such as photosynthesis, phenylpropanoid biosynthesis, and isoflavonoid biosynthesis, while under salt stress, they were mainly enriched in photosynthesis, isoflavonoid biosynthesis, and glutathione metabolism. Further analysis of the enriched proteins demonstrated that alfalfa seedlings enhanced their resistance to salt-alkali stress by up-regulating key proteins in the isoflavonoid biosynthesis pathway (HI4OMT and CYP81E). The upregulation of key enzymes such as HI4OMT and CYP81E significantly enhanced the accumulation of isoflavonoids, which facilitated reactive oxygen species scavenging and maintained cellular homeostasis in plants. Furthermore, under alkaline stress, key enzymes (PAL, CAD, and COMT) in the phenylpropanoid biosynthesis pathway were markedly upregulated, promoting the synthesis of lignin and flavonoids. This process strengthened cell wall integrity and antioxidant capacity, enabling plants to adapt to high-pH environments and cope with alkaline stress. Under salt stress, alfalfa seedlings upregulated critical enzymes (PRDX6, GPX, and GST) in the glutathione metabolism pathway, maintaining redox homeostasis and enhancing ROS scavenging, thereby improving salt stress tolerance. 【Conclusion】In summary, this proteomic study elucidated key proteins and metabolic pathways involved in the response of alfalfa seedlings to salt-alkali stress, providing a theoretical foundation for understanding the molecular mechanisms of salt-alkali tolerance in alfalfa. Simultaneously, this study provided potential candidate proteins and metabolic pathways for breeding salt-alkali tolerant alfalfa. Subsequent research could further validate the functions of these key proteins and employ advanced biotechnological breeding approaches to develop new alfalfa varieties with enhanced salt-alkali tolerance, thereby addressing the challenges posed by soil salinization-alkalization to alfalfa production.

Identification of Key Drought-Responsive Genes in Upright Medicago ruthenica Sojak cv. Zhilixing Based on Transcriptome Sequencing and WGCNA

MU YingTong, LU JingShi, ZHANG YuTong, SHI FengLing

Scientia Agricultura Sinica. 2025, 58(21): 4528-4543. doi:10.3864/j.issn.0578-1752.2025.21.020

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【Background】Drought stress is one of the major abiotic factors limiting global agricultural productivity. Systematic elucidation of transcriptional regulatory mechanisms during drought and rehydration processes is crucial for improving crop drought tolerance and advancing molecular breeding strategies. Medicago ruthenica Sojak cv. Zhilixing, a perennial leguminous forage species, exhibits strong ecological adaptability and drought resistance.【Objective】This study aimed to identify key regulatory modules and core functional genes involved in drought and rehydration responses through transcriptome analysis and co-expression network construction, thereby revealing the underlying molecular mechanisms. 【Method】Four treatment stages were established, including normal irrigation (Group A), mid-term drought stress (Group B), late-stage drought stress (Group C), and post-rehydration (Group D), to simulate the transcriptional response of M. ruthenica under progressive drought and recovery conditions. High-throughput RNA sequencing was performed to obtain gene expression profiles, followed by weighted gene co-expression network analysis (WGCNA) to construct expression modules. Principal component analysis (PCA) and KEGG pathway enrichment were conducted to assess expression variation and functional clustering. Modules and hub genes closely associated with drought tolerance were identified, and six representative genes from the MEmagenta and MEdarkgreen modules were selected for qRT-PCR validation to assess data consistency and biological relevance. 【Result】PCA revealed clear separation of samples across PC1 and PC2 dimensions, indicating stage-specific effects of drought and rehydration treatments on gene expression patterns. Differential expression analysis showed the greatest number of up- and down-regulated genes during mid-stage drought (Group B), suggesting rapid activation of stress- responsive mechanisms. In the late-stage drought (Group C), fewer differentially expressed genes were observed, while pathways related to fatty acid degradation and carbohydrate metabolism were significantly enriched, suggesting a shift toward homeostatic regulation. During rehydration (Group D), most gene expression levels gradually recovered, although some signaling and defense pathways remained active, indicating ongoing adaptive modulation.WGCNA identified four modules significantly correlated with specific treatments (|r| > 0.6). The MEdarkgreen module (r = 0.93), highly expressed in Group C, was enriched in MAPK signaling, endoplasmic reticulum (ER) stress response, lipid metabolism, and flavonoid biosynthesis. Hub genes BZIP17 and IRE1B were implicated in ER stress signaling, protein folding regulation, and transcriptional reprogramming, indicating their key roles in prolonged drought adaptation. The MEmagenta module (r = 0.82), highly expressed under normal conditions (Group A), was enriched in ABA and JA signaling pathways, as well as flavonoid metabolism. Its core genes, ABF4 and MYC2, are involved in stomatal regulation and secondary metabolic control, functioning as crucial regulators during the early drought response. Additional genes such as NAC072 and CLPD contribute to chloroplast protein stability and reactive oxygen species scavenging, supporting their multifaceted roles in stress mitigation. KEGG enrichment results were highly consistent with module functions, confirming the reliability of functional annotations. qRT-PCR analysis showed that all six selected genes were significantly upregulated during mid and late drought stages (Groups B and C) and downregulated upon rehydration (Group D), mirroring RNA-seq expression patterns and validating the biological relevance of the identified modules and genes. 【Conclusion】This study reveals dynamic transcriptional regulation patterns in M. ruthenica under drought stress and rehydration. Two key modules, MEmagenta and MEdarkgreen, were identified as strongly associated with drought adaptation, encompassing hub genes such as ABF4, MYC2, BZIP17, and IRE1B. These genes play central roles in signal transduction, metabolic adjustment, and stress response, representing core components of the molecular mechanisms underlying drought adaptation in M. ruthenica. The findings provide a theoretical foundation and candidate targets for elucidating drought-responsive pathways and advancing molecular breeding of drought-resilient forage crops.

