Location of Soybean Mosaic Virus in Seed-Parts of Individual Dormant and Germinating Mottled and Non-mottled Soybean Seeds

Soybean mosaic virus (SMV) was detected in individual embryos of both dormant mottled (50%) and non-mottled (50%) seeds of certified soybean cvs Davis. Essex. Hutchinson, Ransom, Stonewall, and Young by using the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). SMV was not detected in individual strained seed coats of mottled or non-stained seed coats of non-mottled dormant seeds and endosperms of either type of seed. SMV was not detected m individual stained and nonstained seed coats, epicotyls, and endosperms of mottled and non-mottled seeds at 72 h post-germination. However, the virus was detected in individual hypocotyls at an average level of 31% in post-germinated seeds (mottled and non-mottled) of all cultivars. Symptomless glasshouse-grown 6–8-week-old seedlings from mottled and non-mottled seeds of certified soybean cultivars occurred twice as often as those showing early symptoms. However, almost half of these symptomless plants were found to be SMV-infected by DASELISA. Virus-free soybean plants grown to maturity from non-mottled seeds also produced mottled seeds.     

Symptom Severity, Yield, Seed Mottling and Seed Transmission of Soybean Mosaic Virus in Susceptible and Resistant Soybean: The Influence of Infection Stage and Growth Temperature

The effect of soybean mosaic virus (SMV) infection on symptom severity, yield, seed mottling and seed transmission in soybean in relation to the growth stage at infection and subsequent temperature was investigated using a susceptible (Harosoy), a moderately resistant (Evans) and a highly resistant (Merit) cultivar. Disease symptoms were more severe with early infection. A greater reduction in plant growth and seed yield, and higher percentages of mottled seeds and seed transmission of SMV also occurred with early infection. Virus titer was higher in younger plants than in older ones and also higher in plants infected at the ealier stage than at the later stage of growth. Merit (a highly resistant cultivar previously reported to be immune to seed mottling) inoculated at the early stage of plant growth resulted in infection and production of some mottled seeds. Temperature affected all parameters investigated. The effect of temperature was greater in the susceptible cultivar than in the resistant one. The optimal temperature for symptom severity, yield, seed mottling and seed transmission was 20 °C. Virus titer was highest at 30 °C in all three cultivars. Maturity of susceptible cultivar was delayed by infection.     

SMV种传特性及其田间流行的种子传毒率预测

SMV感染供试品种种子传毒率最高为29.60％,最低1.04％。SMV弱毒株系的传毒率高于其强毒株系。大豆品种营养生长的V_4时期以前感染SMV种子传毒率最高,花期感染种传率显著下降。供试品种中,有初花期感病种子即不传毒;有盛花期感病仍有很低的种传率;亦有结荚初期感病还有很低种传率的品种。SMV流行的收获种子传毒率高低主要取决于大豆营养生长和花期田间病株率的高低。根据品种间早期感染SMV的最高种传率不同、花期感染种传率显著下降的特性,建立了SMV田间流行的种子传毒率预测模型。     

A tissue culture method for the preservation of Soybean mosaic virus strains

The activity and longevity of Soybean mosaic virus (SMV) in soybean callus culture were investigated with 11 SMV strains which are distinguished by differential reactions on soybean cultivars [Glycine max (L.) Merr.]. Dual cultures (soybean callus and SMV) were initiated by direct culture of SMV-infected leaves from susceptible soybean plants on Msoy and MS agar medium. Established SMV-callus cultures were maintained at 25 °C under light, subcultured to fresh MS medium at 2-month intervals or as necessary, and assayed periodically for virus infectivity. The infected calluses on MS medium grew better and stayed active longer than those on Msoy medium. At 10–15 °C, calluses and SMV were viable and active for 13–15 weeks or longer without subculture. The infectivity of SMV from callus cultures was comparable with that of SMV from infected plants, and remained stable for more than a year through five successive subcultures. Callus tissues of dual cultures were uniformly infected by SMV, thus ensuring infectious subcultures by random transfers. Production of in vitro inoculum can be significantly increased by multiple subcultures. Biological integrity of the SMV cultures was maintained with no change of viral virulence and pathotype. The method is of value for preserving a collection of SMV strains in a highly infectious and readily available form and reduces the chance of contamination or loss in viability.     

