一种豇豆重花叶病毒RT-PCR检测方法的研究

对一种豇豆重花叶病毒(Cowpeas evere mosaic virus,CPSMV)反转录PCR分子检测方法进行了研究。利用从美国菌种保藏中心和德国微生物毒株保藏中心引进的CPSMV标准毒株,设计了一对特异性简并引物(CPSMVf/CPSMVr),建立了CPSMVRT-PCR检测方法。该方法具有良好的扩增反应及特异性,灵敏度可达1pg的总病叶组织RNA量。CPSMV双抗夹心酶联免疫法(DAS-ELISA)灵敏度为10μg叶组织。该方法适用于豇豆等豆类蔬菜种子及种苗中对豇豆重花叶病毒的快速检测。     

豇豆疫霉LAMP检测方法的建立

本研究以豇豆疫霉Phytophthora vignae Purss核糖体内转录间隔区(internal transcribed spacer,ITS)序列为靶标片段,设计4条特异性引物,建立了环介导等温扩增(LAMP)检测方法。特异性检测结果表明:8株不同地理来源的豇豆疫霉菌株LAMP检测均为阳性(绿色),扩增产物用2.0%琼脂糖凝胶电泳出现特有的梯形条带,而其他11种卵菌近缘种及12种常见病原真菌和细菌共42个菌株均未观察到这些现象。灵敏度分析显示:该方法检测灵敏度在DNA水平上可达到100fg/25μL。采用LAMP方法对福建建瓯和宁德采集的62份疑似豇豆疫病病株样本进行检测,并用组织分离方法进行验证。结果表明,LAMP和组织分离方法的检出率分别为67.7%(42/62)和61.3%(38/62)。综合以上结果,LAMP方法具有特异性强、灵敏度高、快速高效、操作简单的特点,适合基层部门用于田间豇豆疫霉快速检测。     

菜豆普通花叶病毒长豇豆分离物外壳蛋白基因的序列分析及原核表达

 根据普通生物学和血清学特性,长豇豆病毒病的病原被鉴定为黑眼豇豆花叶病毒(Blackeye cowpea mosaic virus,Bl CMV)、豇豆蚜传花叶病毒(Cowpea aphidborne mosaic virus,CABMV)和黄瓜花叶病毒(Cucumbermosaic virus,CMV)。     

超高效液相色谱串联质谱法测定豇豆中新烟碱类农药残留

本研究建立了超高效液相色谱-串联质谱法(UPLC-MS/MS)同时检测豇豆中9种新烟碱类杀虫剂的多残留分析方法。豇豆样品用QuEChERS方法前处理, 用Kinetex Biphenyl色谱柱分离, 以甲醇和含0.1 mmol/L甲酸铵、0.001%甲酸的水作为流动相进行梯度洗脱, 使用超高效液相色谱串联质谱检测分析。定量限为0.01 mg/kg, 标准工作曲线在0.001~1 mg/kg范围内表现出良好的线性关系, 决定系数(R2)均大于0.99。目标杀虫剂在豇豆中的平均回收率为66.9%~109.8%, 相对标准偏差为1.2%~10.0%。应用该方法对某地16个农贸市场中采集的71份豇豆样本进行检测, 所有样品均符合我国所规定的豇豆(或豆类)中的最大残留限量标准要求。该方法简便、准确、灵敏度高, 适用于新烟碱类杀虫剂在豇豆中的残留监测。     

