Detection of potato virus Y in early-harvested potato tubers by cDNA hybridization and three modifications of ELISA1

The standard ELISA method for detection of PVY in dormant potato tubers was compared with recently developed methods: (1) a hybridization technique, using a complementary DNA-probe to PVY^N; (2) three modifications of ELISA (standard monoclonal, Flegg & Clark polyclonal or monoclonal). Sap of six Dutch cultivars secondarily infected with PVY was subjected to these methods directly after lifting and after six subsequent one-week intervals during storage at 20°C. During storage the detectability of PVY decreased, irrespective of the diagnostic method applied. However, decrease was less in susceptible than in resistant cultivars. Although the hybridization technique yielded slightly better results than the standard ELISA procedure, it was time-consuming and involved hazardous reagents. The modifications of ELISA investigated were not, in general, more sensitive than standard ELISA.     

Breakdown of potato virus X resistance gene NX: selection of a group four strain from strain group three

Isolate DX of potato virus X (PVX) caused the typical reactions of a group three strain, systemic top necrosis in presence of potato hypersensitivity gene Nx and mosaic with gene Nb. When tubers harvested from DX-infected plants of the Nx: Nb cultivars Maris Piper and Pentland Dell were, grown on, most progeny plants were healthy, but some had systemic top necrosis caused by DX while others had mosaic symptoms in upper leaves and severe necrosis in lower ones. Infective sap from the plants with mosaic and necrosis always caused similar symptoms when inoculated to Pentland Dell. Only group four PVX strains can cause systemic infection without top necrosis in Nx:Nb cultivars, so the affected plants all contained a group four strain. That the severe necrosis of their lower leaves resulted from a shock reaction rather than from isolate DX also being present was indicated because the plants partially recovered, inoculations from near to the dilution end-point failed to demonstrate any separation of strains and behaviour on back-testing to Pentland Dell was unchanged after passage twice through tubers of this cultivar. Also, although DX caused severe necrotic symptoms in tubers of both Nx: Nb cultivars, these symptoms did not develop in tubers of plants with mosaic and necrosis. When cultured serially in Nicotiana glutinosa , the group four strain eventually reverted to group three.     

The movement of potato virus Y (PVY) in the vascular system of potato plants

Potato virus Y (PVY) is responsible for major viral diseases in most potato seed areas. It is transmitted by aphids in a non-persistent manner, and it is spread in potato fields by the winged aphids flying from an infected source plant to a healthy one. Six different PVY strains groups affect potato crops: PVY^C, PVY^N, PVY^O, PVY^N:O, PVY^NTN, and PVY^N-Wi. Nowadays, PVY^NTN and PVY^N-Wi are the predominant strains in Europe and the USA. After the infection of the leaf and accumulation of the virus, the virus is translocated to the progeny tubers. It is known that PVY^N is better translocated than PVY^O, but little is known about the translocation of the other PVY strains. The translocation of PVY occurs faster in young plants than in old plants; this mature plant resistance is generally explained by a restriction of the cell-to-cell movement of the virus in the leaves. The mother tuber may play an important role in explaining mature plant resistance. PVY is able to pass from one stem to the other stems of the same plant through the vascular system of the mother tuber, but it is unknown whether this vascular link between stems is permanent during the whole life of the plant. Two greenhouse trials were set up to study the spread of PVY in the vascular system of the potato plant. The PVY-susceptible cultivar Charlotte was used for both trials. It was demonstrated that all stems growing from a PVY-infected tuber will become infected sooner or later, and that PVY^N-Wi translocates more efficiently to progeny tubers than PVY^NTN. It was also demonstrated that the progressive decay of the mother tuber in the soil reduces the possibility for virus particles to infect healthy stems through the vascular system of the mother tuber. This new element contributes to a better understanding of the mechanism of mature plant resistance.     

Distribution of Potato virus Y in potato plant organs, tissues, and cells

The distribution of Potato virus Y (PVY) in the systemically infected potato (Solanum tuberosum) plants of the highly susceptible cultivar Igor was investigated. Virus presence and accumulation was analyzed in different plant organs and tissues using real-time polymerase chain reaction and transmission electron microscopy (TEM) negative staining methods. To get a complete insight into the location of viral RNA within the tissue, in situ hybridization was developed and optimized for the detection of PVY RNA at the cellular level. PVY was shown to accumulate in all studied leaf and stem tissues, in shoot tips, roots, and tubers; however, the level of virus accumulation was specific for each organ or tissue. The highest amounts of viral RNA and viral particles were found in symptomatic leaves and stem. By observing cell ultrastructure with TEM, viral cytoplasmic inclusion bodies were localized in close vicinity to the epidermis and in trichomes. Our results show that viral RNA, viral particles, and cytoplasmic inclusion bodies colocalize within the same type of cells or in close vicinity.     

