Evaluation of the enzyme-amplified ELISA for the detection of potato viruses A,M, S,X, Y,and leafroll

Various parameters,e.g. types of microtiter plate for DAS-ELISA (double antibody sandwich-enzyme-linked immunosorbent assay), use of fresh or frozen amplifier solutions for enzyme-amplified-ELISA, and use of sodium diethyldithiocarbamate (NaDIECA) in sample buffer in cocktail-ELISA were evaluated for the detection of potato viruses A, M, S, X, Y and leafroll from potato foliage. Dynatech Immulon immunoplates provided higher readings for all viruses. Fresh amplifier solution in amplifed-ELISA was superior to frozen solutions. Amplified ELISA gave only marginal improvement in the sensitivity over the standard DAS-ELISA. Addition of NaDIECA in sample buffer did not improve the detection of viruses in DAS-, amplified-, or cocktail-ELISA. Cocktail-ELISA can reduce antigen incubation time to as short as 15 min for PVA, PVM and PVX; 1 hr for PVY and PLRV; and 2–4 hr for PVS using pre-coated plates. Although amplified-ELISA is slightly more sensitive than DAS-ELISA for certain potato viruses, it is not suitable for large-scale indexing of potato viruses in Seed Certification Laboratories, in view of the additional steps needed in carrying out this procedure.     

Potato viruses in China

Potato (Solanum tuberosum L.) is globally the fourth largest staple crop after rice, wheat and maize. China is the largest potato producer worldwide, accounting for 26.3% and 22.2% of the global total area and yield, respectively. Viral diseases have been a limiting factor for sustainable production of potato in China. This article provides update and comprehensive information on the most important viruses, their effect on yield and their control in China. Meristem culture is currently the most widely used technique for production of virus-free potato plants and these plants are used as nuclear stock for the production of certified seed tubers. Although a significant increase in tuber yield has been achieved using certified seed potatoes, lack of application of a national seed potato production scheme has hindered progress. Conventional breeding using virus-resistant cultivars introduced from other countries as parents has already led to the production of Chinese cultivars. Use of virus-resistant wild Solanum species and genetic transformation techniques will provide new virus-resistant varieties in the future to further enhance control of potato viruses in China.     

Solanum demissum P.I. 230579, a True seed diagnostic host for potato virus Y

Seedlings ofSolarium demissum P.I. 230579 in the 5-to 10-leaf stage growing under 18-hour days and at 20 to 24°C developed dark, irregular, slightly elongated local lesions 3 to 5 days following inoculation with any one of three strains of potato virus Y (PVY). Local lesions did not develop after similar inoculations with any one of two or more strains each of potato viruses A, M, S, X, or spindle tuber and a single strain each of potato calico and potato yellow dwarf. Inoculations from diseased specimens infected with PVY plus potato viruses A, M, S, and X, singly or in some combinations did not affect the efficiency of P.I. 230579 in detecting PVY in the mixed infections. Plants of P.I. 230579 developed significant numbers of local lesions from PVY inoculations at temperatures from 16 to 27°C and at inoculum dilutions through 1:100. Excised individual leaves of P.I. 230579 can be used to detect PVY. Leaves of plants exposed to air pollutants are unsuitable for assay purposes.S. demissum P.I. 230579 is homozygous for the local reaction to isolates of PVY and is a valuable aid for indexing aphid or mechanically inoculated potato clones or seedlings for resistance to the virus. It is a superior diagnostic host for differentiating PVY from other viruses commonly found in potatoes in the United States.     

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.     

Distribution of tobacco rattle virus and potato virus X in leaves,roots, and fruits and/or seeds of naturally-infected weeds

Weeds in tobacco rattle virus (TRV)-infested potato fields were examined for TRV and potato virus X (PVX) with the enzyme-linked immunosorbent assay. Questionable positive reactions were evaluated with immunosorbent electron microscopy. Of 615 weeds, representing 28 species, sampled over a two-year period, four species contained only TRV, three were infected only with PVX and eight had both viruses. Tobacco rattle virus was located only in the roots of five species and PVX was found only in the foliage of five species. Seeds and/or fruits of three species contained TRV while two contained PVX. Only one weed species,Solanum sara choides, contained both viruses in all plant portions tested. Potato virus X was not detected in weeds within a field planted with PVX-free potato seed.     

Effect of the ring rot pathogen and latent potato viruses on ring rot symptoms and yield of potatoes

The ring rot bacterium,Corynebacterium sepedonicum (Spieck. and Kotth.) Skapt. and Burkh., and latent potato viruses (potato virus S and potato virus X) were investigated for their effect on ring rot symptom development on potato plants in the greenhouse and on symptom development and yield of potatoes in the field. Both virus-free (VF) and virus-infected (VI) Red Pontiac stem cuttings root-inoculated with ring rot bacteria in the greenhouse developed typical (T) ring rot symptoms, and symptom severity did not differ between VF and VI plants. In a field study, both VF and VI Russet Burbank seed pieces knife-inoculated with ring rot bacteria produced plants with atypical (A) and T ring rot symptoms as well as a combination of both types. The data suggest that more A than T symptoms develop on VI plants and more T than A symptoms develop on VF plants. Combined infection with the ring rot pathogen and the latent potato viruses resulted in greater yield losses of total and marketable Russet Burbank tubers than infection with the bacterial or viral pathogens alone.     

