Hosts and symptoms of Plum pox virus: Herbaceous hosts

Plum pox virus (PPV) is polyphagous and epidemic. Apart from cultivated and wild Prunus species, a large number of herbaceous plants can be hosts of the virus. New herbaceous host species are continuously being reported following artificial inoculation studies. Some of these herbaceous hosts, Chenopodium foetidum , Nicotiana clevelandii , N. benthamiana and Pisum sativum are very useful for concentrating and purifying the virus. The list of plants that have been found to be infected with PPV in their natural environment is shorter than the list of plants which can be experimentally infected. The role of weed species in PPV survival and spread in orchards is poorly understood. It is widely accepted that annual plants or weeds are not important in the epidemiology of PPV.     

Non-structural plum pox potyvirus proteins detected by immunogold labelling

Plum pox potyvirus (PPV) induces in infected Nicotiana clevelandii cells characteristic crystalline inclusions known as nuclear inclusions (NI) when located in the nucleus and as dense material (Dm) when located in the cytoplasm. Crystalline inclusions contain protease (NIa) and RNA-dependent RNA polymerase (NIb) proteins. It is now well established for all potyviruses that cylindrical inclusions contain CI helicase ATPase protein (Martin et al., 1992). The intracellular location of other non-structural PPV proteins remains unknown. Using Escherichia coli expression vectors, specific antibodies were obtained against P1, P3, 6K2 and NIb PPV proteins for which antibodies were not yet available. As expected, NIb antiserum labelled crystalline inclusions. P1, P3 and 6K2 proteins were present in both types of crystalline inclusions found in the nucleus and in the cytoplasm of PPV-infected leaves of N. clevelandii, suggesting that nuclear inclusions and dense material were composed of the same proteins. This composition is discussed.     

Plum pox potyvirus on sour cherry in Moldova

Plum pox potyvirus (PPV) was identified in six cultivars of sour cherry in the collection orchard of the Moldavian Horticultural Research Institute. Study of biological properties of the sour cherry isolate in herbaceous indicators showed its similarity or identity with the PPV isolate of Van Oosten and a significant difference from isolates widespread in Moldova. A purified viral preparation was used to develop antiserum with a working titre of 1:1024. Comparative serological examination of the sour cherry and conventional plum PPV isolates using ELISA, ISEM and SDS-PAGE of the protein capsid could not differentiate these isolates. The sour cherry isolate was transferred to plum resulting in weak but distinctive PPV symptoms in susceptible cv. Sopernitsa.     

A potyvirus isolated from Senna occidentalis

A potyvirus causing severe mosaic symptoms was isolated from Senna occidentalis (syn. Cassia occidentalis ) in the Yemen Republic and Ethiopia. It was transmitted mechanically and by Myzus persicae in a non-persistent manner. The flexuous, rod-shaped particles had a mean length of 830 nm, and pinwheels and scrolls were observed by electron microscopy of thin sections of infected Nicotiana clevelandii leaves. Its host range was narrow with only a few legume species, Nicotiana clevelandii and N. benthamiana susceptible to experimental infection. This virus was purified from N. clevelandii and the coat protein had a molecular mass of 34-5 kDa. It reacted positively in ELISA with monoclonal antibody 197 that is specific for potyviruses, but was not decorated by antibodies to any other potyvirus tested when examined by electron microscopy. The virus has been tentatively named cassia severe mosaic potyvirus.     

Significance of the heterologous encapsidation of zucchini yellow mosaic potyvirus in transgenic plants expressing plum pox potyvirus capsid protein1

Transgenic Nicotiana benthamiana plants expressing the coat protein of an aphid-transmissible strain of plum pox potyvirus (PPV-D) were infected with an aphid non-transmissible strain of another potyvirus, zucchini yellow mosaic potyvirus (ZYMV-NAT). Non-viruliferous Myzus persicae could acquire and transmit ZYMV-NAT from these plants but not from infected N. benthamiana control plants (not transformed, or transformed by the vector alone). Immunosorbent electron microscopy experiments using the decoration technique revealed that ZYMV-NAT virus particles in the infected transgenic plants expressing the PPV coat protein could be coated not only with ZYMV antibodies but also, on segments of the particles, with PPV antibodies. This suggests that aphid transmission of ZYMV-NAT occurred through heterologous encapsidation, and reveals a potential risk of releasing genetically engineered plants expressing viral coat proteins into the environment.     

Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins1

Eleven monoclonal antibodies specific to plum pox potyvirus (PPV) coat protein were obtained by hybridoma technology from Spanish PPV isolates. In addition, two monoclonal antibodies specific for PPV cylindrical inclusions (CIP non-structural proteins) were obtained. The monoclonal antibodies specific for PPV coat protein were assayed by DASI ELISA against 81 PPV isolates. At least nine different epitopes were found and 21 distinct serological patterns of reaction (serogroups) were established using nine selected monoclonal antibodies against the collection of PPV isolates, indicating the high variability of coat protein among PPV isolates. Changes in epitope composition were observed after aphid and mechanical transmission, indicating the occurrence of mixtures of isolates in field trees. Monoclonal antibody 5B reacted with all PPV isolates assayed, with very high affinity, using DASI ELISA. This method was compared with immunocapture-PCR on field samples in spring, and showed very good coincidence of results. The efficiency of PPV detection can be slightly increased using monoclonal antibodies specific to cylindrical inclusions mixed with monoclonal antibodies against structural proteins, and using mixtures of monoclonal antibodies against different epitopes of coat protein. ELISA-I and immunoprinting-ELISA were able to detect CIP and PPV in extracts and tissue section, respectively, of woody plants. Two monoclonal antibodies offer the possibility of distinguishing between Marcus and Dideron PPV types (M or D). These D-specific monoclonal antibodies can be used in routine tests with high affinity.     

A potyvirus isolated from solanaceous hosts

A potyvirus was isolated from Datura stramonium, Lycopersicon esculentum (tomato) and Solanum nigrum in the Yemen. It was transmitted mechanically and by Myzus persicae in a non-persistent manner. Its flexuous rod-shaped particles had a mean length of 719 nm and some of its pinwheel inclusion bodies in infected Nicotiana clevelandii leaves were unusual in that they were dichotomously branched. The virus infected various solanaceous species, but the symptoms it induced were distinct from those of pepper veinal mottle (PVMV) and potato Y viruses. Its particles were purified from N. glutinosa and their coat protein had an atypically high molecular mass a potyvirus of 41·5 kDa. They showed a distant serological relationship to those of PVMV and potato virus V in ISEM decoration tests, but did not react with antisera to particles of any other potyvirus tested. The virus has been tentatively named tomato mild mottle virus.     

Characterization of an isolate of beet mosaic virus from South Kazakhstan

A filamentous virus isolated from a sugar-beet plant showing systemic mosaic collected in South Kazakhstan was identified as an isolate of beet mosaic virus (BMV-K). BMV-K was transmitted by the green peach aphid Myzus persicae in a non-persistent manner, and by sap inoculation to 11 out of 19 species from seven families tested. The virus could not be transmitted to Nicotiana tabacum, N. debneyi, N. glutinosa and N. clevelandii, cither mechanically or with M. persicae. The thermal inactivation point of BMV-K in sugar-beet sap was 55-60 C, dilution end point 1:1000 and longevity in vitro 2 days at 20 C. A purification procedure produced 1-5-3 mg of purified virus from 100 g of infected Stellaria media plants. Purified virus contained a single protein species of molecular weight 34 700 Da. In ELISA tests, BMV-K reacted positively with BMV-specifc antisera obtained from Japan. Germany and Portugal. By competitive DAS- ELISA, the virus isolate was shown to be closely serologically related to all the three isolates of BMV, and very distantly related to bean yellow mosaic and soy bean mosaic viruses.     

