Ineractions between a mild and a severe strain of potato spindle tuber viroid in doubly infected potato plants

Potato (Solarium tuberosum) plants co-infected with a mild and a severe strain of potato spindle tuber viroid (PSTVd) were analyzed by return-polyacrylamide gel electrophoresis for the presence of both strains in vegetative and reproductive plant parts. Both strains were detected in the anthers, flowers, inflorescences, leaves, ovaries, ovules, petals, pistils, roots, sepals, stolons and tubers. Only mild strain was detected from pollen in the cultivars tested. True potato seed (TPS) were not doubly infected when they were obtained from co-infected maternal parent plants pollinated with pollen from healthy plants. Also, when maternal plants infected with severe strain were pollinated with pollen from healthy plants or from those infected with the mild strain, TPS were not doubly infected. A small number of TPS with double infection was obtained when co-infected or mild-strain-infected plants were pollinated with pollen containing the severe strain (singly or doubly infected). The number of TPS containing mild strain predominated in a ratio of 7:1 over TPS containing the severe strain. This study indicates that segregation of strains from doubly-infected plants probably takes place during pollen formation and persists through seed development.     

湖南省马铃薯主产区马铃薯病毒种类及流行分析

马铃薯是世界第四大粮食作物,其病毒病危害严重。2010年对湖南马铃薯主产区采集的66个病毒标样进行了RT-PCR检测,结果表明,检测出的马铃薯病毒有马铃薯Y病毒(PVY)、马铃薯卷叶病毒(PLRV)、马铃薯X病毒(PVX)、马铃薯S病毒(PVS)、马铃薯A病毒(PVA)和马铃薯纺锤块茎类病毒(PSTVd)。其中PVS的检出率最高,为54.5%,其次是PVX,检出率为45.5%,PVY的检出率为39.4%,PSTVd和PVA的检出率均为21.2%,PLRV的检出率为18.2%。2~4种病毒的复合侵染现象较为普遍。PVY中重组型PVY占85.7%。     

Detection of potato virus X in tubers with the enzymelinked immunosorbent assay (ELISA)

Summary Tubers of field-grown plants of ten Dutch potato cultivars, secondarily infected with potato virus X (PVX) or free from this virus were submitted to ELISA after storage at 4°C. About 40 weeks after lifting PVX could be detected reliably. Extinction values with the apical parts of the tubers were slightly higher than those with the basal parts.     

Advantages of a soluble dye in blot immunobinding assays for virus detection in potatoes

Summary A technique is described in which plant sap is blotted onto small pieces (8×11 mm) of nylon membrane and virus particles bound to the paper are detected by a modification of the enzymelinked immunosorbent assay (ELISA). The detectable product of the assay is a soluble yellow dye, the absorbance of which increased with the virus content of the plant sap. Leaf or tuber sap from plants secondarily-infected with either potato leafroll virus or potato virus Y could be clearly distinguished from that of healthy plants and a majority of tubers primarily infected with PVY were also detected.     

Detection,distribution and long-term persistence of potato spindle tuber viroid in true potato seed from heilongjiang,China

Return-polyacrylamide gel electrophoresis (R-PAGE) and tomato bioassay followed by R-PAGE were compared for the detection of potato spindle tuber viroid (PSTVd) from individual true potato seed (TPS). Both methods detected PSTVd from single TPS. TPS extract formulated as sap or nucleic acids in two different buffers did not affect the percentage of viroid detection on tomato plants. There was some evidence of viroid inhibitor in TPS extracts but not in nucleic acid extracts of TPS. Because R-PAGE is more rapid than the tomato bioassay followed by R-PAGE, the former was used to determine the extent of PSTVd in TPS of China’s Keshan Potato Research Institute breeding material. Over 1700 individual TPS were tested. Twenty-four of the 46 seedlots tested (inbred and outcrossed) contained PSTVd. The viroid was detected in 70% of lots from inbred lines compared to 38% of lots from outcrosses. TPS (20 lots) stored in paper bags at room temperature as far back as 1965 were also tested, and PSTVd was detected in TPS stored for 21 years.     