Phosphate Transporter MsPT5 Regulates Phosphate Uptake and Utilization in Alfalfa

HUANG HongMei, WANG SiQi, YANG QingChuan, GUO ChangHong, WANG Xue

Scientia Agricultura Sinica. 2025, 58(21): 4544-4556. doi:10.3864/j.issn.0578-1752.2025.21.021

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【Objective】This study aimed to explore the function of MsPT5, a phosphate transporter in alfalfa, so as to provide the theoretical basis and genetic resources for analyzing the response of alfalfa to low phosphate stress and cultivating phosphate nutrition efficient alfalfa. 【Method】Alfalfa was subjected to both full phosphate and low phosphate stress treatments, and root systems were selected for transcriptome sequencing. Weighted gene correlation network analysis was used to screen for the key gene MsPT5 in response to low phosphate stress in alfalfa; the experiment was carried out to clone the CDS sequence of MsPT5 gene, and the transmembrane domain of MsPT5 protein was analyzed using DNAMAN and TMHMM online websites; Super1300: GFP plant expression vector was used as the backbone, and the overexpression vector Super1300:MsPT5-GFP was constructed through enzyme digestion and homologous recombination; the recombinant vector Super1300:MsPT5-GFP was transformed into Agrobacterium GV3101 using the freeze-thaw method, and Agrobacterium was injected into tobacco for subcellular localization observation; Super1300:MsPT5-GFP was transferred into Arabidopsis through Agrobacterium mediated inflorescence infection, and homozygous strains were screened for inorganic phosphate content determination, biomass statistics, and arsenate phenotype detection; the tissue culture method was employed to obtain MsPT5 transgenic alfalfa, and the positive plants were screened for biomass statistics, phosphate content, and protein content determination. 【Result】MsPT5 was a member of the PHT1 family, with 12 transmembrane domains located on the cytoplasmic membrane; the physiological indicators of Arabidopsis thaliana showed that the inorganic phosphate content of MsPT5 overexpressing strains was 23.02 and 22.30 nmol·mg^-1, respectively, while the inorganic phosphate content of the wild-type control was 19.97 nmol·mg^-1. The biomass of MsPT5 overexpressing strains were 0.047 g/plant and 0.054 g/plant, respectively, while the biomass of the wild-type control was 0.026 g/plant. Overexpression of MsPT5 could increase the inorganic phosphate content and biomass of Arabidopsis; the results of arsenate phenotype detection showed that the MsPT5 overexpression material exhibited a significant arsenic toxicity phenotype; the physiological indicators of alfalfa showed that the biomass of MsPT5 transgenic alfalfa was 19.39 g/plant, 18.62 g/plant, and 16.65 g/plant, respectively, while the biomass of the wild-type control was 15.14 g/plant. The phosphate content of MsPT5 transgenic alfalfa was 0.37%, 0.39%, and 0.38%, respectively, while the wild-type control had a phosphate content of 0.30%. The protein content of MsPT5 transgenic alfalfa was 21.37%, 21.54%, and 19.91%, respectively, while the protein content of the wild-type control was 18.04%. The biomass of MsPT5 transgenic alfalfa was significantly increased, while the phosphate and protein content were significantly higher than the control. 【Conclusion】 MsPT5 was an important gene in alfalfa's response to low phosphate stress. Overexpression of MsPT5 could increase plant phosphate absorption capacity and phosphate content, and improve alfalfa yield and quality. Therefore, MsPT5 was important for cultivating high-yield and high-quality alfalfa.

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