番茄斑萎病毒NASBA检测方法的建立

为建立一种快速检测番茄斑萎病毒(Tomato spotted wilt virus,TSWV)的方法,以TSWV-CP1/TSWV-CP2为引物对TSWV的N基因进行PCR扩增及序列测定,在TSWV N基因的高度保守区设计特异性扩增引物NA-P1/NA-P2进行核酸序列依赖性扩增(NASBA)反应,并对NASBA方法的特异性和灵敏度进行验证。结果表明,建立的NASBA方法最佳反应时间为1.5 h。该方法特异性较好,只有TSWV阳性样品中出现了预期大小为235 bp的扩增产物,与烟草环斑病毒(Tobacco ring spot virus,TRSV)、番茄黑环病毒(Tomato black ring virus,TBRV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、番茄花叶病毒(Tomato mosaic virus,ToMV)、番茄黄化曲叶病毒(Tomato yellow curl virus,ToYCLV)无交叉反应。灵敏度验证中,实时荧光RT-PCR的灵敏度最高,为1.56×10~(-5)ng/μL感病植物RNA模板,NASBA次之,为1.56×10~(-4)ng/μL,普通RT-PCR最低,为1.56×10~(-3)ng/μL。在对9份实际样品检测中,NASBA的阳性检出率与实时荧光RT-PCR、普通RT-PCR相同,均为33%,高于ELISA检测的22%。表明NASBA方法适用于实际样品检测,可对TSWV进行快速检测。     

利用RT-PCR方法检测甜瓜种子中南瓜花叶病毒

通过设计特异性引物SQCPF/SQCPR,扩增南瓜花叶病毒(Squash mosaic virus,SqMV)CP基因的保守序列,进行RT-PCR,对扩增得到的特异性片段进行序列测定,测序结果显示扩增产物序列与GenBank中登录的SqMV外壳蛋白基因存在87%～96%的一致性;建立了适用于检测甜瓜种子中南瓜花叶病毒的RT-PCR结合ELISA方法,通过在酶联板的反应孔中直接进行反转录合成cDNA,能扩增到预期大小的DNA条带,且检测灵敏度高于DAS-ELISA方法10倍以上。用建立的RT-PCR结合ELISA方法检测进境甜瓜种子携带的SqMV,在42份样品中检出4份阳性样品。     

进境欧洲水仙种球上病毒的检测与鉴定

为明确2013年福建口岸截获的进境欧洲水仙种球上携带的病毒种类,应用血清学、电镜观察和分子生物学检测方法对其可能携带的病毒进行了检测与鉴定。结果显示,水仙花叶病毒(Narcissus mosaic virus,NMV)、水仙黄条病毒(Narcissus yellow stripe virus,NYSV)、水仙潜隐病毒(Narcissus latent virus,NLV)、水仙迟季黄化病毒(Narcissus late season yellows virus,NLSYV)和南芥菜花叶病毒(Arabis mosaic virus,ArMV)5种病毒为阳性,其中NLSYV、ArMV为强阳性;NMV、NYSV、NLV和NLSYV为阳性的样品中含有大小约500~750 nm×12 nm的线状病毒粒体,ArMV为阳性的样品中含有直径约30 nm的球状病毒粒体;经序列测定和分析,扩增到的目的片段大小与各病毒预期扩增片段大小一致,并与已报道的各病毒序列高度同源;病毒复合侵染检测结果显示,该批水仙种球39.7%的样品存在2种以上病毒复合侵染。研究表明,该批进境欧洲水仙种球上携带有NMV、NYSV、NLV、NLSYV和ArMV,且复合侵染现象明显。     

豇豆病毒病的鉴定

1983年9月,从新疆石河子豇豆病毒病植株上分离到1株病毒分离物Cp-1。汁液摩擦接种试验的结果证明,它可以侵染3种豆科植物和2种藜科植物。在豇豆上引起系统花叶,叶片皱缩下卷,并有疱疹等症状;在绿豆上表现为局部棕褐色环纹;在苋色藜、昆诺阿藜上引起局部病斑。体外抗性测定,失毒温度为55—60℃,稀释限点为10~(-3)—10~(-4)。体外保毒期3—4天。Cp-1易汁液摩擦接种,桃蚜、棉黑蚜均可传毒,可通过种子传毒,种传率为10.6％。病毒粒体线条状,大小为12—15×700—750毫微米。病株叶片细胞内有风轮状及环状内含体。该分离物与豇豆蚜传花叶病毒(CABMV)的抗血清形成明显的沉淀线。根据以上性状,分离物Cp-1属于马铃薯Y病毒组的豇豆蚜传花叶病毒。在新疆各地采取21个标样,经血清学和寄主范围测定,有13个标样属该病毒,约占62％。说明豇豆蚜传花叶病毒在新疆种植的豇豆上是普遍存在的。     