Properties of viruses of the potyvirus group. 1. A simple method to purify bean yellow mosaic virus,pea mosaic virus,lettuce mosaic virus and potato virus YN

Bean yellow mosaic virus, pea mosaic virus, lettuce mosaic virus, and potato virus Y^N were purified by homogenizing and clarifying infected leaves in a mixture of 0.1 M tris-thioglycollic acid buffer pH 9, carbon tetrachloride and chloroform, followed by differential centrifuging applying moderate centrifugal forces.Samenvatting Het bonescherpmozaïekvirus B25, het erwtemozaïekvirus E198, het slamozaïekvirus en het aardappelvirus Y^N konden worden gezuiverd door geïnfecteerde bladeren te vermalen in een mengsel van 0,1 M tris-thioglycolzuur pH 9, tetrachloorkoolstof en chloroform, en de geklaarde suspensie vervolgens differentiëel te centrifugeren bij matige centrifugaalkrachten.     

Enzyme-linked immunosorbent assay (ELISA) for the detection of potato viruses A and Y in potato leaves and sprouts

With the enzyme-linked immunosorbent assay (ELISA) potato virus A (PVA) could be detected reliably in potato sprouts, especially when these were young and sappy. The detection of this virus in leaves of glasshouse-grown potato plants was less reliable. The tobacco veinal necrosis strain of potato virus Y (PVY^N) was readily demonstrated in foliage of glass-house-grown potato plants using an antiserum to this strain. Plants infected with the common strain (PVY^O) did not react in ELISA with this antiserum. In young sappy sprouts, using the PVY^N antiserum, PVY^N could be detected reliably when samples with PVY^O were excluded, as the reaction of samples infected with the latter virus was intermediate between PVY^N-diseased and PVY-free samples. PVY was also detected in plants inadvertently infected during the experiments.     

Some experiments and considerations on the identification of witches' broom viruses,especially in clovers,in the Netherlands and Italy

Witches' broom phenomena (witches' broom growth + antholysis) observed inTropaeolum majus and in clovers in the Netherlands and in several wild and cultivated plants in Italy were demonstrated to be due to virus infection. The virus(es) could be transmitted by grafting, through dodder and by means of leaf-hoppers (Euscelis spp. primarily). The virus isolates found in both countries did not differ appreciably in the symptoms they caused. Many of the deviations concerned have been known for a long time as teratological phenomena. In the extensive literature considerable confusion exists concerning the identity of several witches' broom virus diseases. They constitute a good example of the problem of virus variability. A continuous splitting of witches' broom viruses on the basis of slight differences in symptom expression or of vector specificity does not seem justified. Since no intrinsic properties of the virus(es) concerned are known, the only conclusion that can be drawn from the information available is that the Dutch and the Italian isolates show a great similarity to aster yellows virus as well as to tomato big bud, stolbur and other witches' broom viruses.     

Serological identification of some soil-borne viruses causing diseases in fruit crops in the Netherlands

Samenvatting Tot voor kort was slechts weinig bekend over de identiteit van de virussen, die verantwoordelijk zijn voor de lepelbladziekte van rode bes en de Eckelraderziekte van kers. Een antiserum werd gemaakt tegen het lepelbladvirus en toetsingen werden uitgevoerd. Het lepelbladvirus bleek een stam van het Schotse raspberry ring spot-virus te zijn. Ook een virus uit Eckelraderzieke kersen bleek met het antiserum tegen lepelbladvirus te reageren. Het antiserum tegen lepelbladvirus reageerde met alle getoetste isolaties van dit virus tot een verdunning van 1/512, doch met een viertal isolaties uit Eckelraderzieke kersen tot een verdunning van slechts 1/128. Hoewel het lepelbladvirus en het virus uit Eckelraderzieke kersen dus duidelijk verwant zijn, bleek, ook uit premunitieproeven, dat er verschillen zijn. De verwantschap tussen het virus uit Eckelraderzieke kersen en het raspberry ring spot-virus is uit verschillende proefresultaten gebleken.Een derde virus, afkomstig uit aardbeiplanten van het ras Red Gauntlet, geïmporteerd uit Schotland, bleek te reageren met een antiserum tegenHarrisons beet ring spot stam van het tomato black ring-virus. Dit antiserum werd verstrekt door Dr.C. H. Cadman, Schotland.     

