单管实时荧光RT PCR方法同时检测大豆种子中的菜豆荚斑驳病毒和烟草环斑病毒

 本研究建立了大豆种子中菜豆荚斑驳病毒（BPMV）和烟草环斑病毒（TRSV）单管双重实时荧光PCR检测方法。将含有相同浓度的分别带有BPMV和TRSV CP基因的质粒溶液作为阳性对照,以受两种病毒侵染的大豆种子作为待测样品进行实时荧光PCR检测,结果表明能从同一管中同时检测出这两种病毒而不发生交叉反应。尽管在阳性对照中,二者的检测限相当,均可达到35 pg/mL,但在实际应用中,两种病毒由于在大豆种子中的浓度不一致而存在一定的差别。该方法快速、灵敏、简便,同时特异性更强,在出入境检验检疫中具有广泛的应用前景。     

Screening rice varieties for resistance to rice yellow mottle virus disease

Abstract

Rice yellow mottle virus (RYMV) is one of the major constraints to rice production in Kenya. The reactions of 30 varieties of rice to an isolate of RYMV were studied. The appearance and development of symptoms in the various varieties were scored on a 0–5 scale. The observed reactions ranged from highly susceptible to highly resistant. Thirty‐six per cent of the varieties showed a high level of resistance while 23.3% were highly susceptible. The intermediate reactions were 16.7% resistant, 6.6% susceptible and 16.7% moderately susceptible.     

诱导剂对番木瓜环斑病毒病的防治效果

为了探讨水杨酸、茉莉酸甲酯和壳聚糖对番木瓜环斑病毒病的病情指数和防治效果的影响,在番木瓜上喷施不同浓度的诱导剂,然后在叶腋处接种番木瓜环斑病毒,观察和记录番木瓜的病情指数,计算其防治效果.结果表明,诱导剂提高了番木瓜对环斑病毒病的抗性,其中水杨酸的效果优于茉莉酸甲酯和壳聚糖.水杨酸、茉莉酸甲酯和壳聚糖的最佳施用浓度分别为50 mg/L、0.05 mmol/L和1％,最佳防治效果则分别为77.19％、53.11％和22.95％.水杨酸、茉莉酸甲酯、壳聚糖与对照之间的防治效果差异极显著,3种诱导剂在最佳浓度下防治效果差异极显著.50 mg/L水杨酸对番木瓜环斑病毒病防治效果最佳,在生产上施用具有一定的经济效益.     

黄瓜绿斑驳花叶病毒病的鉴定与防治

黄瓜绿斑驳花叶病毒病是1种检疫性有害生物,主要为害葫芦科瓜类作物,在引种和调运种子、种苗时应加强对种苗进行检测,最好实行产地检疫。通常采用生物学、电镜观察、血清学和分子生物学进行鉴定。控制黄瓜绿斑驳花叶病毒病的有效方法是调运无病的种苗和瓜果,其次是合理轮作减少毒源。     

Subterranean clover mottle virus: another virus from Australia with encapsidated viroid-like RNA

A new virus, subterranean clover mottle virus (SCMoV) was found at several locations in Western Australia in Trifolium subterraneum L. (subterranean clover) with severe disease symptoms. Paiticles of the virus were shown to share many physical properties with members of the sobemo-virus group. However, in addition to the single-stranded RNA of M_J about 1.5 × 10⁶ which is characteristic of sobemoviruses, SCMoV particles also contained circular and linear viroid-like RNAs of two size classes, one about 400 and the other about 300 nucleotides long. At present it is not clear if the two types of viroid-like RNAs are components of the same SCMoV strain or of closely related strains of the virus which are able to confect subterranean clover.
SCMoV was shown to be serologically related to lucerne transient streak virus (LTSV). However, the relationship was remote and whereas most high-titred antisera to LTSV reacted with SCMoV particles, antisera to SCMoV failed to recogaize those of LTSV. Neither SCMoV nor LTSV are serologically related to velvet tobacco mottle virus (VTMoV) or Solanum nodiflorum mottle virus (SNMV). However, particles of all four viruses have many physical and chemical properties in common, including RNA complements consisting of both virus-like and viroid-like RNAs. Because of their affinities and unique RNA complements, we suggest that SCMoV, LTSV, VTMoV and SNMV may warrant inclusion in a new taxonomic group.     

