马铃薯病毒外壳蛋白融合基因转化马铃薯及其抗病性分析

将多种病毒的有效核酸片断拼接成融合基因转入马铃薯可获得多抗马铃薯材料。针对马铃薯生产中分布广泛、危害严重并经常混合感染的马铃薯X病毒(PVX)、马铃薯Y病毒(PVY)、马铃薯卷叶病毒(PLRV)和马铃薯S病毒(PVS),开展了利用基因工程方法获得兼抗4种马铃薯病毒转基因马铃薯材料的研究。试验在前期获得含4种马铃薯病毒外壳蛋白基因片段的质粒pART27-XSYV-rh的基础上,通过根癌农杆菌(Agrobacterium tumefaciens)介导转化马铃薯(Solanum tuberosum)品种‘陇薯3号’,PCR扩增和PCR-Southern杂交证明,4价融合基因已整合到马铃薯基因组中。qRT-PCR分析表明,该融合基因在转基因植株中能正常表达。3株转基因植株的抗病性鉴定结果表明,2株对4种病毒同时具有抗性;1株对PLRV侵染表现阳性,对另外3种病毒同时具有抗性。     

Cryotherapy of Potato Shoot Tips for Efficient Elimination of Potato Leafroll Virus (PLRV) and Potato Virus Y (PVY)

Viral diseases constitute a major constraint to high yield and high quality production of potato. Potato leafroll virus (PLRV) and Potato virus Y (PVY) are among the most damaging potato viruses and are prevalent in most potato growing areas. In the present study, attempts were made to eliminate PLRV and PVY by three cryogenic protocols, i.e., encapsulation-dehydration, encapsulation-vitrification and droplet. Results showed that both PLRV and PVY could be efficiently eliminated by cryogenic treatments with 83–86% and 91–95% of frequencies of virus-free plantlets obtained for the former and latter, respectively. Frequencies of virus-free plantlets produced by cryogenic treatments were higher than those by meristem culture (56% for PLRV and 62% for PVY) and thermotherapy (50% for PLRV and 65% for PVY), and similar to those by thermotherapy followed by meristem culture (90% for PLRV and 93% for PVY). Survival (75–85%) and regrowth (83–89%) from cryo-treated shoot tips were higher than those from meristem culture (50–55%) and thermotherapy followed by meristem culture (40–50%), but similar to those from thermotherapy (80–87%). The morphology of the plantlets regenerated from cryo-treated shoot tips was similar to that of non-treated plantlets. Thus, cryotherapy would provide an alternative method for efficient elimination of potato viruses, and can be simultaneously used for long-term storage of potato germplasm and for production of virus-free plants.     

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%。     

Occurrence and Distribution of Potato Pests and Diseases in Kenya

Potato plays an important role in food security in Kenya but yields are low (<10 t/ha), and this is partly attributed to the lack of healthy planting material. This study is the first wide-scale survey to determine the occurrence and distribution of common potato pests and diseases in Kenyan seed (certified and quality declared) and ware crops. Potato crops growing on 101 farms in 21 districts were examined. Approximately 36% of plants in farmers’ fields sampled both during the long rains (main potato-growing season) and short rains seasons displayed virus-like disease symptoms. Six viruses (potato leafroll virus (PLRV), Potato virus A (PVA), potato virus M (PVM), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY)) were detected using double antibody sandwich enzyme-linked immunosorbent assay in potato samples. Sequencing of polymerase chain reaction products from PVY-infected plants revealed the presence of recombinant strains of PVY (NTN and Wilga). Four aphid species, Macrosiphum euphorbiae, Aphis gossypii, Myzus persicae, and Aphis fabae, colonized potato in all districts, occurring in greater numbers west of the Great Rift Valley than to the east. There was a positive correlation between virus incidence and aphid numbers in the long rains (main) potato-growing season. PLRV, PVM, PVS, PVX, and PVY were detected in solanaceous weeds. Ralstonia solanacearum was detected in soils from 13 farms in 8 of the 18 districts surveyed. Approximately 38% of soil samples were infested with Meloidogyne spp. Phytophthora infestans isolates belonging to the US 1 and 2_A1 genotypes were identified. Although many economically important diseases are present in Kenya, the lower aphid incidence in districts east of the Great Rift Valley may indicate that these districts are more suitable for seed potato production.     