应用RT-PCR技术检测马铃薯A病毒

参考GenBank中马铃薯A病毒(potato virus A,PVA)的保守序列,利用Primer6.0引物设计软件设计并合成了一对特异性引物PVAF、PVAR,以此引物利用RT-PCR方法对PVA保守序列基因进行了特异性扩增。结果表明:引物PVAF、PVAR能从已知的感染PVA病毒的植株中扩增出834bp的cDNA特异性片段;该RT-PCR的检测灵敏度为1pg的病毒核酸,特异性强,重复性好,可用于PVA病毒的快速检测。     

Effect of haulm treatments on the formation of microsclerotia ofVerticillium dahliae Kleb. on potato

Summary In four pot experiments, potato plants of cv. Element were artificially infected withV. dahliae. At an early and a late harvest haulms were killed chemically, by burning or by various other treatments, including cutting them into pieces of different lengths and keeping the debris on the soil surface or covering with soil. After 4 weeks the plant material was air-dried and the number of microsclerotia per mg was determined. At the early harvest, in two experiments, the chemical treatment yielded more microsclerotia than the cutting treatments. Covering colonised haulm tissue with non-sterilised soil was effective in inhibiting microsclerotia formation. Shorter haulm pieces led to fewer microsclerotia at the later harvest if the material was kept on the soil surface. The variation in microsclerotial yield and in treatment effects among the different experiments was large.     

用NASH方法检测马铃薯纺锤块茎类病毒

利用荧光素标记制备马铃薯纺缍块茎类病毒（PSTVd)特异性探针，采用核酸斑点杂交（NASH）技术对大田马铃薯和马铃薯试管苗进行检测，经放射自显影表达检测结果，结果清晰，重复性好，该方法可检测类病毒最低为0．33pg，灵敏性很高。     

部分发展中国家马铃薯纺锤块茎类病毒病情况调查

马铃薯类病毒病是威胁世界马铃薯生产的主要病害之一,更影响着发展中国家马铃薯产业的发展。2008~2010年,黑龙江省农业科学院植物脱毒苗木研究所连续3年举办了马铃薯病害检测技术国际培训班,对部分发展中国家从事马铃薯工作的人员进行培训,在培训过程中进行了马铃薯类病毒方面的问卷调查,调查结果显示:这些发展中国家对马铃薯纺锤块茎类病毒比较了解,发病较重的国家不多,但总体检测水平不高,甚至不进行马铃薯类病毒的检测,多数国家不重视马铃薯纺锤块茎类病毒病的防治工作。因此,马铃薯纺锤块茎类病毒病是发展中国家的隐患。     

Carrot Pathogen ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum’ Haplotype C Detected in Symptomless Potato Plants in Finland

‘Candidatus Liberibacter solanacearum’ (CLso) haplotype C, a bacterial pathogen transmitted by the carrot psyllid Trioza apicalis, causes yield losses in carrot production. Due to concerns that this pathogen might also threaten potato (Solanum tuberosum) production, the occurrence of CLso in cultivated and volunteer potatoes in Tavastia Proper and Satakunta regions of Finland was studied. Volunteer potato plants were found in 13 of the 27 inspected carrot fields. Of the 148 potato samples tested by PCR, eight volunteer potato plants and one cultivated potato grown at the edge of a carrot field were found to be CLso positive. The PCR products obtained from these potatoes with primers OA2/OI2c, LpFrag4-1611F/LpFrag4-480R and CL514F/CL514R all showed 100% sequence identity to CLso haplotype C. This is the first observation of CLso haplotype C in field-grown potatoes. In addition, transmission experiments were performed. Attempts to transmit CLso into potato with carrot psyllids were not successful; however, CLso haplotype C was transmitted from infected carrots to potato plants by leaf grafting and by phloem connection formed by dodder, a parasitic plant, and found to survive in the potato plants for several weeks after transmission. However, the bacterial colonisation progressed slowly in the potato phloem and the amount of bacteria detected was low. The plants produced from the daughter tubers of the CLso-positive potato plants were all CLso negative, suggesting that CLso haplotype C was not able to pass to the daughter plants. None of the CLso-positive potatoes inoculated in greenhouse or collected from fields showed symptoms characteristic of zebra chip disease, associated with CLso haplotypes A and B.     

