Comparison of four techniques for plum pox virus detection

Journal of Plant Diseases and Protection - Twenty-four stone fruit trees showing typical symptoms of plum pox virus (PPV) were tested for PPV using ELISA, RT-PCR, real-time RT-PCR and RT-LAMP. The...     

Rapid detection of plum pox virus by reverse transcription recombinase polymerase amplification

Journal of Plant Diseases and Protection - Plum pox virus (PPV) is one of the most destructive viral pathogens infecting stone fruit trees worldwide. As PPV causes a viral disease that requires...     

Detection of plum pox virus in Spain

Until recently, plum pox (sharka) virus (PPV) was never detected in Spain on any of the material analysed by the enzyme-linked immunosorbent assay (ELISA). It was only in June 1984 that the virus was first detected by two different antiscra in Japanese plum trees ( Prunus salicina ), cv. Red Beaut, showing typical symptoms of the disease. The detection was later confirmed by graft-transmission to GF-305 peach seedlings, and also by immunoelectron microscopy. The PPV was experimentally transmitted from GF-305 to GF-305 by aphids and from GF-305 to herbaceous plants by mechanical inoculation. Thus far (January 1985), PPV has been detected basically in Japanese plum trees in Sevilla, Murcia, Valencia and Castellón, in apricot in Castellón, and in peach trees in Sevilla and Lérida.
De nombreuses analyses par la méthode ELISA n'ont, pendant longtemps, pas permis de détecter le plum pox virus (agent de la sharka) en Espagne. Ce n'est qu'en juin 1984 que la présence du virus a été confirmée, par l'utilisation de deux antiséra différents, chez des pruniers japonais ( Prunus salicina ) cv. Red Beaut qui manifestaient des symptôines typiques de la maladie. La détection a été confirmée par greffage sur des plants du pêcher GF-305, ainsi que par microscopie immuno-électronique. La transmission du PPV de GF-305 à GF-305 a été réalisée à l'aide de pucerons et de GF-305 à des plantes herbacées mécaniquement. A cette date (janvier 1985). le PPV n'a été détecté quc dans des prunicrs japonais dans, les provinces de Sevilla, Murcia, Valencia et Castellón, ainsi que dans des abricotiers en Castellón et dans des pêchers en Sevilla et en Lérida.     

Plum pox virus detection in dormant plum trees by PCR and ELISA

An immunocapture polymerase chain reaction (IC-PCR) protocol and ELISA were compared for their effectiveness in detecting plum pox virus (PPV) in dormant plum material. Although the IC-PCR was about one thousand times more sensitive than ELISA, PPV was detected by ELISA in 71–80% of bark samples collected in December, January and March 1996/97 from pot-grown rootstock trees inoculated with PPV the previous March, compared with 85–86% detection in the same samples by IC-PCR. In similar samples from one-year-old shoots taken from infected branches of orchard trees, 66–81% were positive by ELISA compared with 81–87% by IC-PCR. With bulked samples taken from the fibrous roots of the pot-grown trees, PPV was detected in 92–100% of samples by IC-PCR in winter compared with only 38–65% by ELISA. These results were confirmed in samples from the roots and shoots of the same trees in 1997/98. Three samples per shoot would have been sufficient to detect PPV by ELISA in 87 of the 88 infected shoots tested during the two winters. However, infected shoots are irregularly distributed in diseased trees and PCR assays of root samples offer the potential for improving the reliability of identifying trees infected with PPV.     

First report of Plum pox virus on plum in Finland

In July 2014, leaves showing symptoms of a viral infection were collected from a plum tree serving as a mother tree in a Finnish nursery and found to be infected by Plum pox virus (PPV). A subsequent survey revealed additional infected trees originating from the infected mother tree. This paper provides the first report of PPV, the causal agent of the most destructive viral disease of Prunus, in Finland.     

Differentiation of Mediterranean plum pox virus isolates by coat protein analysis

Six isolates of plum pox potyvirus from different Mediterranean countries were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), peptide mapping and Western blotting after improved purification of virions using a protease inhibitor cocktail that reduced coat protein degradation. One isolate (Spanish isolate 3.3 from plum) differed from the others in possessing a smaller coat protein (approximately 34 instead 36 kDa) with a possible deletion in the surface-exposed amino-terminal region. Infectivity of the viruses after proteolysis, assessed using a local lesion host, was significantly reduced. Protease digestion conditions were established to generate a 28 kDa resistant core of the viral coat protein. Such conditions (longer incubation times or an increase in the enzyme concentration) differed from the milder ones reported for other potyviruses. Implications of the results in relation to the production and screening of virus-specific monoclonal antibodies are discussed.     