Rice yellow mottle,a mechanically transmissible virus disease of rice in Kenya

In Kenya around Lake Victoria rice is affected by a hitherto undescribed virus for which the name Rice Yellow Mottle Virus (RYMV) is proposed. The virus was easily transmitted mechanically to rice and toOryza barthii andOryza punctata, but not toOryza eichingeri, barley, bulrush millet, durum wheat, finger millet, maize, oats, rye, sorghum, wheat, sugarcane, 20 other monocotyledonous and 9 dicotyledonous plant species.The disease is characterised by stunting and reduced tillering of the rice plant, crinkling, mottling and yellowish streaking of the leaves, malformation and partial emergence of the panicles, and sterility. In severe cases the plant may die. RYMV is stable and highly infective.The vector is the beetleSesselia pusilla Gerstaeker (fam. Chrysomelidae, Galerucinae). The beetle has been identified by Mr John A. Wilcox, New York State Museum and Science Service, New York.S. pusilla transmitted the virus for at least five successive days after feeding on an infective plant.Purified virus preparations consisted of polyhedral particles about 32 m in diameter. The sedimentation coefficient was 116S.Samenvatting In Kenya wordt rijst, voornamelijk Sindano, verbouwd op familiebedrijfjes aan de kust van de Indische Oceaan en langs het Victoria Meer, en door pachters van het meer naar het binnenland gelegen Mwea Irrigation Settlement. In de omgeving van Kisumu (Nyanza Province), waar rijst geteeld wordt volgens slechte cultuurmethoden, wordt sinds 1966 een tot dusver onbekende ziekte, hier rice yellow mottle genoemd, waargenomen.In het veld vallen de zieke planten onmiddellijk op door de gelige verkleuring van de bladeren. De jongste bladeren zijn gevlekt of geel-groen gestreept (Fig. 1). De uitstoeling is gering en de plant gedrongen (Fig. 2). De zaadopbrengst wordt sterk verlaagd door het optreden van steriliteit.Ongeveer 7 dagen na sap-inoculatie van Sindano-zaailingen verschijnen de eerste symptomen. Na inoculatie in een jong stadium van de plant krullen de eerstgevormde bladeren spiraalvormig (Fig. 3). Later worden de bladeren geel en necrotisch. Jong geïnfecteerde planten kunnen zelfs afsterven. De pluimen komen onvoldoende uit de bladschede, zijn misvormd en dragen veelal kleine en misvormde bloembakjes, die meestal loos zijn (Fig. 4). Sindano rijst, 3 weken voor het in pluim schieten geïnoculeerd, liet een zeer sterke opbrengstvermindering zien.In sap, verkregen van wortels van zieke rijstplanten, in de guttatievloeistof en in irrigatiewater van ernstig zieke rijstvelden kon het virus gemakkelijk worden aangetoond. Overdracht van RYMV door zaad en grond werd niet waargenomen.RYMV was nog infectieus na een verhitting gedurende 10 min bij 80°C. De verdunningsgrens hing af van het tijdstip waarop het bladmateriaal van de zieke planten getoetst werd en liep uiteen van 10^–10 (2–3 weken na inoculatie) tot 10^–6 (4–5 weken na inoculatie). Sap bewaard bij kamertemperatuur was nog infectieus na 33 dagen, maar had deze eigenschap verloren na 51 dagen. Bewaard in een koelkast bleef het sap zeker 71 dagen infectieus.A1 de getoetste rijstrassen werden na sap-inoculatie door het virus geïnfecteerd, evenalsOryza barthii enOryza punctata (Fig. 5). Niet vatbaar waren:Oryza eichingeri, baardtarwe, eleusine gierst, gerst (Proctor), haver (M.F.C. 15/67 en Lampton), mais (Hybrid 611B, Hybrid 612 en Hybrid 613B), parelgierst, rogge, sorghum (H726 en H6060), suikerriet (NCo 310 en Q 45), tarwe (Kenya Kudu en Wisconsin), evenals 20 andere monocotyle en 9 dicotyle plantesoorten, waaronder de doorgaans bij plantevirusonderzoek gebruikte toetsplanten.Het kevertjeSesselia pusilla Gerstaecker (fam. Chrysomelidae, Galerucinae; zie Fig. 6) bracht het virus over. Een enkel insekt was in staat het virus gedurende tenminste 5 dagen over te brengen.RYMV was gemakkelijk te zuiveren. Hierbij werd uitgegaan van 16,5–20 g jonge duidelijk zieke bladschijven van jonge 2–3 weken tevoren geïnoculeerde rijstplanten (Sindano) door uitschudden met chloroform en uitzouten met ammoniumsulfaat, iets gewijzigd naar Proll en Schmidt (1964). De elektronenmicroscopische beelden van de gezuiverde virussuspensie laten één type veelkantige deeltjes zien met een diameter van ongeveer 32 m (Fig. 7). De S₂₀-waarde is 116S (Fig. 8 en 9).Ter bestrijding van de ziekte moet op korte termijn allereerst de cultuurmethode van de rijst op de familiebedrijfjes verbeterd worden. Het gehele jaar in verschillende groeistadia voorkomen van rijst op een klein gebied en veelal zieke opslag op juist geoogste velden, dragen belangrijk bij tot de verspreiding van de ziekte. Het gebruik van kortgroeiende rijst-varieteiten wordt aanbevolen om overlapping van de groeiseizoenen te voorkomen.This paper is published with permission of the Chief of Technical Services, Ministry of Agriculture, Kenya.     

