An investigation of the reliability of ELISA as a practical test for detecting potato leaf roll virus and potato virus Y in tubers

Enzyme-linked immunosorbent assay (ELISA), involving mechanical sampling of tubers, was compared with a growing-on test in which virus was assessed visually, and with ELISA on leaves. The percentage infection of potato leaf roll virus and potato virus Y on samples of potential seed tubers sent for advisory test during the winter, was determined by each method. The tuber ELISA underestimated the incidence of both viruses, and was less accurate than the growing-on or leaf ELISA. The effect on the reliability of tuber ELISA of the amount and distribution of virus in the tuber, the compromises made to make the test fast and simple, and the quality of anti-serum and composition of the extraction buffer are discussed.     

生物素标记cDNA探针杂交检测梨树上3种潜隐病毒研究

 本研究合成了苹果茎沟病毒(ASGV)、苹果褪绿叶斑病毒(ACLSV)和苹果茎痘病毒(ASPV)的生物素标记cDNA探针,对斑点杂交和免疫印迹杂交检测这3种病毒的效果进行了分析。以含ACLSV和ASGV克隆片段的大肠杆菌菌液为样品时,斑点杂交检测灵敏度分别为80 cfu/μL和50 cfu/μL。以离体培养砂梨植株为材料,采用斑点杂交法检测砂梨离体培养植株粗提液中的3种病毒,均产生强的杂交信号,且特异性好。比较斑点杂交和ELISA检测病毒含量相对较低的热处理再生植株中ASGV和ACLSV,结果表明斑点杂交具有较高的灵敏度。组织印迹杂交检测砂梨离体植株ASGV和ACLSV的结果显示,这2种病毒在离体植株的各部位均有分布,自基部至茎尖各部位印迹均产生很强的杂交信号。     

Biological characterisation of various geographical isolates of potato virus Y inducing superficial necrosis on potato tubers

A range of selected PVY isolates that induce superficial necrosis on potato tubers, originating from several countries, were compared with standard strains of PVA, PVV and PVY. Biological properties (e.g., host range, aphid transmissibility and relationships based on cross-protection between virus isolates) were studied. PVY^NN isolates differ from the normal PVY^C, PVY^N and PV^O strains by their ability to infect Capsicum annuum but not Chenopodium amaranticolor and C. quinoa. All PVY^NN isolates are transmissible by Myzus persicae , without any significant differences from one standard strain. These additional data confirm that these tuber-necrosing isolates belong to PVY. However, they could be ranged in a homogeneous and distinct group inside the PVY^N group, based on the differences revealed in the host range, in addition to the specific ability naturally to induce necrosis on tubers.     

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.     

Detection of quarantine viruses of potato by ELISA

According to EC regulations, imported material of tuber-forming Solarium spp. has to be tested for the absence of defined quarantine viruses. In order to allow an efficient and timesaving application of the quarantine inspection procedure, antisera have been developed and scrutinized for their use by the ELISA technique. The viruses concerned were Andean potato latent virus (APLV), Andean potato mottle virus (APMV), arracacha virus B oca strain (AVB-O), potato virus T (PVT) and tobacco ringspot virus Andean potato calico strain (TRSV-Ca). The results show that all viruses can be reliably detected by double-antibody sandwich (DAS) ELISA. The detection limits were in the range ≤ 1–32 ng ml⁻¹. The strain specificity by DAS-ELISA of the antisera for APLV and APMV was overcome by mixing antisera from the different strains and strain groups, respectively. Strains C and Lm of APMV seemed to be serologically identical. A comparison of the suceptibility of several wild species of tuber-forming Solanum with that of several cultivars of Solanum tuberosum showed a higher frequency of infection in the wild species.     