Herbaceous host plants for the sharka (plum pox) virus

A number of 75 species belonging to 18 families were tested for their susceptibility and sensitivity to the sharka virus of plum using sap from infectedNicotiana clevelandii leaves; 27 species out of 6 families were found to be new hosts. OnlyRanunculus arvensis may serve as a new test plant. Common weeds and garden plants were among the newly found host plants.Lamium amplexicaule andZinnia elegans became systemically infected. In the glasshouse the virus was transmitted byMyzus persicae from peach seedlings toL. amplexicaule and vice versa. If transmitted in the field as easily as in the glasshouse, elimination of the virus might be very difficult.Samenvatting Uit 18 families werden 75 plantesoorten getoetst op hun vatbaarheid en gevoeligheid voor het sharka-virus van de pruim. De planten werden geïnoculeerd met het sap van geïnfecteerde topbladeren vanNicotiana clevelandii; 27 soorten uit 6 families bleken vatbaar voor het virus. AlleenRanunculus arvensis is wellicht een bruikbare toetsplant. Onder de pas gevonden waardplanten van het sharka-virus bevinden zich ook enkele algemeen voorkomende onkruiden en tuinplanten.Lamium amplexicaule enZinnia elegans werden systemisch door het virus geïnfecteerd. In de kas kon het virus met behulp vanMyzus persicae worden overgebracht van perzikzaailingen naarL. amplexicaule en omgekeerd. Indien de overdracht in de natuur even gemakkelijk verloopt als in de kas, dan kan dit het uitroeien van het sharka-virus in besmette gebieden ernstig bemoeilijken.     

Cross reactions of an antiserum to plum pox potyvirus1

In the early spring of 1992, plum pox-like viruses (PPLVs) were detected by standard ELISA in some Prunus species. The isolates reacted positively with plum pox potyvirus (PPV) antisera in immunosorbent electron microscopy and Western blot analysis. In Western blot analyses, bands associated with the coat protein subunits of the PPLVs were 48–56 kDa, whereas bands associated with the coat protein subunits of known PPV isolates were 32–37 kDa in size. Also, the PPLVs differed from known PPV isolates in their symptoms on woody and herbaceous indicators, and in their herbaceous host range. None of these PPLVs appears to be an isolate of PPV.     

Sensitivity of the detection of plum pox potyvirus by molecular assays1

Recently, plum pox potyvirus (PPV) has been found in Basilicata, southern Italy, on plum, apricot and peach. In 1992-09, we started a large-scale survey to verify the effectiveness of diagnostic methods used during seasons when it is difficult to reveal any presence of the virus. The assays were carried out by dot-blot hybridization on stone-fruit cultivars normally planted in this area. The virus was found, by dot-blot hybridization, to be present in seven cultivars of peach, four of apricot and one of plum. All plants were 8–10 years old and, except for two apricot cultivars, were not displaying any apparent symptoms in spring 1992. Five peach cultivars, intended for use as primary sources of propagation material, were then selected for further study, and assayed in 1992–11 by ELISA and RT-PCR. ELISA tests on these selected peach cultivars were consistently negative, while PCR tests were consistently positive. However ELISA tests gave positive results when repeated in 1993–05. These results not only suggest that primary propagation material should be tested by techniques more sensitive than ELISA, but also question the usefulness of carrying out tests during any phenological phases of the plant.     

Inclusion bodies in plants infected with sharka (plum pox) virus

Sharka virus was found to give rise to the formation of inclusion bodies in nucleus and cytoplasm of host cells, as is known for several other viruses of the potato virus Y group. In inoculatedNicotiana clevelandii needle-shaped inclusion bodies were found loosely distributed in the nucleus 10 days after the first external symptoms appeared. In the cytoplasm, bundles of needles and granular inclusions arose 14 and 18 days, respectively, after external symptoms became visible. The intranuclear needles disappeared shortly before or after the first appearance of granular cytoplasmic inclusions. Inclusion bodies abound in parenchyma cells of fruits from sharka-diseased plum trees, but they did not occur in fruits from sharka-free trees, with or without pseudo-pox symptoms. Thus, inclusion bodies can be of value in the diagnosis of sharka and be of great help in differentiating between plum pox and pseudo-pox.     

