Monoclonal antibodies for the detection and differentiation of faba bean necrotic yellows virus isolates

Murine monoclonal antibodies (MAbs) were produced for the detection of faba bean necrotic yellows virus (FBNYV), an isometric ssDNA virus belonging to a new, yet unnamed genus of plant viruses. A total of 19 FBNYV-specific MAbs were obtained from three fusion experiments and characterised by determining their immunoglobulin types and titres as well as their corresponding epitopes. At least six distinct epitopes were revealed on FBNYV particles of different virus isolates. Only two MAbs reacted with SDS-dissociated FBNYV virions in triple antibody sandwich (TAS)-ELISA and with viral capsid protein in Western blots. Almost all MAbs were more sensitive in detecting FBNYV in viruliferous aphids by TAS-ELISA than polyclonal anti-FBNYV IgG by double antibody sandwich ELISA and permitted virus detection in individual aphids even following short acquisition access feeding periods. Coat protein variation among FBNYV isolates and serological relatedness to taxonomically similar viruses was studied by determining the cross reactivity of these MAbs with several field isolates of FBNYV as well as with milk vetch dwarf (MDV), banana bunchy top (BBTV), and subterranean clover stunt (SCSV) viruses. Whereas none of the MAbs reacted with BBTV, only one reacted with SCSV, indicating that FBNYV and SCSV share a common epitope. By contrast, 16 of the 19 MAbs reacted with MDV, suggesting that FBNYV and MDV are serologically closely related and strains of the same virus. When all 19 MAbs produced were tested against a total of 107 samples of FBNYV collected during virus surveys in Egypt, Ethiopia, Jordan, Morocco and Syria, five MAbs showed differential reactions. While the majority of the samples reacted with all 19 MAbs, about 20% of the 107 FBNYV samples did not react with one and/or other of these five MAbs, permitting the differentiation of seven serotypes of FBNYV and suggesting a considerable coat protein variation in FBNYV isolates from the countries surveyed. The MDV isolate from Japan and five FBNYV samples from Ethiopia appeared to be the least closely related to typical FBNYV isolates by not reacting with three and four, respectively, of the five differentiating Mabs.     

Faba Bean Necrotic Yellows Virus Naturally Infects Phaseolus Bean and Cowpea in the Coastal Area of Syria

In an attempt to identify possible summer hosts of the faba bean necrotic yellows virus (FBNYV), a field survey was conducted in the coastal area of Syria. Using a monoclonal antibody to FBNYV in indirect ELISA, FBNYV was detected in a large number of samples from Phaseolus vulgaris L. and in a few samples from Vigna unguiculata (L.) Walp. in which it caused severe symptoms. This is the first report of natural infection of P. vulgaris and V. unguiculata with FBNYV.     

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D ‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM . About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.     

A functional histidine-tagged replication initiator protein: implications for the study of single-stranded DNA virus replication in planta

Replication initiation of nanoviruses, plant viruses with a multipartite circular single-stranded DNA genome, is triggered by the master Rep (M-Rep) protein. To enable the study of interactions between M-Rep and viral or host factors involved in replication, we designed oligohistidine-tagged variants of the nanovirus Faba bean necrotic yellows virus (FBNYV) M-Rep protein that allow affinity purification of enzymatically active M-Rep from plant tissue. The tagged M-Rep protein was able to initiate replication of its cognate and other FBNYV DNAs in Nicotiana benthamiana leaf disks and plants. The replicon encoding the tagged M-Rep protein multiplied and moved systemically in FBNYV-infected Vicia faba plants and was transmitted by the aphid vector of the virus. Using the tagged M-Rep protein, we demonstrated the in planta interaction between wild-type M-Rep and its tagged counterpart. Such a tagged and fully functional replication initiator protein will have bearings on the isolation of protein complexes from plants.     