Modification of serological techniques and their evaluation for detection of potato viruses in seed certification related activities

Development of alternative serological techniques to ELISA for detection of potato viruses offers advantages for monitoring virus incidence and for seed potato certification systems. Several trials showed that multiplex tissue print immunoassay (TPIA) and dot blot immunoassay (DBIA) might represent fast, practical, and sensitive alternatives for the detection of: Potato leaf roll virus (PLRV), Potato virus S (PVS), Potato virus X (PVX) and Potato virus Y (PVY), from green and/or tuber tissues. In TPIA, the specific precipitation patterns in infected tissues of leaf petioles or stem cross sections, observed with each virus, allowed identification of the specific virus or mixed infections in a single multiplex assay. For detection of PVY in green tissues, DBIA was shown to be over 50 times more sensitive than ELISA. TPIA and ELISA from the tuber stem end or from eyes might be used for rapid detection of PVY and PVS in seed potato tubers without prior germination. PVS was evenly distributed in potato tuber tissue, while PVY was localized in the vascular tissue beneath the epidermis, with irregular distribution along the periphery of the potato tuber. For laboratories in developing countries lacking time and facilities for tests based on tuber germination, monitoring for PVS and PVY using TPIA in tuber tissue may be a suitable alternative to ELISA.     

Occurrence of Viruses Affecting Potato Crops in Khartoum State-Sudan

The incidence of Alfalfa mosaic virus (AMV), Potato leafroll virus (PLRV), and Potato virus Y (PVY) in potato crops derived from various types of seed potatoes was assessed visually and confirmed by direct tissue blot immunoassay, over two winter growing seasons (1999/2000, 2000/2001) at three locations, Elnaiya, Elshehinab, and Shambat in Khartoum State, the main potato growing region in Sudan. Virus infection was most prevalent in 2000/2001. In general, crops grown directly from imported certified seed potatoes and from “improved seed”, produced in Sudan from imported basic seed, showed the lowest levels of PLRV and PVY compared with crops grown from Sudanese farm saved seed. For AMV, however, only crops grown directly from imported certified seed potatoes had low levels of AMV. Crop location also affected virus incidence, although this varied with year. For AMV, levels were similar at all locations in 1999/2000, but were greatest at Elnaiya in 2000/2001. For PVY, levels were greatest at Elnaiya in 1999/2000 and Shambat in 2000/2001. For PLRV, no symptoms were observed in 1999/2000 and virus levels were similar for all locations in 2000/2001. This study reports for the first time the occurrence of AMV in potatoes grown in Sudan.     

Nucleotide Sequence Variability at the 5′-UTR of Potato PVY Isolates

Potato virus Y (PVY) isolates from potato were divided into two major groups – I and II – on the basis of nucleotide sequence variability at the 5′-UTR of the virus genome. In this paper, a consensus sequence for each group was compiled by examining PVY isolates of potato. The consensus sequences of the two groups differed from each other by as much as 31% of nucleotide sequence divergence. However, sequences of most individual members within each group (except for one, the 605 strain) were conserved and varied by less than 1% in group II and up to 6% in group I. These results confirm that the two virus groups are markedly distinct but that the 5′-UTR sequence of individual members within each group is highly conserved. These findings imply that the 5′-UTR can be reliably applied for major grouping of isolates but that its use for discrimination among individual isolates within each group is limited. Isolates of both group I and II included members of the O/N-NTN virus types. Classification of PVY isolates in potato is discussed.     

Population Growth of Myzus persicae on Potato Plants Infected with Different Strains and Variants of Potato virus Y

Potato virus Y (PVY) is one of the most economically important viruses affecting the potato crop. Several strains of the virus, including PVY^O, PVY^N, recombinant isolates; PVY^N:O (PVY ^N-Wi) and PVY^NTN and several variants of PVY^O have been reported from North American potato-production areas. The green peach aphid, Myzus persicae Sulzer, is a colonizer of potatoes and is considered the most important vector of PVY. The objective of this study was to measure the population growth of M. persicae on potato plants infected with different strains and genetic variants of PVY. The initial population of ten winged adults of M. persicae was allowed to develop on a potted plant for 12 days. Results clearly indicated that infections by different strains and genetic variants of PVY did not influence the population growth of M. persicae on potato plants during this period.     

Naturally occurring viral infections inSolanum brevidens andS. fernandezianum

Summary Two species of wild potato,S. brevidens andS. fernandezianum, were surveyed for six potato-infecting viruses in their natural habitats in the Puerto Montt Region and Robinson Crusoe Island of Chile, respectively. Potato viruses S (PVS), M (PVM) and X (PVX) and potato yellowing virus (PYV) were found in some clones of the Chilean Potato Germplasm Collection, whereas only one population ofS. brevidens out of six was virus-infected, namely by PVS. The cultivated potatoes on Robinson Crusoe Island were infected by PVS, PVM and PVX and infested byAulacorthum solani andMyzus persicae, whereasS. fernandezianum was infected by PYV and infested byA. circumflexum, A. solani andAphis spp.     