The isolation of sharka (plum pox) virus from leaves and fruits of plum with herbaceous plants

Samenvatting Uit bladeren, zowel als uit vruchten van sharka-zieke pruimebomen werd een virus overgebracht opChenopodium foetidum enNicotiana clevelandii. C. foetidum reageerde met duidelijke chlorotische of okerachtige lesies.N. clevelandii werd wel door het virus geïnfecteerd, maar bleek ongeschikt als toetsplant. Het virus werd met sap vanC. foetidum naarN. clevelandii en met de bladluisMyzus persicae vanN. clevelandii naar perzik overgebracht en terug. Zo kon worden aangetoond, dat het geïsoleerde virus het sharka virus is. Een nog niet geïdentificeerd virus kon ook van pruim opC. foetidum worden overgebracht, doch er werden geen symptomen waargenomen op de geinoculeerde bladeren.     

李痘病毒单克隆抗体及TAS-ELISA试剂盒的研制

将纯化的李痘病毒(Plum pox virus,PPV)制剂免疫BALB/c小鼠,用SP2/0骨髓瘤细胞与经李痘病毒免疫的BALB/c小鼠的脾细胞融合,有限稀释法克隆和间接ELISA法筛选出2株稳定分泌李痘病毒单克隆抗体的杂交瘤细胞株3F1,7A8。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分别为IgG1、IgG3。间接ELISA方法测定腹水效价分别为3F1:1.0×106,7A8:1.0×105。以多克隆抗体为包被抗体、单克隆抗体为检测抗体的TAS-ELISA试剂盒与李痘病毒的D株系、M株系的病毒分离物均有反应,与同属的马铃薯A病毒、莴苣花叶病毒、西瓜花叶病毒2号、马铃薯Y病毒坏死株系不发生交叉反应。     

Detection of plum pox virus by isolation of double-stranded ribonucleic acid (dsRNA)

Double-stranded RNA (dsRNA) associated with plum pox virus (PPV) in Nicotiana clevelandii and Prunus domestica has been isolated. While dsRNA was detected in N. clevelandii in considerable amounts by electrophoresis, only small amounts were found in P. domestica. This may be due to viscous substances in the leaves of this woody host. Different PPV strains (NAT - not aphid-transmissible; AT - aphid-transmissible) showed specific patterns in electrophoresis gels. When PPV was assayed in N. clevelandii by dsRNA detection or by standard ELISA or ISEM, all three methods were found to be efficient, with none being superior. ELISA, as a simple and fast routine method, is still the method of choice. DsRNA detection will be suitable for plant disease agents undetectable by ELISA and ISEM.     

Additional data on the ultrastructure of inclusion bodies evoked by sharka (plum pox) virus

Electron microscopy of ultrathin sections of leaves ofNicotiana clevelandii infected with sharka virus revealed several types of cytoplasmic inclusions. Pinwheels and lamellar aggregates were frequent. Pinwheels showed a central core with a threadlike structure in the middle. Lamellar aggregates showed a striation with a periodicity of 55 Å on their surface and they were associated with the endoplasmic reticulum. Irregular crystalline structures were found less frequently. Microbodies were common in the cytoplasm of sharka virus infectedN. clevelandii plants, but they also abounded in healthy controls.Nuclear inclusions were present only for short periods after infection.In leaf extracts irregularly shaped inclusions were found. They had a regular striation of 55 Å. Sometimes nearly parallel stripes were seen on their surface, always at an angle of 80 degrees with the striation.Samenvatting In ultradunne coupes van bladeren vanNicotiana clevelandii, geïnfecteerd met het sharkavirus bleken schoepenradvormige insluitsels (pinwheels) en lamellaire aggregaten algemeen voor te komen. De schoepenradvormige insluitsels vertoonden een centrale holte met daarin een draadvormige structuur (Fig. 2). De lamellaire aggregaten vertoonden een streping met een periodiciteit van 55±5 Å, zowel in ultradunne coupes als in bladextracten (Fig. 1). De lamellaire aggregaten bleken vaak geassociëerd met het endoplasmatisch reticulum (Fig. 3). Kristallijne structuren (Fig. 4) werden slechts incidenteel waargenomen. Microbodies waren wel algemeen, maar zij kwamen ook voor in gezonde controleplanten (Fig. 5).Kerninsluitsels werden met de elektronenmicroscoop aangetoond gedurende de periode, dat zij ook lichtmicroscopisch zichtbaar zijn (Fig. 6). Zij bezaten een duidelijke structuur (Fig. 7).Gewezen wordt op een mogelijke relatie tussen de kerninsluitsels en de kristallijne structuren in het cytoplasma. Wellicht zijn de naaldvormige insluitsels, die met de lichtmicroscoop in de kern en het cytoplasma kunnen worden waargenomen, identiek aan de elektronenmicroscopisch waargenomen kerninsluitsels en kristallijne structuren in het cytoplasma.In bladextracten van planten vanNicotiana clevelandii, geïnfecteerd met het sharkavirus, werden onregelmatig gevormde insluitsels gevonden. Ze vertoonden een fijne, precies evenwijdige streping met een periodiciteit van 55±5 Å. Soms bevonden zich op het oppervlak van het insluitsel ook nog wat onregelmatige, grovere strepen. Beide strepingen vormden steeds een hoek van 80 graden (Fig. 1).     