Production and properties of monoclonal antibodies to Solanum nodiflorum mottle sobemovirus

Black raspberry necrosis virus (BRNV) reaches only very low concentrations in herbaceous plants and is difficult to maintain in culture. However, in a mixed culture with an unrelated virus, Solanum nodiflorum mottle (SNMV), in the genus Sobemovirus, the concentration of BRNV particles increases about 1000‐fold. In attempts to produce monoclonal antibodies (MAbs) to BRNV for diagnostic use, purified virus particles from the mixed virus culture were used as immunogen and the resultant antibodies screened against cultures of SNMV alone, BRNV+SNMV and healthy plant extracts. None of the virus‐specific MAbs obtained in this way was specific to BRNV but six were specific to SNMV. Although the original objective was not achieved, the SNMV MAbs were characterised and used to study serological properties of SNMV and other Sobemoviruses. Characterisation of the six SNMV MAbs showed that four were IgG3, one IgG1 and the other IgG2b. SNMV was detected by all six MAbs in ELISA, by five in Western blotting, by three in agarose gel double diffusion tests, but only one was suitable for trapping virus particles in immuno‐electron microscopy (IEM). In Western blotting using virus in sap extracts of Nicotiana clevelandii, each of the five MAbs detected a single major band of Mc. 31 000 in sap containing SNMV, and additional bands of lower mass attributed to degradation of coat protein. In various serological tests, no cross‐reactions were detected between SNMV and seven other viruses from the genus Sobemovirus. However, in IEM but not in Western blotting, significant cross‐reactions were observed between SNMV and Velvet tobacco mottle virus, another species from the genus Sobemovirus. The significance of these different findings is discussed.     

Transmission characteristics and some other properties of bean yellow vein-banding virus, and its association with pea enation

Bean yellow vein-banding virus (BYVBV) has been found occasionally in mixed infection with pea enation mosaic virus (PEMV) in spring-sown field beans (Vicia faba minor) in southern England. Glasshouse tests confirmed that, like PEMV, BYVBV is transmissible by manual inoculation and by aphids in the persistent manner. However, BYVBV can be transmitted by aphids only from plants that are also infected with a helper virus, usually PEMV. Thus after separation from PEMV by passage through Phaseolus vulgaris it was no longer aphid-transmissible. It became aphid-transmissible again only after re-mixing in plants with PEMV or with a substitute helper, bean leaf roll virus (BLRV). It was not transmitted by aphids that fed sequentially on plants singly infected with PEMV and BYVBV. Thus the interaction between BYVBV and PEMV (or BLRV) that enables BYVBV to be transmitted by aphids seems to occur only in doubly infected plants. However, it was not transmitted by aphids from plants doubly infected with BYVBV and broad bean wilt virus (BBWV). BYVBV and PEMV were transmitted more readily by Acyrthosiphon pisum than by Myzus persicae; neither virus was transmitted by Aphis fabae. Phenol extracts of BYVBV-infected leaves were more infective than phosphate buffer or bentonite-clarified extracts and were sometimes infective when diluted to 1/1000. The infectivity of BYVBV in phosphate buffer extracts of leaves singly infected with BYVBV, unlike that in extracts of leaves doubly infected with BYVBV and PEMV (or BLRV), was destroyed by treatment with organic solvents. BYVBV infected 11 of 28 plant species that were inoculated with phenol extracts; seven of the infected species were legumes. No transmission of BYVBV was detected through seed harvested from infected field bean plants. Isometric particles c. 30 nm in diameter were seen in extracts of plants doubly infected with BYVBV and PEMV but not in extracts of plants infected with BYVBV alone. Leaves of plants infected with BYVBV, alone or with PEMV, contained membrane-bound structures c. 50–90 nm in diameter associated with the tonoplast in cell vacuoles. These structures were not found in healthy leaves. BYVBV has several properties in common with other known aphid-borne viruses that are helper-dependent and transmitted in a persistent manner. Possibly, as suggested for some of them, aphid transmission of BYVBV depends on the coating of its nucleic acid with helper virus coat protein.     

Acquisition, Retention and Transmission of Faba Bean Necrotic Yellows Virus by Two of its Aphid Vectors, Aphis craccivora (Koch) and Acyrthosiphon pisum (Harris)

Faba bean necrotic yellows virus (FBNYV) belongs to a new group of plant viruses that have unusually small isometric virions and a multipartite ssDNA genome. It is the causal agent of some virus diseases affecting several food and fodder legumes in west Asia and north Africa. FBNYV is persistently transmitted by various aphid species of which Aphis craccivora appears to be the most significant natural vector. In attempts to obtain a better understanding of factors involved in FBNYV spread under field conditions, the interactions of the virus with A. craccivora and Acyrthosiphon pisum were studied. The two species were efficient vectors and very similar in their minimum acquisition (AAP) and minimum inoculation access feeding periods which ranged from 15 to 30min and 5–15min, respectively. Following an AAP of 72 h and daily serial transfers of individual aphids to single plants, many individuals retained and transmitted the virus throughout their life span (up to 32 days) but at erratic efficiencies. In this persistence experiment A. pisum was a more efficient vector than A. craccivora. For both aphid species no decrease in transmission efficiency was observed, suggesting that nymphs acquired large amounts of FBNYV virions which were not depleted in their hemocoel during the experiment. Based on log-pro-bit analysis, median latency period (LPso) values of 108.8h and 105.0h were calculated for FBNYV in A. craccivora and A. pisum , respectively. FBNYV was not lost during moults and was not passed on to the par-thenogenetic offspring by viruliferous adults. Aphids which acquired FBNYV as adults were strikingly poor vectors as compared to nymphs.     