Detection of tobacco rattle virus in different parts of tulip by ELISA and cDNA hybridisation assays

Different parts of tulips cv. Apeldoorn were assayed for the presence of tobacco rattle virus (TRV) by means of ELISA, cDNA hybridisation and immuno-electron microscopy. Assays were periodically performed during the growing season and upon storage of the bulbs, During the growing season in the field the relative TRV concentrations detected by ELISA and cDNA were highest mainly in the basal stem-parts and basal leaf-parts, respectively. When, during storage, infected bulbs were divided into a number of sections, TRV could be detected only in some of the sections, irrespective of the test used. However, nearly all sprouts of infected bulbs, stored at 5°C for 7 months, appeared to contain detectable amounts of TRV upon testing with ELISA and cDNA. Thus, testing of sprouts may offer a possibility to develop a routine test for TRV in tulip bulbs in due course.Samenvatting Verschillende delen van tulp cv. Apeldoorn werden getoetst op de aanwezigheid van tabaksratelvirus (TRV) met behulp van ELISA, cDNA-hybridisatie en immuno-elektronemicroscopie. Tijdens het groeiseizoen en de bewaring van de bollen werden regelmatig toetsingen uitgevoerd. Gedurende het groeiseizoen op het veld werden de relatief hoogste TRV concentraties voornamelijk gevonden in het basale deel van de stengel en het okselgedeelte van het blad met respectievelijk ELISA en cDNA-hybridisatie. TRV bleek gelokaliseerd aanwezig te zijn in een of meer stukjes van een gedeelde bol, onafhankelijk van de gebruikte toetsmethode. Bijna alle spruiten van geïnfecteerde bollen die gedurende 7 maanden bij 5°C bewaard waren, bleken bij het toetsen met behulp van ELISA en cDNA aantoonbare hoeveelheden van TRV te bevatten. Het toetsen van spruiten biedt de mogelijkheid te zijner tijd een routinetoets voor TRV in tulpebollen te ontwikkelen.     

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.     

The distribution of potato spindle tuber viroid in potato plants and tubers1

Potato spindle tuber viroid (PSTV) in potato plants was investigated by ‘return’ gel electrophoresis. The experiments were carried out under quarantine conditions in the greenhouse with primarily and secondarily infected plants. The PSTV content in different plant parts was estimated by the intensity of the viroid band in polyacrylamide gel. The results showed a decrease of viroid content from the upper to the lower parts of the plant. In both primarily and secondarily infected plants, PSTV was reliably detected in the top leaves, but less so in the lower leaves. In four out of ten secondarily infected plants, PSTV was found in the roots. In dormant tubers, the bands were more intense with samples obtained from the rose end and the heel than from those obtained from the medullary tissue. With one exception, all 64 tubers from 26 primarily infected plants were infected with PSTV.     

检测兰花病毒的斑点酶联法的建立

本文建立了齿兰环斑病毒（Odntoglossum ringspot virus,ORSV）和建兰花叶病毒（Cymbidium mosaic virus,CyMV）两种兰花主要病毒的斑点酶联法检测技术（Dot-ELISA）。用Dot-ELISA对提纯病毒和田间病叶进行检测,结果表明;检测ORSV和CyMV提纯病毒的灵敏度分别为0.82μg/mL和0.244μg/mL;病叶汁液的稀释倍数分别为1280倍和2650倍。该方法具有操作简便、成本低等优点,适合于兰花生产中的病毒检测。     

Partial infection with potato virus YN of tubers from primarily infected potato plants