Resistance to potato viruses M,S, X and the spindle tuber virus in tuber-bearingSolanum species

On two separate occasions, some available accessions of tuber-bearingSolanum species were tested for resistance to potato viruses. Challenge inoculations were made mechanically with infective sap; and assessment was by sub-inoculation to differential host plants, supplemented by serological tests. In 1956–57, four accessions remained free from potato virus S (PVS), and nine remained free from PVM. In 1968–70, one or more clones of 11 accessions showed resistance to PVS—one of them,S. megistacrolobum, a diploid, apparently being hypersensitive. Resistance to PVX was found in seven accessions, and to PVM in two. There appeared to be resistance to the potato spindley tuber virus in five accessions.     

Effect of temperature and latent viruses on atypical ring rot symptoms of Russet Burbank potatoes

The ring rot bacterium,Corynebacterium sepedonicum (Spieck. and Kotth.) Skapt. and Burkh. [Clavibacter michiganense subsp.sepedonicum (Spieck et Kotth.) Davis et al.], and latent potato viruses (potato virus S and potato virus X) were investigated for their effect on atypical (ATYP) ring rot symptom development on Russet Burbank potato plants at different temperatures. Plants grown at 21 C from stem cuttings root-inoculated withC. sepedonicum developed typical wilting and chlorotic symptoms of ring rot that were equally severe on virus-free (VF) and virus-infected (VI) plants. All VF and VI plants grown at 15 C from inoculated stem cuttings exhibited ATYP symptoms that included extreme stunting, resetting, and chlorotic symptoms of ring rot. More severe ATYP symptoms developed on VI than on VF plants. Up to 5 wk after inoculation withC. sepedonicum, ATYP symptom severity ratings of both VF and VI plants increased and declined thereafter. The ATYP severity ratings were highly correlated with fresh weight of plants with high severity ratings being associated with low fresh weights. These results emphasize the need to determine the role of temperature and viral pathogens on ring rot symptomology in existing and newly developed potato cultivars and thereby enable better field detection of bacterial ring rot.     

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.     

Association of viral,bacterial and fungal pathogens with vascular discoloration of Russet Burbank potato tubers

Russet Burbank potatoes from the 1979 and 1980 crop years, collected from Chicago, IL repack warehouses and retail markets, were sampled for vascular discoloration. The amount of discoloration varied among sampling months and states of origin and decreased from 7.7% in 1979 to 1.6% in 1980. Highest levels of vascular discoloration were detected in December and January samples. Vascular discolored and non-discolored (control) tubers were assayed for the presence of potato leafroll, potato virus X, and beet western yellows viruses by the enzyme-linked immunosorbent assay (ELISA). Tubers were also assayed for 2 subspecies ofErwinia carotovora and forVerticillium albo-atrum andV. dahliae. Potato leafroll virus was detected in 31 of 831 vascular discolored tubers.V. albo-atrum was detected in 1 of 180 discolored tubers. Beet western yellows virus was not detected in discolored or non-discolored tubers. Two subspecies ofE. carotovora and potato virus X were equally common in discolored and non-discolored tubers.     

The Inter-genebank potato database and the dimensions of available wild potato germplasm

The Association of Potato Inter-genebank Collaborators (APIC) constructed a database of all wild potato holdings of the most important potato genebanks in Europe, the United States, Peru, and Argentina. The Inter-genebank Potato Database (IPD) now contains data of 11,819 wild potato accessions conserved in seven potato genebanks. The collector’s number is the key identifier used to merge all databases into the IPD. A total of 7,112 different wild potato accessions were identified, which comprise 5,306 accessions with known collector’s numbers. The IPD passport database showed that almost 30% of accessions held in APIC genebanks are from Argentina, a country that comprises less species diversity than Peru and Bolivia. These latter countries are represented by 24% and 20% of accessions, respectively. APIC genebanks maintain 188Solarium taxa out of more than 230 recognized by the latest comprehensive treatment of potatoes(Solarium sect.Petota) by Hawkes (1990). About 60% of the accessions comprise only 20 taxa represented by 785 to 92 accessions each. Conversely, 2% of the accessions comprise 72 taxa and are represented by five or fewer accessions each. About 70 taxa are not available in any genebank. The IPD evaluation database comprises 5,603 records with data from more than 33,000 evaluations of wild potato accessions. Fifty-five traits are summarized, including the reactions of the accessions to 12 pathotypes or races or strains of 12 fungi, four bacteria, 12 viruses, one viroid, 13 nematodes, and seven insects; response to heat and cold stress; and content of dry matter, starch, vitamin C, amylase, reducing sugars, and glycoalkaloids. About 30% of the wild potato populations screened showed various levels of resistance to most of the diseases and pests evaluated. The IPD database is available on the Internet at www.potgenebank.org.     