Resistance to <Emphasis Type="Italic">Potato virus Y</Emphasis> in a Multitrait Potato Breeding Scheme without Direct Selection in Each Generation

In an experimental breeding scheme to improve late blight (Phytophthora infestans) and white potato cyst nematode (Globodera pallida) resistance of tetraploid potato over three generations of crossing and selection, 15 clones survived the final selection, and these were derived from 15 great-grandparents. There was no direct selection for resistance to Potato virus Y (PVY), but 14 out of the 15 great-grandparents were resistant to PVY and three had extreme resistance. Thirteen of the 15 descendants had PVY resistance and one extreme resistance. This was within the range expected for a random (unselected) sample from the genotypes of the great-grandparents. Hence, we found no evidence for any positive or negative association between PVY resistance and the attributes selected. The conclusion is that laborious selection is not required in every generation when many parents have PVY resistance, including some with more than one copy of a PVY resistance gene or resistance at more than one locus. However, in the future, determining the major virus resistance genes present in potential parents in each generation using diagnostic molecular markers would prevent susceptible × susceptible crosses being made and maximise the number of resistant × resistant ones.     

Multiplex detection ofPotato virus S, Potato virus X, andPotato virus Y by non-radioactive nucleic acid spot hybridization in potato tissue culture plantlets

Potato plantlets initiated into tissue culture must be tested for numerous viruses prior to propagation for seed potato production. Ideally, one plantlet is tested for all pathogens of concern and, if found pathogen-free, this plantlet is propagated for production of seed potatoes. Commercially available ELISA kits are generally used for the pathogen tests, but the commercial kits have some limitations. For example, the protocols differ for different viruses, so multiple extractions must be completed, increasing the time and expense of testing. This is a significant problem with tissue culture plantlets, for which there is limited material available to test and an ever-increasing number of pathogens that must be tested for, including viruses in the potyvirus, carlavirus, potexvirus, luteovirus, pomovirus, tobravirus, tospovirus, alfamovirus, and tymovirus groups. We have optimized a non-radioactive nucleic acid hybridization (NASH) assay for the simultaneous detection of carlavirusPotato virus S (PVS), potexvirusPotato virus X (PVX) and potyvirusPotato virus Y (PVY) in potato tissue culture plantlets. This assay requires a single extraction from a small portion of a tissue culture plantlet for the detection of viruses from three different families.     

Potato Carlavirus S (PVS) resistance of potato clone B6603-12

The potato (Solarium tuberosum L.tuberosum) breeding line B6603-12 was shown to be resistant to Potato Carlavirus S (PVS) on the basis of non detection of PVS by ELISA in plants that were: 1) either graft or mechanically inoculated with composite isolates of PVS; and 2) interplanted among PVS infected cultivars in fields for twelve growing seasons.     

Census Report of the <Emphasis Type="BoldItalic">Potato Virus Y</Emphasis> (PVY) Population in Swiss Seed Potato Production in 2003 and 2008

The status of the Potato virus Y (PVY) in Swiss seed potato production was investigated in the years 2003 and 2008 by analysing 385 leaf samples of field-grown, suspicious potato plants collected in four representative seed control fields. Serological investigations by ELISA showed that in c. 84% of the PVY-positive samples in both years, viruses belonging to the PVY^N group were found. All 124 serologically positive PVY samples collected in 2003 and a selection of 81 isolates of 2008 were further typified by molecular tests and by biological assays on tobacco and potato plants. These tests largely confirmed the predominance of the PVY^N group and, within this group, the prevalence of recombinant PVY^NTN, with 81.4% and 70.4% in 2003 and 2008, respectively. The percentage of PVY^N-Wilga (PVY^N-Wi) increased from c. 6% to 17% between the two years. PVY^O was detected only in 10.5% and 4.9% of all molecularly analysed samples in 2003 and 2008, respectively. The persistent predominance of recombinant PVY^NTN in Swiss seed potatoes indicates that this strain group is now widespread, representing a considerable threat to Swiss seed potato production.     