湖北甘薯病毒病的检测与鉴定

2013—2015年采集了湖北黄冈、鄂州、武汉、荆州以及宜昌等5个地区的甘薯病毒病样品,通过双生病毒通用引物PCR扩增、ds RNA技术和序列分析等方法,鉴定了这5个地区甘薯病毒病的病原。结果显示,甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)、甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)和甘薯卷叶病毒(Sweet potato leaf curl Georgia virus,SPLCGV)等4种病毒被检出。其中,SPFMV SPLCGV这两种病毒在湖北皆为首次报道。     

Properties of viruses of the potyvirus group. 2. Buoyant density,S value,particle morphology,and molecular weight of the coat protein subunit of bean yellow mosaic virus,pea mosaic virus,lettuce mosaic virus,and potato virus YN

The buoyant densities of bean yellow mosaic virus (BYMV) B25, pea mosaic virus (PMV) E198, lettuce mosaic virus (LMV), and potato virus Y^N (PVY^N) were 1.318, 1.321, 1.330, and 1.326 g/ml, respectively. Their S values were 143, 140, 143, and 145 S. The particle morphology of BYMV B25, PMV E198, and LMV could reversibly be changed by magnesium ions. PVY^N particles were broken in the presence of magnesium ions. The molecular weight of the coat protein subunit of the four viruses was 34,000 daltons. In many preparations also a 28,000 daltons component was present. This must be considered to be a breakdown product, derived from the 34,000 daltons component by proteolytic activity.Samenvatting De zweefdichtheden van het bonescherpmozaiekvirus (BYMV) B25, het erwtemozaiekvirus (PMV) E198, het slamozaïekvirus (LMV) en het aardappelvirus Y^N (PVY^N) bedroegen respectievelijk 1,318, 1,321, 1,330 en 1,326 g/ml. Hun sedimentatiecoëfficiënten waren 143, 140, 143 en 145 S. De deeltjesvorm van BYMV B25, PMV E198 en LMV kon reversibel worden veranderd door toevoeging en verwijdering van magnesium-ionen. Deze induceerden het optreden van breuken in de deeltjes van PVY^N. Het molecuulgewicht van de manteleiwiteenheid van de vier virussen bedroeg 34.000 daltons. In veel preparaten was bovendien een component van 28.000 daltons aanwezig. Deze moet worden beschouwd, als een proteolytisch afbraakprodukt dat ontstaat uit de component van 34.000 daltons.     

The long-term preservation of potato virus Y and watermelon mosaic virus in liquid nitrogen in comparison to other preservation methods

Samples of watermelon mosaic virus (WMMV), consisting of clarified sap of diseased squash plants, were found to be very infective after more than 4 years of storage if the samples had been deep frozen at –18°C, and to have lost most of their infectivity within 4 years of preservation by freeze drying and subsequent storage of the samples at 4°C.Potato virus Y (PVY) inocula, consisting of samples of clarified sap of diseased red peppers, lost their infectivity in less than 4 years if the samples had been deep frozen at –18°C but retained their infectivity for more than 4 years if the samples had been freeze dried and stored at 4°C.A decrease in infectivity of both viruses in the deep frozen at –18°C and freeze dried inocula could be observed even during the first months of storage. However, samples of clarified sap stored in or over liquid nitrogen maintained their activity for at least 22 months for WMMV and 32 months for PVY with no indication of a decrease in infectivity of the viruses. Storage in or over liquid nitrogen seems therefore a very promising long-term preservation method for plant viruses.Samenvatting Watermeloenmozaïekvirus inocula, bestaande uit helder gecentrifugeerd sap van zieke squashplanten, bleken hun infectievermogen zeer goed te hebben behouden na meer dan vier jaar bewaring bij –18°C, maar binnen vier jaar grotendeels te hebben verloren na droogvriezen en bewaren bij 4°C.Overeenkomstig bereide aardappelvirus Y inocula uit zieke paprikaplanten hadden hun infectievermogen binnen vier jaar verloren na bewaring bij –18°C, maar konden hun infectievermogen grotendeels behouden gedurende meer dan vier jaar na droogvriezen en bewaring bij 4°C.Binnen enkele maanden na het begin der bewaringsperiode kon al een afname van het infectievermogen van beide virussen in de diepgevroren (–18°C) en drooggevroren inocula worden gevonden. Helder gecentrifugeerde sapmonsters die in of boven vloeibare stikstof bewaard waren, hadden gedurende tenminste 22 maanden voor het watermeloenmozaïekvirus en 32 maanden voor het aardappelvirus Y hun volle infectievermogen behouden. Bewaring in of boven vloeibare stikstof is daarom de beste van de drie getoetste bewaarmethodes voor opslag op lange termijn van beide virussen. Deze methode lijkt ook veelbelovend voor een groot aantal andere virussen.     