侵染苜蓿的豇豆花叶病毒的研究

 从新疆苜蓿矮缩花叶病株上分离到一种病毒分离物G-14,在测定的9科40种植物中,此分离物可侵染6科19种植物,易由汁液摩擦接种,桃蚜(Myzus persicae)、豆蚜(Aphis craccivora)、棉黑蚜(A.atrata)和苜蓿无网长管蚜(Acyrthosiphon rondoi)等不传毒,可由首蓿叶象岬(Hypera variabilis)传毒;致死温度55~60℃,稀释限点10^-3~10^-4,体外保毒期21天;病毒粒体球状,直径约28nm。电镜观察病组织超薄切片,可见晶格状排列的病毒粒体。经琼脂双扩散血清学测定,该分离物与豇豆花叶病毒(CPMV)抗血清呈阳性反应。由此鉴定G-14为CPMV。提纯病毒经SDS-聚丙烯酰胺凝胶电泳测定有3个蛋白组分。分子量分别为44800,24500,22400道尔顿;琼脂糖电泳测得有二个核酸组分。     

The effect of cropping history and the role of cowpea debris in the epidemiology of cowpea scab

The role of cowpea ( Vigna unguiculata) debris as a primary source of inoculum for Sphaceloma sp., the pathogen of cowpea scab, was studied in field experiments. Three fields were selected in 1993 and three in 1994, in which cowpea had been grown 1, 2 or 3 years previously as part of a crop rotation. Polyethylene mulch was spread over the soil to prevent soil/debris splash in half of the plots. No scab symptoms were observed on the primary leaves. It took an average of 25 days for primary symptoms to be observed in each field, irrespective of mulching. Mulching had a significant effect on disease severity only in the two sites where cowpeas were last grown a year before the trial (sites 1a and 1b). In both years, interaction between time (days after sowing) and site (fields in which cowpea had been grown 1–3 years earlier) had a significant effect on disease incidence while in 1994, interaction between time, site and mulching was also significant ( P  < 0.05). Higher disease incidence was observed in 1994 than in 1993. In all fields, there were increases in disease incidence over time. Rain splashing may have contributed to higher disease incidence in plots without mulch. The presence of scab in the mulched plots in fields last sown to cowpea 3 years before the trial suggests that the pathogen may survive on infected cowpea debris, which acts as one of the sources of primary inoculum. Hence longer periods of crop rotation with nonhosts may be required to control the disease.     

Identification of blackeye cowpea mosaic virus from germplasm of yard-long bean and from soybean,and the relationships between blackeye cowpea mosaic virus and cowpea aphid-borne mosaic virus

Two potyvirus isolates, one from germplasm of yard-long bean (Vigna unguiculata ssp.sesquipedalis) introduced into the Netherlands, and another one from soybean plants (Glycine max) in Indonesia, were compared with two virus isolates of blackeye cowpea mosaic virus (BICMV) from the USA and a Moroccan isolate of cowpea aphid-borne mosaic virus (CAMV). It is proposed that all five isolates be now considered BICMV on the basis of host ranges, symptoms and serology. From our results, and a reassessment of the literature it is suggested to drop the name CAMV in favour of BICMV.Samenvatting Twee potyvirussen, de een in Nederland ingevoerd met genenmateriaal vanVigna unguiculata ssp.sesquipedalis en de ander uit planten van sojaboon (Glycine max) in Indonesië, werden vergeleken met twee isolaten van blackeye cowpea mosiac virus (BICMV) en een Marokkaans isolaat van cowpea aphid-borne mosaic virus (CAMV). Op grond van waardplantenreeksen, symptomen en serologie stellen de auteurs voor om alle vijf isolaten te beschouwen als BICMV. Gebaseerd op de verkregen resultaten en een kritische beschouwing van de literatuur wordt de aanbeveling gedaan om de naam CAMV te laten vallen ten gunste van BICMV.     

