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.     

An analysis of the diphenylamine reaction in sap of healthy and leaf roll virus infected potato plants

Summary An analysis was made of a difference in colour development between saps from healthy and leaf roll virus (PLRV) infected potato plants, manifested in the diphenylamine reaction ofDische. The colour development was found to be caused by sugars. The differences in colour development were due to the sugar concentration which could be different in healthy and diseased leaves, and not to DNA in the latter. We could correlate the increases in sugar concentration with the severity of virus symptoms. The mechanical transmission of the PLRV, as suggested byBrandenburg (1962), could not be confirmed.Samenvatting Een verschil in kleur, dat tussen sap van gezonde en bladrolviruszieke aardappelplanten door de difenylmaine-reactie vanDische kan optreden, werd geanalyseerd. Het bleek dat de kleurontwikkeling werd veroorzaakt door suikers. De verschillen in kleur van de reactiemengsels waren te wijten aan het feit dat de suikerconcentratie in bladeren van zieke planten een hogere waarde kan bereiken dan in bladeren van gezonde planten en niet aan een hoger desoxyribonucleïnezuurgehalte in zieke bladeren. Er bleek een duidelijke correlatie te bestaan tussen de stijging van de suikerconcentratie en de sterkte van de bladrolsymptomen. De mechanische overdracht van het bladrolvirus, zoals die doorBrandenburg (1962) was gesuggereed, kon niet worden bevestigd.     

Incidence and molecular characterization of potato leaf roll virus in seed potato production in Serbia

European Journal of Plant Pathology - The distribution and frequency of potato leaf roll virus in the four most important potato growing regions in Serbia were studied during the seven years...     

Impact of the potato inoculation date on potato virus Y load and viral distribution in daughter tubers at harvest

Aged plants are more difficult to infect than young plantlets. This modification of susceptibility is described as mature plant resistance (MPR). For potato virus Y (PVY), MPR is known to lead to low infection rates of plants inoculated at the postflowering stage and a decrease in the number of infected daughter tubers. However, the impact of inoculation date on the capacity of PVY to accumulate in daughter tubers has not been studied so far. Field and greenhouse experiments were carried out to better understand PVY epidemiology and to help potato growers to evaluate consequences of early/late infections on the quality of their crops. In field trials, potato plants (cv. Bintje) were covered by insectproof nets from planting to harvest except for a 14-day period to expose plants to natural PVY infections. Under controlled conditions, potato plants were mechanically inoculated with PVY at different dates from preflowering stages (early inoculations) to postflowering stage (late inoculations). At harvest, daughter tubers were individually collected and analysed to define proportions and viral load of infected tubers according to the time between virus inoculation and harvest. Our results showed that although the age of plants at the time of inoculation can modify their susceptibility to PVY infection, in return, early and late PVY inoculations lead to similar rates of infected tubers at the plant scale and equivalent viral accumulation in infected tubers. All together, these data revealed that both early/late infections are high risks for the sanitary quality of potato tubers.     

Enzyme-linked immunosorbent assay (ELISA) for the detection of potato viruses S and M in potato tubers

Enzyme-linked immunosorbent assay (ELISA) with alkaline phosphatase successfully detected potato virus S (PVS) and potato virus M (PVM) in secondarily infected tubers of some Dutch potato cultivars. Extinction was higher for PVS than for PVM but values for both declined slightly within 8 weeks of lifting and it is suggested that testing be carried out within this period. Values (A405) of ELISA reactions between healthy and infected tubers were statistically significant and storage at 4° or 20°C had no effect on detectability of the viruses.Samenvatting Aardappelvirus S kon zowel in knollen van oogst 1978, die gedurende 49 weken bij 4°C waren bewaard en waarvan de kiemrust reeds was verbroken, als in knollen van oogst 1979, die bij 4° of 20°C waren bewaard en nog in de kiemrust verkeerden, tot 8 weken na rooien betrouwbaar met ELISA worden aangetoond. Aardappelvirus M kon eveneens met ELISA betrouwbaar worden aangetoond in knollen van oogst 1979, bewaard bij 4° of 20°C, tot 8 weken na het rooien.De extinctiewaarden voor aardappelvirus S waren hoger dan die voor aardappelvirus M. De waarden voor beide virussen vertoonden een daling gedurende de onderzoekperiode (tot 8 weken na rooien). Er kon geen effect van de bewaartemperatuur (4° en 20°C) op de aantoonbaarheid van de virussen worden aangetoond. Geen verschillen werden waargenomen tussen de extinctiewaarden van het sap uit navel- en krooneinden van de knollen, die nog in de kiemrust verkeerden.Guest worker from April–September 1979 as a fellow of the International Agricultural Centre, Wageningen, the Netherlands, from the Agricultural Research Centre, Yanco, NSW 2703, Australia.     