The translocation of potato virus S,isolate “Eersteling”, in potato plants

Samenvatting Uit enige infectieproeven met aardappel-S-virus, isolatie Eersteling, blijkt dat planten van het ras Bintje onder kasomstandigheden zeer moeilijk met deze stam zijn te besmetten. Planten van het ras Mentor zijn vatbaarder, maar kunnen slechts in een jong stadium (twee tot vier weken na het poten) worden besmet.     

Pelargonium flower-break and pelargonium line pattern viruses in the Netherlands; purification,antiserum preparation,serological identification,and detection in pelargonium by ELISA

Two viruses, detected frequently in the Netherlands in pelargonium, were identified by serology and test plant reactions. Antisera were prepared and an ELISA procedure was developed to detect the viruses in pelargonium.One of the viruses, PFBV-N, proved to be pelargonium flower-break virus. With the antiserum to PFBV-N, it could be detected reliably throughout the year inPelargonium zonale Springtime Irene.The other virus, PLPV-N, was serologically closely related to pelargonium line pattern virus (PLPV) and to pelargonium ring pattern virus (PRPV), as were an old virus isolate from Saturnus, collected in the Netherlands in 1971 (L128), and PLPV isolates from Yugoslavia (PLPV-Y) and Denmark (PLPV-D). There were only minor differences in host-plant reactions between the virus isolates. Based on these tests, PLPV and PRPV are considered as isolates of the same virus, for which, for practical reasons, the name pelargonium line pattern virus is proposed.PLPV could be reliably detected by ELISA inP. zonale Springtime Irene and Amanda throughout the year with only a few exceptions. InPelargonium peltatum Tavira, however, reslts were erratic due to uneven distribution of virus in the plant. Best results were obtained when petioles of fully expanded leaves were tested.     

盆栽文心兰上的凤仙花坏死斑病毒的检测与分子鉴定

应用酶联免疫吸附检测法(ELISA)和RT-PCR技术从表现有同心圆褪绿斑的进境盆栽文心兰上检测出凤仙花坏死斑病毒.ELISA检测发现该病毒在文心兰上分布不均匀,病毒主要集中在表现症状的病斑处.同时,根据该病毒S RNA上的核衣壳蛋白(N)基因序列保守区设计了一对特异性引物,利用RT-PCR方法检测得到500bp的预期DNA片段.经HindⅢI和EcolI酶切验证后克隆了该序列片段,序列分析结果表明该病毒的N基因序列和已经发表的凤仙花坏死斑病毒(登陆号为AB109100、AB207803、DQ425096)核苷酸序列同源性为99%,进一步确认进境盆栽文心兰上携带凤仙花坏死病毒.这是INSV首次在中国的报道,也是第一次在文心兰上发现该病毒.     

Investigations of currant viruses in the Netherlands II. Further observations on spoon leaf virus,a soil-borne virus transmitted by the nematodeLongidorus elongatus