The development of mature plant resistance in four potato cultivars against aphid-inoculated potato virus YO and YN in four potato cultivars

Summary Field-grown potato plants of cvs King Edward, Record, Maris Piper and Désirée were inoculated on seven different dates during the growing season of 1987 and 1988 with either potato virus Y^O (PVY^O) or PVY^N, using three viruliferous peach-potato aphids (Myzus persicae) per plant. In each cultivar, the proportion of progeny tubers infected with PVY^O or PVY^N was high in plants inoculated during the four weeks following emergence, the proportion declining to zero or close to zero in the subsequent 4–6 wks.     

Chemiluminescent detection of potato spindle tuber viroid in true potato seed using a digoxigenin labelled DNA probe

Summary The potential use of a simple, sensitive and non-radioactive method for detecting potato spindle tuber viroid (PSTVd) in germinated true potato seeds, based on nucleic acid hybridization with a PSTVd-specific DNA probe labelled with digoxigenin, was investigated. Two simple procedures for the clarification of plant extracts suitable for a non-radioactive detection system were also investigated. The nucleic acid hybridization technique could detect one PSTVd-infected seed in more than 150 healthy seeds. The benefits of this non-radioactive detection system are discussed.     

Evaluation of a vacuum collector for insect pest control in potato

A field scale vacuum insect collector designed for the control of the Colorado Potato Beetle,Leptinotarsa decemlineata (Say) was tested on potatoes in 1990. The vacuum collector was more effective against adults and small larvae than against large larvae. Results suggested that a large proportion of potato aphids can also be removed from plants. The spread of plant diseases PSTVd and PVX, readily transmitted by contact, was not increased by the repeated use of the vacuum collector.     

Survey of potato spindle tuber viroid in seed potato growing areas of the United States

Fourteen United States (U.S.) seed potato certification agencies surveyed all U.S. seed potato growing areas for presence of the potato spindle tuber viroid (PSTVd). The survey included general surveillance, which involved searching for the occurrence of PSTVd in state seed potato certification records from 1990 through 2000, and a field survey, which involved testing selected crops for PSTVd infection by nucleic acid dot blot hybridization during 1999 through 2001. No PSTVd incident was documented in any of the state certification records, nor was PSTVd detected in the field surveys. All U.S. seed-growing areas were determined to be free of PSTVd. It is concluded that PSTVd has been eradicated and freedom from potato spindle tuber viroid has been successfully maintained in all of the seed potato growing areas in the United States.     

Detection of potato viruses A,M, S,X, Yand leafroll and potato spindle tuber viroid from tissue culture plantlets using single leaf discs

Using enzyme-linked immunosorbent assay (ELISA) and dotimmunobinding assay (DIBA) for potato viruses A (PVA), M (PVM), S (PVS), X (PVX), Y^N (PVY^N), Y^O (PVY^O) and leafroll (PLRV) and nucleic acid spot hybridization (NASH) for potato spindle tuber viroid (PSTVd), virus and viroid were detected reliably from single leaf discs (6 mm) of tissue-culture plantlets. Leaf discs taken from leaf positions (1 to 8) (bottom to top) can be used for detection of all viruses except PLRV where the lower leaves had higher concentrations of virus than the leaves from the upper part of the plantlet. Virus cultures were maintained for 1 to 4 years in several potato cultivars. The levels of virus remained reproducible except for PVM concentration, which was found to be very low in cv. Green Mountain. Using densitometry software, the DIBA spots were quantified and results were comparable to A₄₀₅ values obtained by ELISA. PSTVd concentration as measured by densitometry from spots of NASH indicated no loss of viroid over 1–4 years in tissue culture in two potato cultivars.     