Distribution,host range and some properties of a virus disease of sunflower in Kenya

Abstract

Apparently similar viruses were associated with two different disease symptoms in sunflower (Helianthus annuus) in Kenya, one a yellow blotch and the other a leaf crinkle. The virus occurred in all sunflower‐growing regions but the crinkle disease was confined to areas east of the rift valley. Disease incidence varied with locality and season. Mechanical transmission of virus from plants with either disease was possible, but the yellow blotch virus was also transmissible from an important alternate field host, Tridax procumbens by Aphis gossypii to 23 species in three families. Partially purified preparations of virus made from mechanically inoculated seedlings affected with either symptom type contained 26 nm virus particles which, in ISEM tests, reacted with antisera prepared against six members of the luteovirus group.     

Historical and recent tomato black ring virus and beet ringspot virus isolate genomes reveal interspecies recombination and plant health regulation inconsistencies

Tomato black ring virus (TBRV) and beet ringspot virus (BRSV) are closely related but distinct members of subgroup B of the genus Nepovirus. Both viruses have broad host ranges and are transmitted by seed, pollen, and ectoparasitic nematodes. Although 13 TBRV and 3 BRSV genome sequences were already available, no attempt has been made to link sequence data from these recent sequences with those of historical isolates studied in the pre-sequencing era. High-throughput sequencing was used to generate eight new TBRV and BRSV genome sequences from three historical >60-year-old and two >30-year-old isolates, and three more recent isolates. These eight isolates were from the Czech Republic, Germany, and the UK. We compared these with all genomes sequenced previously. Intraspecies recombination (three of four TBRV and two of four BRSV isolates) was frequent amongst the eight new genomes. Interspecies recombination was also present within the RNA1 of TBRV isolates BRSV-3393 SG GB and BRSV-9888 ST GB. No satellite RNAs were associated with the eight new genomes. Two commercial enzyme-linked immunosorbent assay (ELISA) kits used to detect TBRV during routine testing differed in that one detected only TBRV and the other only BRSV, so they are likely to provide incorrect but potentially complementary virus occurrence information. We suggest both ELISA kits, or appropriate molecular tests, be used by biosecurity authorities to avoid this problem. This study illustrates the value of sequencing historical isolates preserved from the pre-sequencing era.     

Brown ring formation and streak mottle,two distinct syndromes in lilies associated with complex infections of lily symptomless virus and tulip breaking virus

Brown ring formation in bulbs of lilies, particularly of Mid-century hybrids, is described as a newly recognized disease. Symptoms of streak mottle in cultivars ofLilium speciosum Thunb., not associated with abnormalities of bulbs, are briefly described with reference to the literature. Sometimes the two syndromes occur in the same crop such as in the Mid-century hybrid Enchantment, showing brown ring formation in bulbs and an indistinct mottling in field plants. Severe leaf mottling appears in Midcentury hybrids andL. speciosum when plants are forced under glass. In both diseases tulip breaking virus (TBV) was always found to occur in complex with lily symptomless virus (LSV), which was consistently detected in apparently healthy plants of the Mid-century hybrid Enchantment and in severalL. speciosum cultivars.The part of TBV involved in the complex diseases described has been demonstrated by serological, electron-microscopical, and inoculation studies with lilies and tulips. LSV was sap-transmitted from lily to tulip but it could not be detected in several randomly taken samples of a dozen field-grown tulip cultivars. Suppression of TBV in plants of Enchantment, grown from bulbs with brown ring formation under field conditions, is discussed. TBV was serologically and electron-microscopically detectable only in plants grown under glass. A similar phenomenon was observed inL. speciosum cultivars under both conditions.Samenvatting Bruinkringerigheid in bollen van Midcentury-hybriden wordt beschreven als een nieuw onderkende virusziekte (Fig. 1, 2 en 3). Streperige vlekkerigheid op bladeren vanL. speciosum cultivars (Fig. 4), niet tegelijkertijd optredend met bolafwijkingen, wordt in het kort beschreven, en deed zich voor zoals in de literatuur wordt vermeld.Soms treden de twee syndromen op in hetzelfde gewas zoals in de Midcentury-hybride Enchantment, die bruinkringerigheid in bollen laat zien en een onduidelijke vlekkerigheid in veldplanten. Ernstige vlekkerigheid op de bladeren komt naar voren in Midcentury-hybriden enL. speciosum wanneer de planten in de kas worden gebroeid.Bij de beschreven ziekten was het complex van tulpemozaïekvirus (syn.: tulpebloembrekingsvirus; TBV) en symptoomloos lelievirus (LSV) steeds aanwezig. Het laatstgenoemde virus werd steeds aangetoond in ogenschijnlijk gezonde planten van de Midcentury-hybride Enchantment en van verscheidene cultivars vanL. speciosum.Het aandeel van het TBV in de beschreven complexe ziekten bleek uit serologisch en elektronenmicroscopisch onderzoek en uit inoculatieproeven met lelies en tulpen (Tabel 1 en 2). LSV werd met sap overgebracht van lelie naar tulp (Fig. 5 en 6). LSV kon niet worden aangetoond in verscheidene willekeurig genomen monsters uit planen met mozaïeksymptomen op de bladeren van een twaalftal te velde geteelde tulpecultivars. In planten met bruinkringerigheid te velde werd een teruglopen van de concentratie van het TBV waargenomen. TBV was serologisch en elektronenmicroscopisch lechts aantoonbaar in lelie-planten die in de kas groeiden. Een overeenkomstig verschijnsel werd waargenomen inL. speciosum-cultivars.     