Sources of genetic resistance in cowpea (Vigna unguiculata (L.) Walp.) to cowpea aphid-borne mosaic potyvirus

Fifty-one cowpea (Vigna unguiculata (L.) Walp.) genotypes were tested by mechanical inoculation with seven geographically diverse isolates of cowpea aphid-borne mosaic potyvirus to identify resistant sources. Of 51 genotypes three, TVu-401, TVu-1582 and TVu-1593 were found immune to all the seven isolates. Forty-five genotypes gave different reactions to individual isolates. Several immune, resistant and tolerant genotypes against each isolate were identified. A considerable evidence of pathogenic variability among the virus isolates was also observed.     

Identification of cowpea (Vigna unguiculata) cultivars and lines immune to variants of blackeye cowpea mosaic potyvirus

Fifty-one cowpea ( Vigna unguiculata ) cultivars and lines were tested by mechanical inoculation against seven geographically and pathogenically diverse isolates of blackeye cowpea mosaic potyvirus (BICMV), to identify genetic resources with comprehensive BICMV resistance. Five genotypes, IT 80S 2049, Big Boy, Corona, Serido, and Tennessee Cream #8 were immune from all seven isolates, and an additional three genotypes, TVu-2657, TVu-2740, and TVu-3433, were immune from all isolates except PU-7B, an aberrant B1CMV isolate. The diversity among BICMV isolates was illustrated by the range of responses to inoculation among cowpea genotypes, many of which were either immune from or tolerant of individual B1CMV isolates.     

Identification of an aphid-transmitted cowpea mosaic virus

A virus disease of cowpea widespread in North Italy has been found to be caused by a virus which has the following properties: a. rod-shaped particles about 750 mµ in length; b. serological affinity with bean common mosaic virus; c. aphid-transmission; d. seed-transmission in cowpea at a percentage of 0.3–1.59; e. fairly wide host range covering 19 species representing 13 genera and 6 families; f. dilution end-point 1:4000; g. thermal inactivation point 60–62°C; h. longevity in vitro 5 days. The virus is tentatively called “cowpea aphid-borne mosaic virus”.     

豇豆根腐病病原菌鉴定

正豇豆营养价值较高,且在改善土壤肥力等方面具有重要作用。近年来,由于环境恶劣、栽培管理不当、抗病品种缺乏等原因,使得豇豆根腐病发生严重,现已成为制约豇豆生产的重要土传病害。目前,国内外已报道可引起豇豆根腐病的镰孢菌有11种,如茄病镰孢Fusarium solani、尖孢镰孢F. oxysporum、轮枝镰孢F. verticillioides等(https://nt.ars-grin.gov/fungaldatabases/),但是对茄病镰孢种下专化型     

Relationships between co-infection with cowpea aphid-borne and cucumber mosaic viruses and yield of cowpea lines with varying resistance to these viruses

The effect of mixed infection of cowpea aphid-borne mosaic (CAMV) and cucumber mosaic (CMV) viruses on cowpea yield varied with the resistance levels of cowpea lines, since there was a strong interaction between the latter and virus infection types atP ≤ 0.01. In cowpea line TVu 3629, with low resistance to both viruses, mixed infection significantly(P ≤ 0.05) reduced most yield components as compared with single infections and uninoculated controls. However, in line TVu 15656, which is highly resistant to both viruses, the effect of mixed infections was not significantly different from that of single infections or from the control. Between these two extremes were cowpea lines TVu 13683 and TVu 410, which were mildly resistant to CMV and CAMV, respectively; single infections significantly reduced the yield components relative to control, although these were generally not different from mixed infections. CMV, hitherto considered to be not economically important in cowpea in Nigeria, could induce severe yield losses when present in mixed infection with CAMV in cowpea lines with low resistance to both viruses. However, since mixed infection did not affect the yield of the highly doubly-resistant TVu 15656 cowpea line, it may be possible to breed for combined resistance to both viruses.     