General resistance against potato virus Y introduced into a commercial potato cultivar by genetic transformation with PVYN coat protein gene

Summary Transgenic cv. Folva potato plants expressing the coat protein gene of potato virus Y strain N (PVY^N) were produced usingAgrobacterium tumefaciens mediated transformation. Forty independent transformants were selected for resistance screening. Four clones showed complete resistance to mechanical inoculation with all the five PVY isolates tested: the PVY^N isolate from which the coat protein gene was derived, two PVY^O isolates, and two PVY^NTN isolates. Two of the fully resistant clones contained only one copy of the transgene, demonstrating that it is possible by genetic engineering to obtain highly virus resistant potato clones that can also be useful in future breeding programmes.     

Genetic Resistances to Potato Leafroll Virus, Potato Virus Y, and Green Peach Aphid in Progeny ofSolanum etuberosum

Increasing prevalence of potato leafroll virus (PLRV) and potato virus Y (PVY) has been reported in seed and commercial potato production, resulting in the rejection of potatoes for certification and processing. Host plant resistance to PLRV and PVY and their primary vector, green peach aphid,Myzus persicae, could limit the spread of these viruses. Host plant resistance to PLRV, PVY, and green peach aphid has been identified in non-tuber-bearingSolanum etuberosum (PI 245939) and in its backcross 2 (BC₂) progeny. Resistance to green peach aphid involved a reduction in fecundity and adult aphid size. In addition, one BC₂ individual was identified as possessing a genetic factor that was detrimental to nymph survival. PVY resistance was identified in all five BC₂ progenies evaluated in a field screening under intense virus pressure. PLRV resistance was identified in two of the five BC₂ progeny. This resistance was stable in field and cage evaluations with large populations of viruliferous aphids. Based on the segregation of virus resistances in the BC₂ , PVY and PLRV resistances appear to result from the action of independent genetic mechanisms that reduce the levels of primary and secondary virus infection. Two BC₂ individuals, Etb 6-21-3 and Etb 6-21-5 were identified as having multiple resistances to PLRV, PVY, and green peach aphid derived fromS. etuberosum. This germplasm could prove useful to potato breeders in the development of virus-resistant cultivars.     

New isolates of the necrotic strain of potato virus Y (PVYN) found recently in Poland

Summary In comparison to the previously known isolates of potato virus Y^N (PVY^N), some isolates found in Poland since 1984 are more infectious to potato plants, reach faster a higher concentration and induce milder disease symptoms. Potato cultivars resistant to the standard type of PVY_N may be susceptible to the new isolates whereas those that are extremely resistant to PVY remain extremely resistant to the new isolates. The potato cultivar Elipsa is suitable for the differentiation of PVY^N isolates.     

Potato Virus Y NTN : A Coat and P1 Protein Sequences Analysis of a Brazilian Isolate

Potato tubers (Solanum tuberosum L. cv. Monalisa) showing superficial necrotic rings typical of Potato virus Y ^NTN (PVY^NTN) infection were collected from commercial potato fields in the State of São Paulo (Brazil). An isolate named IAC-PVY^NTN was characterized by sequencing 822 and 836 nucleotides from the coat protein and P1 protein coding genes, respectively. The IAC-PVY^NTN was observed as a European/North American recombinant isolate closely related to three Eu-PVY^NTN isolates that share an alternative recombination site in the coat protein cistron near position 144. Therefore, the IAC-PVY^NTN isolate may represent a novel recombinant variant between PVY^N N-605 Swiss (European) and PVY O-139 Canadian (North American). Cluster analysis of the P1 sequences found IAC-PVY^NTN in the sub-cluster of Eu-PVY^N/NTN/N:O. This study supports previous evidence of a common origin for PVY^N/NTN in Europe and Brazil.     

Studies on the etiology of tuber necrotic ringspot disease in potato

Summary Potato virus M (PVM), potato virus S (PVS), potato virus X and tobacco veinal necrosis strain of potato virus Y (PVY^R) were isolated from potatoes showing tuber necrotic ringspot disease (TNRD). Potato mop-top virus, tobacco rattle virus, tobacco necrosis virus and tomato black ring virus could not be isolated from the diseased plants. Because PVM and PVS could be isolated from potato plants that did not show symptoms, these viruses could not be causally related to TNRD. However, TNRD is closely connected with infections by PVY^R which always could be isolated from potato plants with TNRD symptoms.