进境玫瑰鲜切花李属坏死环斑病毒的检测鉴定

近期上海口岸连续多次从进境的玫瑰鲜切花中检出我国检疫性有害生物李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV),通过DAS-ELISA、RT-PCR、SYBR Green I实时荧光RT-PCR和序列分析等4种方法分别检测和复验该病毒,结果均为阳性,由此确定从进境玫瑰鲜切花中检出了PNRSV。同时本文分别检测玫瑰叶片、花瓣、茎干和花萼,结果均带有PNRSV,确定该病毒系统侵染玫瑰。     

Serological differences between red currant spoon leaf virus,virus isolates from Eckelrade-diseased cherry trees and the Scottish raspberry ringspot virus

Antisera were made to red currant spoon leaf virus (SLV), an isolate of the Scottish raspberry ringspot virus (RRV), and two virus isolates from Eckelrade-diseased cherry trees (EV). Different virus isolates, including one from Belgium, were tested against these antisera. The results indicate that we are dealing with a group of virus isolates with different antigenic properties. SLV is very closely related to RRV, being undoubtedly a strain of this virus. Dutch EV isolates differ from SLV and RRV and from each other, the Belgian isolate being closely related to one of the Dutch EV isolates. The serological differences found do not correspond with the geographical distances between the localities where the viruses were collected.Samenvatting Antisera werden bereid tegen het lepelbladvirus van rode bes (SLV), een isolatie van het Schotse raspberry ringspot-virus (RRV) en twee virusisolaties uit kersebomen met Eckelraderziekte (EV). Met behulp van deze antisera werden verschillende virusisolaties getoetst, waaronder één uit België van kers met Eckelraderziekte. De resultaten zijn vermeld in de tabellen 1 en 2. Ze duiden erop, dat we te maken hebben met een groep van virusisolaties met verschillende antigene eigenschappen. SLV is zeer nauw verwant aan RRV en is ongetwijfeld een stam van dit virus. Nederlandse EV-isolaties verschillen zowel van SLV en RRV, als van elkaar. De Belgische isolatie is nauw verwant aan één van de Nederlandse EV-isolaties. De grootte van de gevonden verschillen correspondeert niet met de geografische afstand tussen de plaatsen waar de virusisolaties werden verzameld.     

番茄环斑病毒单克隆抗体的制备及检测

将纯化的番茄环斑病毒(Tomato ringspot virus,ToRSV)制剂免疫BALB/c小鼠,末次免疫后第3天取其脾细胞与SP2/0细胞融合,采用选择性培养基、有限稀释法克隆和间接ELISA方法进行筛选,成功获得了2株分泌ToRSV单克隆抗体的杂交瘤细胞株并分别命名为1H8、1D4.用间接ELISA方法对所获得的2个杂交瘤细胞株进行亚型鉴定均为IgG1亚类.间接ELISA效价测定结果1H8为1:105,1D4为1:106.TAS-ELISA实验结果表明此2株杂交瘤细胞所分泌的单克隆抗体均能与本研究室保存的从德国引进的番茄环斑病毒分离物、从美国ATCC引进的番茄环斑病毒分离物PV-100、PV-174、PV-239发生特异性反应,而不与同属其它3种病毒:烟草环斑病毒(Tobacco ringspot virus,TRSV)、南芥菜花叶病毒(Arabis mosaic virus,ArMV)、马铃薯黑环斑病毒(Potato black ringspot virus,PBRSV)发生反应.     