Differential diagnosis ofVerticillium dahliae in potato with antisera to partially purified pathogen-produced extracellular antigens

Summary An extracellular protein-lipopolysaccaride antigen (PLP) was purified from culture fluids ofVerticillium dahliae. Antiserum produced in rabbits to the PLP detected the antigen in homogenates of tubers, stems and leaves ofV. dahliae-infected potato plants but not in extracts of healthy potato tissue or extracts of potato plants infected by other fungal pathogens. The antigen was not detectable in extracts of potato isolates ofV. tricorpus, V. nigrescens andV. nubilum or various other fungi. The antigen was shown to be different from cross-reactive antigens detected by antisera to mycelial antigens. When used as a tool for specific diagnosis ofV. dahliae infection in potato, antiserum to PLP was more reliable than that prepared against fungal body antigens. Publication of the Agricultural Research Organization No 245-E.     

马铃薯晚疫病种薯带菌的分子检测

从马铃薯块茎上分离纯化得到晚疫病菌、银腐病菌、癌肿病菌、红腐病菌、黑点病菌和镰刀菌等并提取它们的全基因组DNA,用引物08-3和08-4对这些真菌的DNA进行PCR扩增,以检验该引物扩增晚疫病菌DNA的特异性。取马铃薯种薯的芽眼、芽及其他不同部位并用NaOH法提取晚疫病菌DNA,再用引物08-3和08-4进行特异性扩增以检验种薯是否带晚疫病菌。结果表明:(1)引物08-3和08-4有很强的特异性,只能扩增出大小为257bp的马铃薯晚疫病菌DNA片段;(2)用该引物能扩增出种薯芽眼组织中大小为257bp的晚疫病菌DNA,说明通过PCR特异扩增,能直接检测出马铃薯种薯中潜伏的晚疫病菌。(3)本次检测的本地感病品种米拉有10%的种薯带晚疫病菌。该研究为种薯传播晚疫病菌的分子检测提供了依据和方法。     

Detection and quantification ofSpongospora subterranea f. sp.subterranea by PCR in host tissue and naturally infested soils

A polymerase chain reaction (PCR) assay using primers SsF and SsR designed from the internal transcribed spacer (ITS) regions ofSpongospora subterranea f. sp.subterranea was developed for the specific identification and quantification ofS. subterranea. These primers amplified a 434 bp product from DNA ofS. subterranea spore balls, but not from DNA of healthy potato, common scab tuber, and taxonomically related plasmodiophorids. This PCR assay was successfully used for the detection ofS. subterranea in naturally infected symptomatic and asymptomatic potato tubers.Spongospora subterranea in other infected symptomless host plants was detected by PCR. The PCR assay was modified with improved soil DNA extraction methods to detectS. subterranea in soil. The assay was sensitive, and one spore ball per gram of soil could be detected. Following the design of a heterologous competitor DNA template from the sequence of λDNA, a competitive PCR assay for the quantification ofS. subterranea in soil was developed and provided accurate quantification in the range of 1 to 10⁴ spore balls per 0.25 g of soil. In a preliminary survey of naturally infested field soil samples, spore ball concentrations were estimated to vary from ca 0 to 3600 spore balls per 0.25-g soil sample by this competitive PCR assay. The spore ball levels were compared with the powdery scab disease incidence of potatoes in these fields, and a correlationship between spore ball levels and subsequent disease incidence was found. The PCR assays developed in this investigation can be routinely used to detect and quantifyS. subterranea in diseased plant tissue, asymptomatic plant tissue, and infested soil.     

Use of IKI leafroll test to reduce net necrosis storage losses of potatoes

An iodine-potassium-iodide (IKI) starch staining procedure was adapted for use on potato plant leaflets to estimate the frequency of potato leafroll virus (PLRV) infection in potato fields prior to harvest. Large increases in the percentage of tubers with net necrosis occurred during storage among tubers from fields with high PLRV infection rates. Such fields were reliably identified prior to harvest by the IKI test, so their tubers could be processed at harvest to avoid net necrosis storage losses. The test could be performed on hundreds of samples per hour by untrained personnel with commonly available equipment. PLRV infection frequency varied widely in Columbia Basin potato fields. Most infected plants expressed no symptoms but could be detected by the IKI test before harvest. High rates of virus dissemination apparently occur late in the growing season in the Columbia Basin