Soybean RNA interference lines silenced for eIF4E show broad potyvirus resistance

Soybean mosaic virus (SMV), a potyvirus, is the most prevalent and destructive viral pathogen in soybean-planting regions of China. Moreover, other potyviruses, including bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV), also threaten soybean farming. The eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in controlling resistance/susceptibility to potyviruses in plants. In the present study, much higher SMV-induced eIF4E1 expression levels were detected in a susceptible soybean cultivar when compared with a resistant cultivar, suggesting the involvement of eIF4E1 in the response to SMV by the susceptible cultivar. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that soybean eIF4E1 interacted with SMV VPg in the nucleus and with SMV NIa-Pro/NIb in the cytoplasm, revealing the involvement of VPg, NIa-Pro, and NIb in SMV infection and multiplication. Furthermore, transgenic soybeans silenced for eIF4E were produced using an RNA interference approach. Through monitoring for viral symptoms and viral titers, robust and broad-spectrum resistance was confirmed against five SMV strains (SC3/7/15/18 and SMV-R), BCMV, and WMV in the transgenic plants. Our findings represent fresh insights for investigating the mechanism underlying eIF4E-mediated resistance in soybean and also suggest an effective alternative for breeding soybean with broad-spectrum viral resistance.     

Soybean leaf pubescence affects aphid vector transmission and field spread of soybean mosaic virus

A study was undertaken to determine the influence of trichome density on the spread of non-persistently transmitted plant viruses by aphid vectors. A system using soybean plants and soybean mosaic virus (SMV) tested the hypothesis that greater leaf trichome density inhibits probing activity of vector species, leading to reduced virus spread and retarded virus epidemics under field conditions. Probing activity of three important aphid vectors of SMV, Myzus persicae, Rhopalosiphum maidis, and Aphis citricola, was affected by the density of soybean leaf trichomes. Less pubescent and glabrous isolines elicited greater probing activity than did densely pubescent isolines. Among the parameters considered, probe duration was found to be species specific, whereas the following traits were consistent among species for the denser isolines: reduced numbers of probes, greater length of time to first probe, and less time spent probing. Laboratory transmission of soybean mosaic virus was reduced in the more densely pubescent isolines by the vector species tested. Field spread of SMV was negatively correlated with density of pubescence. In our system, we found that denser leaf pubescence retards field epidemics of non-persistently transmitted plant viruses.     

抗除草剂转基因大豆后代遗传分析

分析了来源于农杆菌介导的4个独立的大豆转化系的后代遗传特性。分别采用种子切片GUS染色方法和除草剂涂抹以及喷洒方法检测gus报告基因和抗除草剂bar基因在后代的表达。其中3个转化系T1代gus基因和bar基因能够以孟德尔方式3：1连锁遗传,说明这2个基因整合在大豆基因组的同一位点。这3个转化系在T2代获得了纯合的转化系,并能够稳定遗传至T5代。有一个转化系在T1代GUS和抗除草剂检测都为阴性,但通过Southern杂交证明转基因存在于后代基因组,显示发生了转基因沉默。为了证明转基因沉默是转录水平还是转录后水平,T1代植物叶片接种大豆花叶病毒（SMV）并不能抑制转基因沉默,说明该转化系基因沉默可能不是发生在转录后水平。     

Factors affecting aphid acquisition and transmission of soybean mosaic virus

Myzus persicae transmitted soybean mosaic virus (SMV) most efficiently following 30 or 60 s acquisition probes on infected plants. There were no differences in susceptibility to SMV infection of soybean plants 1 to 12 wk old, but symptoms were more severe in plants inoculated when young than when old. Soybeans inoculated between developmental stages R3 and R6 only showed yellowish-brown blotching on one or more leaves. There were no observable differences in the time of appearance or type of symptoms shown by soybean seedlings inoculated either by sap or by aphids; infected plants became acquisition hosts for aphids 5–6 days after inoculation. There was no change in the efficiency with which M. persicae transmitted SMV from source plants up to 18 wk after inoculation. M. persicae transmitted SMV from leaves of field-grown soybeans when plants were inoculated at developmental stages V6, R2, and R3 and tested as sources 57–74 days after inoculation but not from plants inoculated at R5 and tested as sources 14 to 32 days after inoculation. M. persicae acquired SMV from soybean buds, flowers, green bean pods, and unifoliolate, trifoliolate, and senescent leaves. Middle-aged and deformed leaves were better sources of the virus than buds, unfolding and old symptomless leaves. The results are being incorporated into a computer model of SMV epidemiology.     

Rapid Differentiation of Soybean Mosaic Virus Isolates by Antigenic Signature Analysis

Antigenic differences among 14 soybean mosaic virus (SMV) isolates were shown by signature analysis employing a panel of nine well-characterized monoclonal antibodies. Sets of binding curves were generated by reacting aliquots of serial dilutions of leaf extracts from soybean plants infected with each strain of SMV simultaneously with each antibody in the panel. An iterative alignment procedure allowed comparison of binding profiles from different assays in which the starting concentrations of SMV antigen were unknown. Desiccation of infected soybean leaf tissue altered antigenic signatures by inducing changes in reactivity of epitopes recognized by certain antibodies. The method was useful for the elucidation of subtle antigenic differences amongSMV strains. The procedure requires a minimum amount of tissue processing, the analysis is rapid and sensitive, and it requires neither purification nor knowledge of concentration of virus in ground leaf tissue from infected plants.     