A field experiment was designed to obtain information on the extent of partial infection of Bintje potato tubers with potato virus Y^N. Data from eight fields, in which plants were artificially infected with the virus, showed that tubers with a weight lower than 30 g became infected only about half as frequently as bigger tubers; the latter were divided into three groups of 30–60 g, 60–90 g and over 90 g, but there were no significant differences in the extent of infection in these groups. In the same experiment it was found that the heel end of a tuber is less frequently infected than the rose end. This means that in testing samples of potato tubers for the presence of potato virus Y^N it is preferable to grow the rose end of the tuber.Samenvatting Een veldproef, waarbij aardappelplanten kunstmatig met aardappel-Y^N-virus werden geïnoculeerd met het doel nadere gegevens te verkrijgen omtrent de gedeeltelijke besmetting van knollen met dit virus, leverde de volgende resultaten op. Het percentage besmette knollen met een lager gewicht dan 30 g bleek ongeveer de helft te bedragen van dat van knollen in de gewichtsklassen 30–60 g, 60–90 g en de klasse zwaarder dan 90 g. Tussen de drie laatstgenoemde groepen traden geen betrouwbare verschillen in besmetting op.Verder bleek dat in alle gewichtsklassen de oogstek genomen van de top van de knol meer kans heeft met Y^N-virus besmet te geraken dan de oogstek genomen van het naveleinde van de knol. Voor de nacontrole van aardappelpoot-goed betekent dit, dat het toetsen van een oogstek van de top van een knol voor dit doel beter geschikt is dan de oogstek van het naveleinde. De knolgrootte speelt hierbij blijkbaar geen rol van betekenis, daar knollen lichter dan 30 g in het algemeen niet voor pootgoed worden gebruikt.     

Detection of the root lesion nematode Pratylenchus penetrans in potato tubers

The root lesion nematode Pratylenchus penetrans parasitizes a wide range of economically important crops, including potato (Solanum tuberosum). Damage by P. penetrans impacts not only the potato yield but can also reduce the tuber quality. Detailed information on tuber infection by P. penetrans is scarce for most cultivars and molecular detection of nematodes from infected tubers is needed. The objective of this study was to assess tuber symptomatology due to P. penetrans infection in 10 potato cultivars and to provide an accurate molecular methodology for nematode detection using tuber peels. Sprouts of certified potato seed from cultivars Agata, Agria, Camel, Désirée, Dirosso, Kennebec, Laura, Picasso, Royata, and Stemster were planted in 2 L pots, and soil was inoculated with 4 P. penetrans/g of soil. Sixty days after inoculation, tubers were harvested, inspected for lesions, and the number of nematodes/g of potato peel assessed. Observations of tubers with symptoms showed the presence of P. penetrans in superficial layers of peels around the lenticels and injured necrotic tissue. Different nematode stages were detected in tubers of all inoculated cultivars, varying from 4 to 46 nematodes/g of potato peel. Species-specific primers showed suitable sensitivity and reproducibility for the detection of P. penetrans in tuber potato peel samples. The molecular detection of P. penetrans directly from tuber peels can facilitate routine nematode inspections of potato seed tubers or cull potatoes for nematode detection, and prevent further dissemination of this species.     

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...     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

Detection of spore balls of Spongospora subterranea on potato tubers by enzyme-linked immunosorbent assay

A rabbit was immunized with a homogenate of spore balls (cystosori) of Spongospora subterranea which also contained some potato tuber debris. The resultant polyclonal antiserum contained antibodies which reacted against extracts of spore balls and healthy tubers. Antibodies to the tuber debris were removed by incubating the blood serum with an extract from a S. subterranea -free tuber. In enzyme-linked immunosorbent assay (ELISA), the γ-globulin fraction of the absorbed antiserum reacted with dilute tuber extracts containing the equivalent of as little as 0·08 spore balls per ml. The reaction with spore balls was inhibited in high concentrations of tuber sap. Extracts of peel from symptomless tubers which had been stored in contact with scabbed tubers also reacted with the γ-globulin, presumably because the symptomless tubers became contaminated with spore balls. However, spore balls in soil were only weakly detected when high numbers were present. The γ-globulin did not react with the plasmodial stage of the pathogen, or with 15 other micro-organisms tested, including resting spores of Plasmodiophora brassicae.     