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Zusammenfassung Aus Kartoffelpflanzen (Solanum tuberosum, Sorten Adema, Malacsinka, Mona Lisa, Romano und Rosalie) die Symptome des Tuber necrotic ringspot zeigten, wurden Kartoffel-M-Virus (PVM), Kartoffel-S-Virus (PVS), Kartoffel-X-Virus (PVX) und Kartoffel-Y-Virus (PVY) isoliert. Die Viren wurden mit Hilfe von Differentialwirten, Elektronenmikroskopie, Serologie und Pr?munit?tstests identifiziert. Die untersuchten Kartoffelknollen zeigten deutlich sichtbar nekrotische Ringsymptome (Abb. 1). Von den Nekrosen aufweisenden Knollen und von Kartoffelpflanzen konnten von allen Sorten drei Viren (PVM, PVS, PVY) isoliert werden, von der Sorte Malacsinka zus?tzlich noch PVX (Tab. 2). Entsprechend den Symptomen der Testpflanzen (Tab. 3) identifizierten wir die Virus-isolate aus Kartoffelpflanzen die Symptome des Tuber necrotic ringspot aufwiesen als PVM, PVS, PVX und PVY. Aus keiner kranken Pflanze konnte Kartoffelbüscheltrieb-Virus, Tabakmauche-Virus, Tabaknekrose-Virus und Tomatenschwarzring-Virus isoliert werden. Da PVM und PVS aus Pflanzen, die keine Symptome aufwiesen, isoliert werden k?nnten, k?nnen diese beiden Viren nicht urs?chlich mit den Symptomen der Tuber necrotic ringspot Krankheit (TNRD) verbunden sein. Wir konnten jedoch eine enge Verbindung zwischen TNRD und der Infektion mit PVY feststellen, da dieses Virus von allen Pflanzen, die Symptome des TNRD aufwiesen, isoliert werden konnte. Die Eigenschaften von PVY-Isolaten aus Knollen, Bl?ttern und Wurzeln ?hnelten jenen, die in verschiedenen Testpflanzen gefunden wurden (Tab. 4). Die typischen Symptomreaktionen aufNicotiana debneyi (Abb. 2) undN. tabacum Sorte Xanthi-nc sowie aufSolanum demissum A6-Hybride lassen vermuten, dass die Ursache ein Tobacco veinal necrosis—Stamm des PVY, (PVY^R, siehe Abb. 3) war. Es ist schwierig zu einem endgültigen Ergebnis über die ?tiologie der TNRD zu kommen, weil einige PVY^R-isolate sogar aus symptomlosen Kartoffelknollen gewonnen werden konnten. Die Untersuchungen werden fortgesetzt.

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Résumé Les virus M (PVM), S (PVS), X (PVX) et Y (PVY) sont isolés à partir de plantes de pommes de terre (Solanum tuberosum cvs Adema, Malacsinka, Mona Lisa, Romano et Rosalie) présentant des sympt?mes de taches nécrotiques annulaires. Les virus sont identifiés à l'aide d'h?tes différentiels, de la microscopie électronique, de la sérologie et de tests de protection croisée. Les tubercules examinés présentent des taches nettement visibles de nécroses annulaires (fig. 1). A partir des tubercules nécrosés et des plantes, trois virus (PVM, PVS, et PVY) sont isolés pour tous les cultivars à l'exception de cv. Malacsinka pour lequel PVX est également isolé (tabl. 2). Les sympt?mes révélés sur les plantes tests (tabl. 3) indiquent que les virus isolés à partir des plantes présentant des sympt?mes de nécroses annulaires sur tubercules sont PVM, PVS, PVX et PVY. Les virus du Mop-Top de la pomme de terre, du rattle du tabac, de la nécrose du tabac et des taches noires en anneau de la tomate n'ont pu être isolés d'aucune des plantes malades. Puisque PVM et PVS peuvent être isolés de plantes de pommes de terre ne présentant pas de sympt?mes, ces deux virus ne peuvent être directement responsables des sympt?mes de taches nécrotiques annulaires sur tubercules (TNRD). Cependant, une relation étroite entre TNRD et la contamination par PVY est établie, puisque ce virus peut être isolé de toutes les plantes présentant des sympt?mes de TNRD. Le tableau 4 regroupe les caractéristiques de quel-ques cultures pures de PVY isolées à partir de tubercules, feuilles et racines et semblables à celles trouvées dans différentes plantes h?tes. Les sympt?mes typiques surNicotiana debneyi (fig. 2) etN. tabacum cv. Xanthi-nc, ainsi que surSolanum demissum-A6-hybride laissent à penser que l'agent responsable est une souche nécrotique des nervures du tabac de PVY (PVY^R, voir fig. 3). Il est difficile de conclure de fa?on définitive, quant à l'étiologie de la maladie des taches nécrotiques annulaires, car PVY^R peut être également isolé de tubercules ne présentant aucun sympt?me. Des études axées sur l'étiologie de TNRD sont en cours.