Transmission of plum pox potyvirus in Spain1

Transmission tests were conducted in the laboratory to determine which are the aphid species responsible for the great natural spread of plum pox potyvirus (PPV) observed in the field in Spain. Woody hosts were used in these tests and different transmission techniques were compared. The aphid species tested were Myzus persicae, Aphis gossypii, A. spiraecola, A. fabae, Hyalopterus pruni and Brachycaudus prunicola. Although the transmission rates obtained were, in general, quite low, it can be stated that, except for B. prunicola (pending confirmation of results), all species tested transmitted PPV under the conditions of the trial.     

Purification of plum pox (sharka) virus with the use of Triton X-100

Plum pox virus was purified by adding up to 5% non-ionic detergent Triton X-100 to extracts clarified by low-speed centrifugation. After stirring for 1/2 h, the suspensions were subjected to 2 cycles of differential centrifugation followed by sucrose density-gradient centrifugation. Purity of the product was confirmed by electron microscopy and equilibrium density-gradient centrifugation in CsCl. The virus sedimented in the analytical ultracentrifuge as a single peak with a sedimentation coefficient of about 170 S at infinite dilution. Virus so purified showed an absorption spectrum with a minimum at 247 nm and a maximum at 263 nm. The modal length of the virus particles in purified preparations was 764 nm. Antiserum prepared had a specific titer of 4096.Samenvatting In een mortier werden systemisch geïnfecteerde bladeren vanNicotiana clevelandii gehomogeniseerd in een overmaat buffer. Na centrifugeren bij laag toerental werd aan de bovenstaande vloeistof Triton X-100 toegediend tot een concentratie van 5%. De suspensie werd een half uur geroerd en vervolgens onderworpen aan differentieel centrifugeren, centrifugeren door een 20% suikeroplossing en centrifugeren in een 10–50 % lineaire suikergtadient (Fig. 2).De infectiositeit van het virus bleek zowel afhankelijk van de wijze van homogeniseren als van de samenstelling en de hoeveelheid van de buffer tijdens het homogeniseren (Tabel 1 en 2). Door het gebruik van Triton X-100 werden veel hogere virusopbrengsten verkregen dan met behulp van ether/tetrachloorkoolstof (Fig. 1). Het gezuiverde virus, vertoonde een absorptiespectrum met een minimum bij 247 nm en een maximum bij 263 nan (Fig. 3) en sedimenteerde in de analytische ultracentrifuge als een enkele pick metaeen sedimentatie coëfficiënt van ongeveer 170 S (Fig. 4). De hoge mate van zuiverheid werd tevens aangetoond met behulp van evenwichtscentrifugering in CsCl en elektronenmicroscopie (Fig. 5). De gemiddelde lengte van de virusdeeltjes in gezuiverde preparaten was 764 nm. Een antiserum kon worden verkregen met een titer van 4096.     

南瓜花叶病毒胶体金免疫层析试纸条的研制

使用柠檬酸三钠还原法制备25nm粒径的胶体金颗粒,在pH7.6,蛋白用量13μg/mL条件下,制备形成稳定胶体金蛋白复合物;使用微定量喷头在硝酸纤维素膜上喷好病毒检测线和质控线,组装制成免疫层析检测试纸条。结果表明经过条件优化后制备的南瓜花叶病毒试纸条特异性好,可在10min内检测出结果,且对南瓜花叶病毒阳性材料的检测灵敏度可达到稀释104倍。