Quantitative studies on the uptake and retention of potato leafroll virus by aphids in laboratory and field conditions

Enzyme-linked immunosorbent assay (ELISA) was adapted for the efficient detection and assay of potato leafroll virus (PLRV) in aphids. Best results were obtained when aphids were extracted in 0.05 M phosphate buffer, pH 7.0, and the extracts incubated at 37 °C for 1 h before starting the assay. Using batches of 20 green peach aphids (Myzus persicae), about 0.01 ng PLRV/aphid could be detected. The virus could also be detected in single aphids allowed a 1-day acquisition access period on infected potato leaves. The PLRV content of aphids depended on the age of potato source-plants and the position of source leaves on them. It increased with increase in acquisition access period up to 7 days but differed considerably between individual aphids. A maximum of 7 ng PLRV/aphid was recorded but aphids more usually accumulated about 0.2 ng PLRV per day. When aphids were allowed acquisition access periods of 1–3 days, and then caged singly on Physalis floridana seedlings for 3 days, the PLRV content of each aphid, measured subsequently, was not strongly correlated with the infection of P. floridana. The concentration of PLRV in leaf extracts differed only slightly when potato plants were kept at 15, 20, 25 or 30 °C for 1 or 2 wk, but the virus content of aphids kept on leaves at the different temperatures decreased with increase of temperature. PLRV was transmitted readily to P. floridana at all temperatures, but by a slightly smaller proportion of aphids, and after a longer latent period, at 15 °C than at 30 °C. The PLRV content of M. persicae fed on infected potato leaves decreased with increasing time after transfer to turnip (immune to PLRV). The decrease occurred in two phases, the first rapid and the second very slow. In the first phase the decrease was faster, briefer and greater at 25 and 30 °C than at 15 and 20 °C. No evidence was obtained that PLRV multiplies in M. persicae. These results are compatible with a model in which much of the PLRV in aphids during the second phase is in the haemocoele, and transmission is mainly limited by the rate of passage of virus particles from haemolymph to saliva. The potato aphid, Macrosiphum euphorbiae, transmitted PLRV much less efficiently than M. persicae. Its inefficiency as a vector could not be ascribed to failure to acquire or retain PLRV, or to the degradation of virus particles in the aphid. Probably only few PLRV particles pass from the haemolymph to saliva in this species. The virus content of M. euphorbiae collected from PLRV-infected potato plants in the field increased from early June to early July, and then decreased. PLRV was detected both in spring migrants collected from the plants and in summer migrants caught in yellow water-traps. PLRV was also detected in M. persicae collected from infected plants in July and August, and in trapped summer migrants, but their PLRV content was less than that of M. euphorbiae, and in some instances was too small for unequivocal detection.     

Seed transmission of broad bean stain virus and Echtes Ackerbohnenmosaik-Virus in field beans (Viciafaba)

When grown in a glasshouse during spring or autumn field bean (Vicia faba minor) seedlings infected with seed-borne broad bean stain virus (BBSV) or Echtes Ackerbohnenmosaik-Virus (EAMV) usually showed symptoms on some leaves within 4 wk of emergence. Symptoms caused by each virus were indistinguishable. The viruses were transmitted as often through unblemished seeds as through seeds with necrotic patches or stains on the seed coat, and sometimes as often through large as through small seeds. Soaking seeds for 24 h in solutions of 8-azaguanine or polyacrylic acid did not decrease transmission. Both viruses were detected in nearly mature seeds by inoculation to Phaseolus vulgaris but neither virus was detected in fully ripened seeds by inoculation or serological tests. The percentage of seeds from field plots that produced infected seedlings when sown in a glasshouse was closely related to the percentage of parent plants that showed symptoms of BBSV and/or EAMV at the end of flowering. The relationship seemed similar in different cultivars. On average EAMV was transmitted through more seeds than BBSV, probably because more parent plants were infected with EAMV. Inspection of seed crops for symptoms of BBSV and EAMV at the seedling stage and again at the end of flowering is probably the most practicable way of identifying progeny seed lots with little or no infection.     