西瓜感染黄瓜绿斑驳花叶病毒后糖的变化与倒瓤关系的研究

 By high performance liquid chromatography (HPLC), the contents of glucose, fructose and sucrose, and the sweetness were analyzed in watermelon inoculated with Cucumber green mottle mosaic virus (CGMMV).The relationship between sugar change and blood-flesh of watermelon was determined. The results indicated that the content of glucose was apparently increased compared to that of the control before maturity (within 28 days after pollination), but reduced to 24.8% of the control after maturity (at 35 days after pollination). The content of fructose was higher than that of the control within 14 days after pollination, and then decreased with a significant difference. In all cases, the content of sucrose increased with the growth of watermelon. However, compared with the control, the sucrose content of watermelon inoculated with CGMMV was lower. The ratios of glucose, fructose and sucrose in the total sugar were abnormal. Coincidence with the changes of the total sugar, the fruit sweetness before maturity was higher than that of the control, whereas decreased sharply after maturity (lower than that of the control). The inner pulp of the mature fruit appeared to be water-soaked and dirty red with no edibility. In a word, after inoculation with CGMMV, the changes of sugars and sweetness affected the watermelon quality.     

侵染昆明玫瑰的李坏死环斑病毒的鉴定及其分子检测

采集昆明地区种植的花叶症状明显的玫瑰样品,运用现代分子生物学技术与常规技术相结合的方法,初步鉴定引起玫瑰花叶病的主要病毒病原为李坏死环斑病毒.该病毒引起玫瑰植株的系统花叶、畸形和皱缩等症状;电镜下病毒粒体为球形,直径为22～23nm;ELISA检测发现该病毒在植株芽、花粉和顶部叶片的浓度最高;同时,根据外壳蛋白的保守区利用Primer 5.0设计该病毒的特异引物,对该病毒进行分子检测,得到450bp的预期DNA片断,并在此基础上,进行了巢式RT-PCR的分子检测,表明巢式RT-PCR的检测能力最强.并通过序列的同源性分析得知该病毒的外壳蛋白与已知PNRSV的同源性为98.0%,进一步证明了该病毒为李坏死环斑病毒.     

Parietaria mottle virus, a possible new ilarvirus from Parietaria officinalis (Urticaceae)

A previously undescribed virus, probably a new member of the ilarvirus group, was isolated from Parietaria officinalis showing symptoms of yellow mosaic or mottling. This virus, for which the name parietaria mottle virus (ParMV) is proposed, differs in host range from other ilarviruses. ParMV was purified from Chenopodium quinoa by sap clarification with chloroform, and differential and sucrose density gradient centrifugation.
Purified particles were quasi-isometric to ovoid with diameters of about 24, 29 and 36 nm; no bacilliform particles were detected. Buoyant density in caesium chloride, determined on glutaraldehyde-fixed virus, was 1.35 g/cm³.
An antiserum to ParMV was obtained with a titre of 1:32 in agar gel double-diffusion tests. ParMV did not react with eight other viruses or virus strains belonging to the ilarvirus group or to pelargonium zonate spot virus.
Polyacrylamide gel electrophoresis indicated that ParMV contains a single major protein species of estimated molecular weight 24300 Da and four RNA species of estimated molecular weight 1 37, 1.19, 0.86 and 0.36 × 10⁶ Da.     

香石竹斑驳病毒(CarMV)云南分离物CP基因的克隆和序列分析

 香石竹斑驳病毒(CarMV)是侵染香石竹的主要病毒。关于该病毒的株系和不同地区分离物的病毒核酸序列研究报道较少。     

进境玉米种子携带玉米褪绿斑驳病毒的检测与鉴定

 Imported maize seeds were planted in a quarantine greenhouse and two seedlings with chlorotic mottle symptoms were observed. The ELISA results showed that the two seedlings reacted positively with antibody against Maize chlorotic mottle virus (MCMV). The positive samples were further identified by RT-PCR method and the 711 bp size target bands were amplified specifically. The similarity range of nucleotide sequence of both RT-PCR product and six MCMV coat protein genes was 97%-99%. The amino acid sequence homology was 97.88%-99.89%. Based on the above results, the virus was identified as MCMV. It was the first time that MCMV was intercepted from imported maize seeds.