Krantz: A Russet cultivar for the irrigated sands

Krantz is an oblong to slightly blocky, russet-skinned potato cultivar with medium-late maturity and a vigorous vine. It is resistant to hollow heart, has high resistance to common scab and possesses high field resistance to late blight. It is moderately resistant toVerticillium wilt, susceptible to early blight, viruses S and X, and exhibits symptoms of bacterial ring rot. Krantz is adapted to the irrigated sands, but will growth crack in heavier soils.     

The infection pressure of potato leafroll virus and potato virus Y in relation to aphid populations in Cyprus

Summary The infection pressure of two viruses, potato leafroll (PLRV) and potato virus Y (PVY), both common in seed potatoes grown in Cyprus, was determined in three experiments in 1982–83. Virus-free bait plants, of potato and four other species, were exposed weekly to field infection during the growing season (March–June), and then returned to an aphid-free glasshouse for symptom expression. Only tobacco plants produced clear symptoms enabling reliable assessment of PVY infection pressure. When assessed with ELISA or by tuber indexing, the potato plants were efficient baits for both viruses whose infection period commenced at emergence (mid March to early April) and ended within 6–7 weeks. The seasonal trend of aphid populations, determined with Moericke traps or 100-leaf counts, correspond to that of virus spread. Correlation and regression analysis of aphid and virus data implicated the alate form ofMyzus persicae as the principal vector of both viruses.     

Potato plants transformed with a potato virus Y P1 gene sequence are resistant to PVYO

The cloned P1 sequence of PVY^O was transferred in sense orientation into the potato cultivar Pito usingAgrobacteriummediated transformation. Sixteen of the putatively transformed plants (NPTII positive) were assayed for PVY^O resistance. No PVY^O was detected in four plants, representing two lines, 21 days after two sap-inoculations and 35 days after graft-inoculation, and the plants remained symptomless, whereas other tested plants showed mosaic symptoms and had high PVY titers similar to those of the control plants. No line was resistant to PVY^N and potato viruses A and X. Southern analysis confirmed the presence of the transgene(s) in the two PVY^O-resistant and one susceptible line examined, but no signal was detected in nontransformed Pito. These results suggest a high level of protection against PVY^O in potato transformed with P1 sequence of PVY^O.     

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。     

马铃薯茎尖脱毒效果影响因素的研究

以克新2号、克新16号和克新17号等8个马铃薯品种为试验材料,对马铃薯茎尖脱毒效果的影响因素进行了研究。结果表明:不同种类的植物生长调节剂对茎尖的生长有不同的影响,茎尖在⑤号培养基(MS+1.0mg·L-1KT+0.5mg·L-1IAA+0.5mg·L-1GA3)上可不产生愈伤组织,直接成苗,是最适宜的培养基;6～8℃低温预处理和37℃热空气处理对茎尖脱毒有利;茎尖取材来源对成苗率没有影响,对脱毒效果影响较大,但脱毒效果依品种而异;为避免造成损失,需在15个月内准备出用于更换的基础苗。     

Rapid detection of potato viruses by dot-ELISA

Summary The dot-ELISA, an immunosorbent assay on nitrocellulose (NC) membranes, was applied to potato viruses. The sensitivity of the test, as determined by visual assessment of the colour reactions in serial dilutions, allowed a reliable detection of the potato viruses M, S, X, Y, A and of potato leafroll virus in leaves of non-potato hosts and potato plants. Tuber extracts taken from the rose end resulted in higher dilution end points than those from the heel end. The test can be performed within two to three hours by using stored NC-membranes precoated with specific γ-globulin. The possibility of storing antigen treated NC-membranes for some days allows the assay to be quickly applied to samples taken in the field.     

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.     

The incorporation of resistance toGlobodera Pallida intoSolanum Tuberosum germplasm adapted to North America 1

Potato seeds of 24 families consisting primarily ofSolanum tuberosum ssp.andigena with someS. vernei germplasm that were segregating for resistance to potato cyst nematodes (Globodera pallida races P₄A and P₅A andG. rostochiensis race R₁A) were obtained from the International Potato Center. These seeds produced 468 clones with sufficient tubers for evaluation for resistance toG. pallida races P₄A and P₅A in pot tests at the International Potato Center. Twenty-six of these clones, selected for a high degree of resistance toG. pallida races P₄A and P₅A, were crossed with neotuberosum ×Solanum tuberosurn hybrids that had been selected for resistance toG. rostochiensis race R₁A (golden nematode) and the viruses PVX and PVY. The resultant progenies possessed a high degree of resistance to all three races of the potato cyst nematode. From these progenies, 23 clones were selected for resistance to the three races (G. pallida P₄A and P₅A,G. rostochiensis R₁A) of potato cyst nematodes. The better adapted clones with resistance to the 3 races of potato cyst nematodes will be used in the next cycle of back-crossing.