Evidence for Complementation of Plant Potyvirus Pathogenic Strains in Mixed Infection

Several strains of soybean mosaic virus (SMV) can be differentiated on the basis of the phenotypic response of various soybean cultivars (e.g., the soybean line Williams ‘82 is susceptible to all SMV strains, whereas the lines P. I. 96983, L78-379, and Davis are functionally immune to SMV strain G2 but susceptible to strains G7 and G7a). Inoculation of the immune lines with G2, followed 2 days later by inoculation with G7 or G7a, resulted in systemic spread of the avirulent SMV G2. Further evidence, suggests that complementation groups of SMV strains may exist.     

Robust RNAi-mediated resistance to infection of seven potyvirids in soybean expressing an intron hairpin <Emphasis Type="Italic">NIb</Emphasis> RNA

Viral pathogens, such as soybean mosaic virus (SMV), are a major constraint in soybean production and often cause significant yield loss and quality deterioration. Engineering resistance by RNAi-mediated gene silencing is a powerful strategy for controlling viral diseases. In this study, a 248-bp inverted repeat of the replicase (nuclear inclusion b, NIb) gene was isolated from the SMV SC3 strain, driven by the leaf-specific rbcS2 promoter from Phaseolus vulgaris, and introduced into soybean. The transgenic lines had significantly lower average disease indices (ranging from 2.14 to 12.35) than did the non-transformed (NT) control plants in three consecutive generations, exhibiting a stable and significantly enhanced resistance to the SMV SC3 strain under field conditions. Furthermore, seed mottling did not occur in transgenic seeds, whereas the NT plants produced ~90% mottled seeds. Virus resistance spectrum screening showed that the greenhouse-grown transgenic lines exhibited robust resistance to five SMV strains (SC3, SC7, SC15, SC18, and a recombinant SMV), bean common mosaic virus, and watermelon mosaic virus. Nevertheless, no significantly enhanced resistance to bean pod mottle virus (BPMV, Comovirus) was observed in the transgenic lines relative to their NT counterparts. Consistent with the results of resistance evaluation, the accumulation of each potyvirid (but not of BPMV) was significantly inhibited in the transgenic plants relative to the NT controls as confirmed by quantitative real-time (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). These results demonstrate that robust RNAi-mediated resistance to multiple potyvirids in soybean was conferred by expressing an intron hairpin SMV NIb RNA.     

Cell-free Translation of Potyviral RNA Including Extracts from Disease Resistant Soybean

Cell-free translation of soybean mosaic virus (SMV), strain G2 or G7, RNAs in the presence of fractionated leaf extracts from the cultivars Davis or Williams‘82 revealed no differences that were associated with disease resistance of the cultivars to the virus strains. Extracts of leaf tissue from Davis (resistant to strain G2) and susceptible Williams‘82 were fractionated into three broad peaks. Apparent proteinase activity was detected in peak II of both soybean cultivars in the cell-free translation of SMV-G2 or -G7 RNAs. Inhibition of translation occurred in the presence of peak extracts from either of the soybean cultivars at levels of 500 μ/ml and/or 1000 μ/ml of protein. No inhibition of proteolytic processing was observed by extracts from either cultivar.     

Increased multiple virus resistance in transgenic soybean overexpressing the double-strand RNA-specific ribonuclease gene PAC1

Viruses constitute a major constraint to soybean production worldwide and are responsible for significant yield losses every year. Although varying degrees of resistance to specific viral strains has been identified in some soybean genetic sources, the high rate of mutation in viral genomes and mixed infections of different viruses or strains under field conditions usually hinder the effective control of viral diseases. In the present study, we generated transgenic soybean lines constitutively expressing the double-strand RNA specific ribonuclease gene PAC1 from Schizosaccharomyces pombe to evaluate their resistance responses to multiple soybean-infecting virus strains and isolates. Resistance evaluation over three consecutive years showed that the transgenic lines displayed significantly lower levels of disease severity in field conditions when challenged with soybean mosaic virus (SMV) SC3, a prevalent SMV strain in soybean-growing regions of China, compared to the non-transformed (NT) plants. After inoculation with four additional SMV strains (SC7, SC15, SC18, and SMV-R), and three isolates of bean common mosaic virus (BCMV), watermelon mosaic virus (WMV), and bean pod mottle virus (BPMV), the transgenic plants exhibited less severe symptoms and enhanced resistance to virus infections relative to NT plants. Consistent with these results, the accumulation of each virus isolate was significantly inhibited in transgenic plants as confirmed by quantitative real-time PCR and double antibody sandwich enzyme-linked immunosorbent assays. Collectively, our results showed that overexpression of PAC1 can increase multiple virus resistance in transgenic soybean, and thus provide an efficient control strategy against RNA viruses such as SMV, BCMV, WMV, and BPMV.