Effect of potato physiology on the interpretation of ELISA results for potato leafroll virus

This paper records the occurrence of an antigen produced in plants infected by potato leafroll virus (PLRV), which is copurified with the virus and induces an immune response in rabbits used for virus antiserum production. The antigen also appeared to be produced in uninfected but physiologically stressed potato plants. These plants reacted with antisera to PLRV in ELISA tests, thereby giving false positive results. We refer to the compound as virus-stress antigen (VSA). The importance of this finding, not only to serological testing for PLRV, but also as a possible explanation for some false-positive reactions that occur with other host-virus combinations, is discussed. The necessity of having detailed information on plants from which samples are taken for testing is emphasized.     

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.     

A test of taxonomic and biogeographic predictivity: resistance to Potato virus Y in wild relatives of the cultivated potato

A major justification for taxonomic research is its assumed ability to predict the presence of traits in a group for which the trait has been observed in a representative subset of the group. Similarly, populations in similar environments are expected to be more alike than populations in divergent environments. Consequently, it is logical to assume that taxonomic relationships and biogeographical data have the power to predict the distribution of disease resistance phenotypes among plant species. The objective of this study was to test predictivity in a group of widely distributed wild potato species, based on hypotheses that closely related organisms (taxonomy) or organisms from similar environments (biogeography) share resistance to a simply inherited trait (Potato virus Y [PVY]). We found that wild potato species with an endosperm balance number (EBN) of 1 (a measure of cross compatibility) shared resistances to PVY more than species with different EBN values. However, a large amount of variation was found for resistance to PVY among and within species. We also found that populations from low elevations were more resistant than those from high elevations. Because PVY is vectored by aphids, we speculate that the distribution of aphids may determine the level of selection pressure for PVY resistance.     

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.     

Crop border and mineral oil sprays used in combination as physical control methods of the aphid‐transmitted potato virus Y in potato

BACKGROUND: The objectives of this work were to determine if the control of potato virus Y (PVY, genus Potyvirus, family Potyviridae) in seed potato could be improved by combining border crops and mineral oil sprays, and if the border crop acts as a barrier or a virus sink. RESULTS: Field tests over 3 years confirmed that mineral oils alone are an effective barrier to PVY, and showed that borders alone act as a PVY sink. Combining the familiar mineral oil and the more recent crop border methods was almost twice as effective in reducing PVY incidence as either one used alone. The combination provided consistently high PVY control compared with the variable and often lower level of control by either method alone. The contribution of the oil to PVY reduction was similar whether it was applied to the border, the center seed plot, or both. Oil application to the border alone should not affect efficacy and would help keep control costs down. CONCLUSION: Combining border and oil provided the best reduction in PVY incidence 3 years out of 3, providing producers with a tool to reduce year‐to‐year variation in the effectiveness of crop borders or oil sprays used separately. © Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri‐Food Canada. Published by John Wiley and Sons, Ltd.     

侵染广东甘薯的甘薯曲叶病毒分子检测与鉴定

甘薯曲叶病是广东甘薯的一种新病害,病株表现为叶片皱缩,向上卷曲,叶脉肿大等症状.PCR检测结果显示,所有病样中均存在菜豆金色花叶病毒属病毒.通过滚环扩增方法获得了3个病毒分离物的全基因组.扩增产物克隆及序列分析结果表明,这3个病毒分离物基因组均仅含有DNA-A,具有菜豆金色花叶病毒属单组分病毒基因组典型特征;其大小分别为2 829 nt (JX286653、JX286654)和2 828 nt(JX286655).三者核苷酸序列同源率为96.0％～98.4％;与已报道的甘薯曲叶病毒19个分离物的同源率均大于89.0％.因此,引起广东甘薯曲叶病的病原被鉴定为甘薯曲叶病毒.本研究是首次在广东发现甘薯曲叶病毒.     

Comparison of three methods for the detection of Potato virus Y in seed potato certification

Journal of Plant Diseases and Protection - Potato virus Y (PVY) is becoming increasingly important in potato growing regions worldwide. The main reason for this is an increase in the incidence of...     

Chenopodium quinoa,a herbaceous test plant for chlorotic leaf spot virus in apple

The chlorotic leaf spot virus which occurs latent in apples, is widespread in Dutch orchards. In the present paper sap inoculation onChenopodium quinoa is recommended for rapid indexing of trees. Young leaves gave satisfactory results when tested during the first 4–6 weeks after expansion of the buds.The best results were obtained when homogenated petals were used as inoculum.Samenvatting Het chlorotische-bladvlekkenvirus komt in Nederland zeer algemeen latent voor in alle appelrassen. Het virus kan door middel van sapinoculatie opChenopodium quinoa worden aangetoond. Een week na de inoculatie reageren de toetsplanten met lokale vlekken op de bladeren. Deze methode is veel sneller dan de indicatie met behulp van houtige toetsplanten. Hoewel gedurende het gehele seizoen virusoverdracht mogelijk is, kan het virus goed worden aangetoond met jong blad in de eerste 4 tot 6 weken na het uitlopen van de bladknoppen. Nog betere resultaten worden verkregen met bloemblaadjes.     