Identification of resistance to Cercospora leaf spot of cowpea

Twelve selected cowpea cultivars were screened for resistance to Cercospora leaf spot (CLS) disease caused by Pseudocercospora cruenta and Cercospora apii s. lat. under artificial epiphytotic conditions in a replicated field trial, with the objective of developing a quantitative measure of disease resistance. CLS incidence, leaf spotting score, lesion density, lesion size, proportion of nodes infected, diseased leaf area, conidia number mg⁻¹ and fascicle density were assessed in 12 cowpea genotypes at crop maturity. Proportion of nodes infected and leaf spotting score were best able to quantitatively differentiate between the levels of resistance, and allow the exploitation of quantitative resistance to the disease. Both lesion density and lesion size were important in determining the final leaf spotting score but the former was epidemiologically more important than the latter, indicated by its correlation to most of the CLS symptom measures. There was differential resistance to the P. cruenta and C. apii s. lat. among the cowpea varieties screened. Among the cowpea lines screened, resistance to P. cruenta was more common than resistance to C. apii s. lat. Nevertheless, P. cruenta was considered the more aggressive and epidemiologically more important than C. apii s. lat. on the varieties tested evidenced by the strong correlation of P. cruenta incidence with acropetal spread of CLS, intensity of leaf spotting, conidia number mg⁻¹ and fascicle density. The highly susceptible varieties namely VRB7, Los Banos Bush Sitao no.1 and CB27 were susceptible to both Cercospora pathogens. The cowpea variety VRB-10 was completely resistant to both pathogens and is a useful source of resistance in CLS breeding programmes.     

Host–parasite relations between cowpea and Alectra vogelii

Summary Although the angiospermous parasitic weed Alectra vogelii is a major biotic constraint to cowpea production in Africa, there is little information on the host:parasite association between them. Accordingly, the dry matter production and partitioning in a cowpea: A. vogelii association was studied over the growth cycle. Cowpea was grown in pots containing 1350, 2700 or 4000 A. vogelii seeds kg⁻¹ top soil and with uninfected controls. Alectra vogelii attachment on to cowpea roots was first detected 30 d after crop emergence, and the first shoots emerged 44 d after crop emergence. New A. vogelii attachments on to cowpea roots continued to be produced throughout the growth of the crop. Alectra vogelii infection did not decrease cowpea dry matter production, but it significantly altered dry matter partitioning by increasing the proportion of root dry matter. Alectra vogelii infection significantly reduced dry matter accumulation in cowpea pods. The loss of dry matter in cowpea pods was largely accounted for by dry matter gain in A. vogelii shoots. The data are discussed in relation to how A. vogelii and other parasitic plants influence dry matter partitioning in their hosts.     

Rhizobacteria‐mediated resistance against the blackeye cowpea mosaic strain of bean common mosaic virus in cowpea (Vigna unguiculata)

BACKGROUND: The present study investigated the effect of seven Bacillus‐species plant‐growth‐promoting rhizobacteria (PGPR) seed treatments on the induction of disease resistance in cowpea against mosaic disease caused by the blackeye cowpea mosaic strain of bean common mosaic virus (BCMV). RESULTS: Initially, although all PGPR strains recorded significant enhancement of seed germination and seedling vigour, GBO3 and T4 strains were very promising. In general, all strains gave reduced BCMV incidence compared with the non‐bacterised control, both under screen‐house and under field conditions. Cowpea seeds treated with Bacillus pumilus (T4) and Bacillus subtilis (GBO3) strains offered protection of 42 and 41% against BCMV under screen‐house conditions. Under field conditions, strain GBO3 offered 34% protection against BCMV. The protection offered by PGPR strains against BCMV was evaluated by indirect enzyme‐linked immunosorbent assay (ELISA), with lowest immunoreactive values recorded in cowpea seeds treated with strains GBO3 and T4 in comparison with the non‐bacterised control. In addition, it was observed that strain combination worked better in inducing resistance than individual strains. Cowpea seeds treated with a combination of strains GBO3 + T4 registered the highest protection against BCMV. CONCLUSION: PGPR strains were effective in protecting cowpea plants against BCMV under both screen‐house and field conditions by inducing resistance against the virus. Thus, it is proposed that PGPR strains, particularly GBO3, could be potential inducers against BCMV and growth enhancers in cowpea. Copyright © 2009 Society of Chemical Industry