Detection of pineapple closterovirus in pineapple plants and mealybugs using monoclonal antibodies

Monoclonal antibodies (MAbs) were produced to the pineapple closterovirus (PCV) in Hawaii. These antibodies were shown to be specific for PCV by decoration of the virus particles in immunosorbent electron microscopy (ISEM) and indirect enzyme-linked immunosorbent assay (ELISA). Several methods of ELISA were compared. An indirect DAS ELISA using a polyclonal antibody to trap virus particles followed by reaction with monoclonal antibody was shown to be the method of choice for detecting PCV in pineapple plants. Pineapple root tissue was found to be most suitable for detecting PCV in crude samples by indirect ELISA. PCV was detected in symptomatic and asymptomatic pineapple plants collected from Oahu and Maui, and pineapple collections in the USDA/ARS National Clonal Germplasm Repository, but was not detected from pineapple plants grown from seed. At least two serotypes of PCV were detected. In addition. PCV was detected from mealybugs collected from wilted pineapple plants, but not from mealybugs of the same species collected from a colony reared on squash. The role of PCV in mealybug wilt of pineapple is being investigated.     

Detection of double-stranded RNA and virus-like particles in Australian isolates of Pythium irregulare

Mycelial extracts of Pythium irregulare were processed using CF-11 chromatography and analysed by gel electrophoresis for double-stranded RNA (dsRNA) content. DsRNA molecules of between 1 and 6 kb were found in 33 of 39 isolates tested. The dsRNA profiles of individual isolates remained unchanged after repeated subculturing of hyphal tips, after storage under water for 2 years, or when single zoospores were used to generate subcultures. However, dsRNA profiles varied between isolates, and even within individual populations; in the one case eight different dsRNA types were recovered upon testing 17 isolates from a single wheatfield. Isometric virus-like particles (VLPs) of approximately 45 nm were found in isolates containing dsRNA, while no such particles could be found in isolates in which dsRNA was not detected. Electrophoretic profiles of dsRNA extracted from partial purifications of VLPs were identical to those of dsRNA extracted from whole mycelium, suggesting that the dsRNA is at least partially encapsidated. Attempts to transmit dsRNA between isolates by hyphal anastomosis or by coinfection in wheat plants were unsuccessful, with only parental types being recovered. Analysis of 29 isolates representing 14 other Pythium species failed to detect dsRNA, even when co-isolated with P. irregulare known to contain dsRNA     

Introgression of a Tombusvirus Resistance Locus from Nicotiana edwardsonii var. Columbia to N. clevelandii

ABSTRACT A new variety of Nicotiana, N. edwardsonii var. Columbia, was evaluated for its capacity to serve as a new source for virus resistance genes. Columbia was developed from a hybridization between N. glutinosa and N. clevelandii, the same parents used for the formation of the original N. edwardsonii. However, in contrast to the original N. edwardsonii, crosses between Columbia and either of its parents are fertile. Thus, the inheritance of virus resistance genes present in N. glutinosa could be characterized by using Columbia as a bridge plant in crosses with the susceptible parent, N. clevelandii. To determine how virus resistance genes would segregate in interspecific crosses between Columbia and N. clevelandii, we followed the fate of the N gene, a single dominant gene that specifies resistance to Tobacco mosaic virus (TMV). Our genetic evidence indicated that the entire chromosome containing the N gene was introgressed into N. clevelandii to create an addition line, designated N. clevelandii line 19. Although line 19 was homozygous for resistance to TMV, it remained susceptible to Tomato bushy stunt virus (TBSV) and Cauliflower mosaic virus (CaMV) strain W260, indicating that resistance to these viruses must reside on other N. glutinosa chromosomes. We also developed a second addition line, N. clevelandii line 36, which was homozygous for resistance to TBSV. Line 36 was susceptible to TMV and CaMV strain W260, but was resistant to other tombusviruses, including Cucumber necrosis virus, Cymbidium ringspot virus, Lettuce necrotic stunt virus, and Carnation Italian ringspot virus.     