Ralstonia solanacearum as a potato pathogen in Serbia: Characterization of strains and influence on peroxidase activity in tubers

Since 2011, the outbreaks of brown rot caused by Ralstonia solanacearum race 3, biovar 2, phylotype IIB-1 (R3/B2/PIIB-1) have significantly compromised potato production in Serbia. During 6 years of monitoring (2013–2018) among 3,524 potato tuber samples, 344 were found positive for brown rot disease. R. solanacearum R3/B2/PIIB-1 was isolated from seven cultivars among 12 monitored, and in five localities among 17 monitored. Cultivar Lady Claire was found to have the highest disease frequency (31.98%). A total of 78 isolates were identified by R. solanacearum-specific primer pairs (PS-1/PS-2 and OLI-1/Y-2), as well as the following tests: restriction fragment length polymorphism analysis, biovar determination, immunofluorescence, biochemical analysis, and pathogenicity. The genetic composition of 36 selected isolates assessed using multilocus sequence analysis with seven genes (adk, gapA, gdhA, gyrB, ppsA, hrpB, and fliC) showed that all isolates originating from Serbian potato were homogeneous. By using the TCS algorithm of concatenated sequences to get insight into the phylogeography of isolates and other R. solanacearum strains deposited in the NCBI database, we showed that their origin is undetermined. Peroxidase (POD) activity was measured in brown rotted potato tubers. A positive correlation was found between POD activity and disease severity rated on the analysed tubers. In general, POD activity increased by 2–22 times in vascular necrotic tissues compared to non-necrotic ones, and depended on disease severity but not on cultivar. Native polyacrylamide gel electrophoresis analysis of POD profiles resulted in a total of 10 distinct POD isoforms, of which PODs 3–5 were highly intensified in response to R. solanacearum.     

Characterization of a potyvirus from eggplant (Solanum melongena) as a strain of potato virus Y by N-terminal serology and sequence relationships

A potyvirus (eggplant mottle virus, EMoV) causing mosaic mottling in eggplant ( Solanum melongena ) was characterized on the basis of biological, serological and partial nucleotide sequence properties. EMoV infected Chenopodium amaranticolor and members of the Solanaceae. Polyclonal antiserum against EMoV showed antigenic relationship with henbane mosaic potyvirus (HMV) and potato Y potyvirus (PVY). Virus-specific antibodies directed to the N-terminal region of EMoV cross-reacted only with PVY. Determination and comparison of nucleotide sequence of the coat protein (CP) and the 3'-untranslated region (UTR) of EMoV with other potyviruses showed that the level of homology was highest with PVY isolates. Comparative sequence analyses of the CP amino acid and 3'-UTR sequences with distinct PVY isolates placed EMoV within the PVY^O subgroup.     

苜蓿花叶病毒ELISA检测方法的建立及其在大豆病毒调查和抗性资源鉴定中的应用

苜蓿花叶病毒(alfalfa mosaic virus, AMV)是一种世界性分布、宿主范围广、具有严重危害性的植物病毒,能引起大豆的严重病害。本研究利用原核表达的AMV CP蛋白制备的抗血清,建立了高效、准确的AMV间接ELISA检测方法,并应用于病害调查和抗性鉴定,结果表明制备的3份抗血清对重组蛋白和AMV感染的大豆植物粗提液的效价均达到256 000倍,血清特异性分析结果显示3份抗血清仅识别感染AMV的大豆叶片,不识别感染大豆花叶病毒(soybean mosaic virus, SMV)的大豆叶片。通过建立的AMV间接ELISA与常规RT-PCR同时对采集的50份疑似感染AMV的大豆样品进行检测,有46份样品检测结果一致,符合率达92%。利用建立的AMV ELISA方法和课题组已建立的SMV ELISA方法对吉林省大豆主产区的大豆样品进行病毒检测的结果表明,病毒检出率为38.30%,SMV的检出率达30.85%,AMV的检出率达17.06%,复合侵染率为9.61%。对接种AMV的40个大豆品种进行抗性鉴定,结果显示40份大豆全部感染AMV,但是病毒载量存在差异,部分品种表现出AM...     

Comparison between ELISA and bio tin-la belled probes from cloned cDNA of potato virus X for the detection of virus in crude tuber extracts

Diseases caused by potato virus X (PVX) are of significant agronomic importance, and early detection is vital. Biotinylated DNA probes were prepared by random-primed labelling from cDNA inserts and used for the detection of PVX in crude extracts of infected tubers. The minimum detection level in these extracts is in the order of femtograms of PVX RNA. Comparison with ELISA showed that the hybridization method is 100–250 times more sensitive. Probes prepared by this method are highly specific for target RNA even in crude tuber extracts.