     

Overexpression of a GmCnx1 Gene Enhanced Activity of Nitrate Reductase and Aldehyde Oxidase,and Boosted Mosaic Virus Resistance in Soybean

Molybdenum cofactor (Moco) is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1) is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L.) Cnx1 gene (GmCnx1) was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR) and aldehydeoxidase (AO) of T₁ generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 μg g^-1 h^-1 and30 pmol L^-1, respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants).In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV). DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.     

大豆花叶病毒研究进展

大豆花叶病毒（Soybean mosaic virus,SMV）病是在世界范围内广泛分布并普遍发生的病毒病害之一,导致大豆严重减产和种质衰退。这引起了国内外许多学者的科研兴趣,研究内容涉及SMV株系划分与发生分布、传播流行方式、寄主上的症状和影响因素、基因组结构组成及各基凶功能、植物生理生化抗性、大豆SMV抗性遗传育种等各方面。本文综述了近年来国内外SMV的科研方向和进展,旨在为进一步的研究提供依据。     

RNAi-mediated <Emphasis Type="Italic">Soybean mosaic virus</Emphasis> (SMV) resistance of a Korean Soybean cultivar

Soybean [Glycine max (L.) Merr.] is an important crop for vegetable oil production, and is a major protein source worldwide. Because of its importance as a crop, genetic transformation has been used extensively to improve its valuable traits. Soybean mosaic virus (SMV) is one of the most well-known viral diseases affecting soybean. Transgenic soybean plants with improved resistance to SMV were produced by introducing HC-Pro coding sequences within RNA interference (RNAi) inducing hairpin construct via Agrobacterium-mediated transformation. During an experiment to confirm the response of transgenic plants (T₂) to SMV infection, no T₂ plants from lines #2 (31/31), #5 (35/35) or #6 (37/37) exhibited any SMV symptoms, indicating strong viral resistance (R), whereas NT (non-transgenic wild type) plants and those from lines #1, #3 and #4 exhibited mild mosaic (mM) or mosaic (M) symptoms. The northern blot analysis showed that three resistant lines (#2, #5 and #6) did not show the detection of viral RNA accumulation while NT, EV (transformed with empty vector carrying only Bar) and lines #1, #3 and #4 plants were detected. T₃ seeds from SMV-inoculated T₂ plants were harvested and checked for changes in seed morphology due to viral infection. T₃ seeds of lines #2, #5 and #6 were clear and seed coat mottling was not present, which is indicative of SMV resistance. RT-PCR and quantitative real-time PCR showed that T₃ seeds from the SMV-resistant lines #2, #5 and #6 did not exhibit any detection of viral RNA accumulation (HC-Pro, CP and CI), while the viral RNA accumulation was detected in SMV-susceptible lines #1, #3 and #4 plants. During the greenhouse test for viral resistance and yield components, T₃ plants from the SMV-inoculated transgenic lines #2, #5 and #6 showed viral resistance (R) and exhibited a more favorable average plant height, number of nodes per plant, number of branches per plant, number of pods per plant and total seed weight with statistical significance during strong artificial SMV infection than did other plant lines. In particular, the SMV-resistant line #2 exhibited superior average plant height, pod number and total seed weight with highly significance. According to our results, RNAi induced by the hairpin construct of the SMV HC-Pro sequence effectively confers much stronger viral resistance than did the methods used during previous trials, and has the potential to increase yields significantly. Because of its efficiency, the induction of RNAi-mediated resistance will likely be used more frequently as part of the genetic engineering of plants for crop improvement.     

Inoculation with a bacterial consortium alleviates the effect of cadmium overdose in soybean plants

Inoculating plants that have inefficient antioxidant systems with plant-associated bacteria allows them to overcome heavy metal intoxication. We monitored protein oxidation, the activity of plant defense system enzymes, and the phenolics content in soybean (Glycine max L.) during a prolonged exposure to cadmium (Cd). The assistance of the bacterial consortium reduced the bioavailability of Cd in a soil containing 10 times the metal’s Standard Maximum Value (SMV). This reduced the accumulation of Cd in the soybeans’ roots and seeds. At 100 SMV, bacterial inoculation resulted in increased Cd bioavailability, which enhanced cadmium uptake by the soybean plants. At both Cd concentrations, oxidative stress was more prolonged in the soybean’s roots than its leaves. In cadmium-polluted soil, glutathion peroxidase activity changed more rapidly in the roots of plants when they had been inoculated. Inhibition of the peroxidases’ activities strengthened the activity of glutathione-S-transferase; increased the phenolics content in plant roots; and alleviated stress in inoculated soybean plants compared to untreated plants. The bacterial consortium may be recommended for a plant protection at 10 SMV Cd in the soil, and for phytostabilization at 100 SMV.