Detection and characterization of Plum pox virus: serological methods

Tremendous progress has been made in the research and development of Plum pox virus (PPV) serological reagents and methods in recent years. Two facts have revolutionised the serological detection and characterization of the virus: the development of the ELISA method in 1977, and the later emergence of specific monoclonal antibody technology. The availability of commercial kits has popularised PPV diagnosis, now making diagnosis possible at large scale for quarantine purposes, eradication programmes and control of the disease in nurseries. The use of the universal monoclonal antibody 5B-IVIA, used in DASI-ELISA, is the most accurate system for routine PPV detection. Likewise, the use of typing monoclonal antibodies gives exact characterization of the main PPV types described: 4DG5 for PPV-D, AL for PPV-M, EA24 for PPV-EA, and TUV and AC for PPV-C. There is, in general, an excellent correlation between serological data obtained with PPV specific monoclonal antibodies and data obtained by molecular PCR based methods. ELISA using a single or a mixture of monoclonal antibodies will remain the preferred method for universal detection and routine screening of PPV for years to come. Today, other serological methods and reagents are also recommended in the EPPO Diagnostic Protocol, increasing the number of reliable tests available for PPV detection. These developments have helped to control sharka disease in recent years. International co-operation in this field has been crucial to the improvement and validation of serological tools for PPV detection and characterization.     

A new virus disease of watercress in England1

In 1983 plants showing symptoms typical of watercress chlorotic leafspot agent (WCLSA) were found in Kent (GB). Later, plants showing similar symptoms were found in Dorset (1986) and Hampshire (1987), the main watercress-producing regions of England. These plants, unlike those infected with WCLSA, contained virus particles which were isometric with diameters of 37-38 nm. An antiserum to the virus was produced, with a titre in immunodiffusion tests of 1/128 and this gave good results in immunosorbent electron microscopy (ISEM) and ELISA tests on crude plant sap. Purified preparations of the virus reacted positively in ISEM and immunodiffusion tests with an antiserum to watercress yellow spot virus, a partially described virus occurring in France. Tests suggest that the crook root fungus Spongospora subterranea f.sp. nasturtii is probably the vector of this virus but other means of transmission cannot be excluded. Virus incidence within watercress beds varied, with little or no infection in the water inlet region of the bed where there are also low levels of crook root infection, higher levels in the central region and highest levels at the outlet region where crook root infection is also highest.     

检测植物病毒的三种血清学方法敏感性的比较

 以芋花叶病毒和豇豆花叶病毒为试验材料,进行了酶联免疫吸附试验,免疫吸附电子显微术和点免疫结合试验检测植物病毒的敏感性的研究。无论是检测感染组织粗汁液还是纯化的病毒,点免疫结合试验均优于酶联免疫吸附试验和免疫吸附电子显微术。在使用羟基吲(口乃木)磷酸盐和氮蓝四唑为碱性磷酸酶的底物时,点免疫结合试验检测纯化的豇豆花叶病毒的可测感度为0.35ng,芋花叶病毒为0.83ng。对三种检测植物病毒的血清学方法进行了比较,并讨论了点免疫结合试验的优点。     

Plum pox virus detection in dormant plum trees by PCR and ELISA

An immunocapture polymerase chain reaction (IC-PCR) protocol and ELISA were compared for their effectiveness in detecting plum pox virus (PPV) in dormant plum material. Although the IC-PCR was about one thousand times more sensitive than ELISA, PPV was detected by ELISA in 71–80% of bark samples collected in December, January and March 1996/97 from pot-grown rootstock trees inoculated with PPV the previous March, compared with 85–86% detection in the same samples by IC-PCR. In similar samples from one-year-old shoots taken from infected branches of orchard trees, 66–81% were positive by ELISA compared with 81–87% by IC-PCR. With bulked samples taken from the fibrous roots of the pot-grown trees, PPV was detected in 92–100% of samples by IC-PCR in winter compared with only 38–65% by ELISA. These results were confirmed in samples from the roots and shoots of the same trees in 1997/98. Three samples per shoot would have been sufficient to detect PPV by ELISA in 87 of the 88 infected shoots tested during the two winters. However, infected shoots are irregularly distributed in diseased trees and PCR assays of root samples offer the potential for improving the reliability of identifying trees infected with PPV.     