Spoon leaf virus (SLV) of red currants spreads very slowly in the field. All common Dutch red currant varieties are susceptible as well as some raspberry varieties and a number of weed species. The virus is soil-borne and is transmitted by the nematodeLongidorus elongatus. In cross-protection tests SLV shows a close relationship to raspberry ringspot virus but there are some phytopathological differences between the two viruses. Soil treatment with DD gives a good control of the virus. Planting material can be indexed reliably by means of sap inoculation onChenopodium quinoa.Samenvatting Uit veldwaarnemingen in een oude Bangertse bessetuin te Blokker bleek dat de infectiehaarden van lepelblad zich uitbreiden. De uitbreiding verloopt langzaam en bedroeg in de genoemde tuin van 1959 tot 1964 slechts 8 tot 10 procent (fig. 1). Proeven en waarnemingen toonden aan dat herinplanting in de infectiehaarden binnen korte tijd leidt tot herinfectie (tabel 1). Alle gangbare Nederlandse besserassen zijn vatbaar voor lepelbladvirus evenals sommige frambozerassen (fig. 2) en een aantal onkruidsoorten (tabel 2). In appels, peren en pruimen kon het virus niet worden aangetoond. Uit premunitieproeven (tabel 5) bleek, dat lepelbladvirus nauwer verwant is met raspberry ringspot-virus (RRV) dan met de RRV-stam van Eckelraderizieke kers. Er werd geen verwantschap gevonden tussen het lepelbladvirus en tomato black ring-virus (TBRV). Lepelbladvirus blijft in de grond achter en blijkt voornamelijk gelocaliseerd te zijn in de bovenste grondlaag ter dikte van 20 cm (tabel 3). Het virus wordt overgebracht doorLongidorus elongatus, een aaltje, dat in de Bangert algemeen voorkomt. Infectie met lepelbladvirus kan worden voorkomen door een grondbehandeling met DD (800 1 per ha). Selectiemateriaal kan op eenvoudige en betrouwbare wijze getoetst worden door middel van sapinoculatie opChenopodium quinoa.     

Relative efficiency of a number of aphid species in the transmission of potato virus YN in the Netherlands

This paper reports the results of live-trapping winged aphids in an Ashby (1976) trap in potato crops in the Netherlands from 1983–1987. During this period, a total of 122 aphid species were trapped. Although only four of those species were able to colonise potato, 26 of them were able to transmit PVY^N from potato to potato test plants. The transmission rates and relative efficiency factors (REF's) of those transmitters were determined. Aphis sambuci, Cryptomyzus galeopsidis, Dysaphis spp.,Hyadaphis foeniculi, Hyalopterus pruni andMyzus cerasi were recorded for the first time as vectors of PVY^N in the Netherlands.The numbers of aphids per species caught per season differed very much, also the virus transmission results of some fluctuated from year to year, e.g.Brachycaudus helichrysi. The REF's in various reports differ greatly, thus the value of a universal REF is doubtful. Assessment of the rate of virus spread in a potato crop is discussed.Samenvatting Van de in Nederland voorkomende aardappelvirussen wordt het aardappelvirus Y^N (PVY^N) als het meest schadelijke bij de pootgoedproduktie beschouwd. Dit virus kan op non-persistente wijze door een aantal bladluissoorten worden overgebracht. De groene perzikluis,Myzus persicae wordt geacht dit het meest efficiënt te kunnen doen.In de periode 1983–1987 is onderzocht welke bladluissoorten, geregistreerd in aardappelpercelen te Wageningen, Y^N-virus kunnen overbengen. In deze periode werden 122 gevleugelde bladluissoorten met behulp van een Ashby-val levend gevangen en op hun vermogen om Y^N-virus van aardappel naar aardappel over te brengen getoetst. 26 soorten hiervan zijn potentiële Y^N-virusoverbrengers. De relatieve efficiëntiewaarden (REF) voor elk van deze soorten werden opnieuw berekend.De REF-waarde voorBrachycaudus helichrysi werd berekend op 0,21, in afwijking van de waarde (0,01) die Van Harten (1983) aan deze toekende na waarnemingen gedurende slechts één seizoen.40% van het totaal aantal geregistreerde bladluizen bestond uitCavariella aegopodii, M. persicae, Metopolophium dirhodum, Rhopalosiphum insertum, R. padi enSitobion avenae. Minder talrijk (6,5%) was de groepB. helichrysi, Brachycandus spp. enMacrosiphum euphorbiae. De groepAphis nasturtii, Myzus certus enPhorodon humuli had een aandeel van 3,2% in de totale vangst. BehalveC. aegopodii zijn de genoemde soorten in staat het PVY^N over te brengen van aardappel naar aardappel. Van de rest zijn echter ook een aantal soorten in staat PVY^N over te brengen maar die zijn slechts in geringe mate gevangen. A. nasturtii, Brachycaudus spp.,M. certus enM. persicae besmetten vaker dan andere soorten aardappelplantjes met PVY^N. Van de soortenAphis sambuci, Cryptomyzus galeopsidis, Dysaphis spp.,Hyadaphis foeniculi, Hyalopterus pruni andMyzus cerasi werd voor de eerste keer in Nederland overdracht van PVY geconstateerd.