植物诱抗剂AHO联合茎尖培养脱除马铃薯S病毒和马铃薯Y病毒

研究了植物诱抗剂3-丙酮基-3-羟基羟吲哚(3-acetonyl-3-hydroxyoxindole, AHO)联合茎尖培养从试管苗中脱除马铃薯S病毒(PVS)?马铃薯Y病毒(PVY)的方法和效率?取PVS侵染的‘定薯3号’和‘定薯4号’以及PVY侵染的‘靖薯3号’和‘靖薯4号’的壮芽, 茎尖剥离后培养至4~5个叶片, 用100 mg/L植物诱抗剂 AHO水剂喷施试管苗, 每隔2 d喷施一次, 共3次, 末次喷施2 d后取茎尖剥离培养, 获得再生试管苗?用电子显微镜负染色?ELISA?荧光定量RT-PCR检测再生试管苗的带病毒情况?结果显示, AHO对4个品种的马铃薯试管苗生长无影响, 用AHO处理后再茎尖剥离培养, 脱毒率均高于未处理的对照; 检测结果还显示AHO处理的马铃薯再生苗的带毒量也低于未处理的对照, 且随处理次数增加带毒量下降?研究结果表明, 利用植物诱抗剂AHO联合茎尖剥离培养方法可以提高脱除PVS?PVY的效率, 获得无病毒核心苗?     

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.     

地高辛标记cDNA探针检测苹果茎痘病毒

 Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP. The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization. The results showed that the probe was sensitive and specific. The probe couldn't hybridize with total RNA of Apple stem grooving virus, Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control, only hybridized with that extracted from dormant shoot infected with ASPV. The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.     

Necrotic diseases caused by viruses in Swedish potato tubers

Potato tuber necrosis in the form of spraing symptoms is caused by infection with Potato mop‐top virus (PMTV) or Tobacco rattle virus (TRV); spraing has become more important in the Swedish potato crop production. In this study, the presence in Sweden of three potato‐infecting viruses associated with necrotic symptoms in tubers was demonstrated: PMTV, TRV and Tobacco necrosis virus (TNV). This study shows that both PMTV and TRV are frequent in Swedish potato fields. PMTV was found in all Swedish counties, except the two most northern ones. TRV was present at several locations in southern and up to the central parts of Sweden. Both viruses were found further north than observed in earlier surveys. PMTV and TRV were analysed in tubers with spraing symptoms and it was not possible to decide visually if the symptoms were caused by either PMTV or TRV. Furthermore, a high occurrence of symptomless tuber infections was observed for several potato cultivars. A unique observation from this study was a mixed infection of both PMTV and TRV in tubers of cv. Berber, where no visual differences on symptom development were detectable for these tubers compared to single infections. During the survey, tubers of cv. Melody were found to display necrotic symptoms in the skin that are characteristic for the ABC disease. This suggested infection by TNV, which also could be confirmed.     

两个马铃薯Y病毒黑龙江马铃薯分离物株系鉴定

为明确分离自黑龙江省克山县马铃薯上的2个病毒分离物KS4和KS7的分类地位,通过RTPCR扩增、克隆获得其基因组序列,利用重组分析程序包和最大似然法分别进行重组分析和系统发育分析。结果显示,分离物KS4和KS7的开放阅读框均有9 186个核苷酸,编码3 061个氨基酸,分离物KS4的核苷酸和氨基酸序列均与马铃薯Y病毒(potato virus Y,PVY)分离物Mb112一致率最高,分别为96.9%和98.4%;分离物KS7的核苷酸序列与PVY分离物12-94一致率最高,为97.4%,其氨基酸序列与PVY分离物SYR-Ⅱ-Be1一致率最高,为97.8%。重组分析表明,分离物KS4和KS7均为分离物N-605和Oz的重组体,其中KS4基因组5′-端的2 392个核苷酸来自分离物N-605,其余核苷酸来自分离物Oz;KS7基因组的第800～2 227个核苷酸和第5 637～8 950个核苷酸来自分离物N-605,其余核苷酸来自分离物Oz。系统发育分析发现,分离物KS4被聚类到N:O株系(PVY~(N:O)),分离物KS7被聚类到NTN株系(PVY~(NTN))b型。