The occurrence of three viruses in hop (Humulus lupulus) in China

Forty-two percent of leaf samples taken from hop cv. Qingdao Dahua growing in plantations in northeastern China in 1983-1984 were infected with hop mosaic virus (HMV) and/or hop latent virus (HLV). Mosaic or line-pattern symptoms were seen in some plants but only in spring and it is uncertain if the symptoms were caused by either virus which appeared in our studies to be similar, both serologically and in host range, to HMV and HLV described in Europe and North America. In host range studies, HMV infected Nicotiana clevelandii systemically without inducing symptoms. Occasionally it caused chlorotic spots in the inoculated leaves of Chenopodium quinoa and brown star-point lesions in inoculated leaves of Phaseolus vulgaris cv. Topcrop, The virus was purified by PEG precipitation from extracts of N. clevelandii plants; yields were about 40-120 mg/kg fresh leaves. The virus contained a single protein (estimated molecular weight 34 200) that was estimated to contain 272 amino acids with no methionine, and a single RNA species (estimated molecular weight 295 x 10⁶) that represented c. 6% of the particle weight. HLV sometimes caused inconspicuous chlorotic spots in inoculated leaves of C. murale and local pinpoint lesions in P. vulgaris in winter only. A virus similar to alfalfa mosaic virus was isolated from leaves of hop cv. Golding and caused systemic necrotic symptoms in C. quinoa and C. amaranticolor , and systemic chlorotic spots in hop cv. Styrian.     

免疫电镜法测定3种Potyvirus组病毒的研究

 对大豆花叶病毒(SMA),花生斑驳病毒(PMV)和豇豆蚜传花叶病毒(CAMV)3种Potyvirus病毒进行了免疫电镜(ISEM)的测定。ISEM法同样证实了这3种病毒在血清学关系上的不同。3种病毒抗血清对同源抗原都具有"捕获"大量病毒粒体的能力,"捕获"数量是异源关系或正常血清的20~50倍.ISEM方法的灵敏度和专化性受到包被抗血清的稀释度和在抗原上处理时间等因素的影响."捕获"病毒粒体最适的抗血清稀释度是10-3或10-4;抗原处理时间,在一定范围内随处理时间的延长而增加"捕获"病毒粒体的数量,处理时间超过10-20小时即不再增加"捕获"粒体数量.同源抗体抗-抗原反应对病毒粒体上有"装饰"作用,而异源关系没有"装饰"作用。"装饰"的免疫电镜法是鉴别同源病毒的重要标准。     

Detection of plum pox potyvirus and analysis of its molecular variability using immunocapture-PCR1

We have developed an immunocapture-PCR (IC-PCR) detection technique for plum pox potyvirus (PPV) which is both simple and highly sensitive. This single-day assay can detect about 2000 virus particles (200 fg of virus) diluted in 100 μl of crude plant sap, which is equivalent to a sensitivity about 2000 times better than that of a standard ELISA assay. RFLP analysis and sequencing of the amplified cDNA fragment indicate that three groups of strains with limited intra-group variability can be discriminated. Two of these groups correspond to the previously described D and M serotypes of PPV. The third group contains, so far, only the El Amar Egyptian isolate. Strains belonging to the D or M serotypes can easily be discriminated by Rsal polymorphism in the amplified cDNA fragment. Synthetic oligonucleotides allowing specific amplification of PPV strains belonging either to the D or to the M serotypes have also be designed.