Involvement of Volatile Substances in Systemic Resistance of Barley Against Erysiphe graminis f. sp. hordei Induced by Pruning of Leaves

Horsegram yellow mosaic disease was shown to be caused by a geminivirus; horsegram yellow mosaic virus (HYMV). The virus could not be transmitted by mechanical sap inoculation. Leaf dip and purified virus preparations showed geminate virus particles, measuring 15-18 * 30 nm. An antiserum for HYMV was produced and in enzyme-linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM) tests HYMV was detected in leaf extracts of fieldinfected bambara groundnut, french bean, groundnut, limabean, mungbean, pigeonpea and soybean showing yellow mosaic symptoms. Bemisia tabaci fed on purified HYMV through a parafilm membrane transmitted the virus to all the hosts listed above but not to Ageratum conyzoides, okra, cassava, cowpea, Croton bonplandianus, Lab-lab purpureus, Malvastrum coromandalianum and tomato. No reaction was obtained in ELISA and ISEM tests between HYMV antibodies and extracts of plants diseased by whitefly-transmitted agents in India such as A. conyzoides yellow mosaic, okra yellow vein mosaic, C. bonplandianus, yellow vein mosaic, M. coromandalianum yellow vein mosaic, tomato leaf curl and cassava mosaic. HYMV was also not found to be related serologically to bean golden mosaic, virus.     

The serological relationships and some other properties of isolates of broad bean wilt virus from faba bean and pea in China

An isolate (N15) of broad bean wilt virus (BB W V) from faba bean in China was compared with some other isolates and strains including the nasturtium ringspot strain (NRSV, BBWV serotype I), parsley virus 3 (PV3, serotype I) and BBWV isolate PV131 (serotype II). In host range studies, N15 infected 12 of 14 species, including soybean and spinach. It was purified from Chenopodium quinoa and pea by a method that yielded up to 8mg/100g tissue. By the same method, NRSV yielded up to 4mg/100 g. Purified preparations of N15 and NRSV contained isometric particles c. 26 nm in diameter which sedimented as three components, N15 at 62, 93 and 117 S, and NRSV at 60, 91 and 116 S. In immunodiffusion tests using antisera to N15 and NRSV, N15 was distinguishable from NRSV but indistinguishable from PV131. In ISEM tests, many more particles of N15 and NRSV were trapped by homologous than by heterologous antiserum; in decoration tests, much antibody attached to homologous particles but none to heterologous particles. In DAS ELISA using N15 antiserum, N15 and six other Chinese faba bean or pea isolates, and a Chinese spinach isolate, were readily detected and were indistinguishable from each other and from PV131; unlike NRSV and PV3, none of the Chinese isolates, nor PV131, was detected using NRSV antiserum. These results indicate that the Chinese isolates belong to BBWV serotype II group.     

Characterization of Potyvirus Isolates from West African Yams (Dioscorea spp.)

In comparative studies on potyviruses from West African yams (Dioscorea spp.) the following isolates were used: Dioscorea greenbanding mosaic virus (DGMV) and a Nigerian yam virus (YV-N), both isolated from Dioscorea rotundata, and a beet mosaic virus isolate from D. alata (BtMV-Y) formerly designated Dioscorea alata ring mottle virus. Naturally infected D. alata containing very few particles of BtMV-Y, contained primarily particles of a second potyvirus (Dioscorea alata virus, DaV) which could not be transmitted but which was included in these studies wherever possible. The normal lengths of DGMV, YV-N, DaV, and BtMV-Y were 754, 772, 805, and 812 nm, respectively. All viruses induced cytplasmic inclusions of the pinwheel type and laminated aggregates. In addition, the nucleoli of BtMV-Y infected cells contained characteristic electron dense inclusions. The buoyant density of purified DGMV and BtMV-Y in CsCl was 1.336 g/cm³ and 1.321 g/cm³, respectively. The sedimention velocities (S_rel) of DGMV, YV-N, and BtMV-Y were 156, 158, and 162 Srespectively. In SDS-polyacrylamide gel electrophoresis the coat protein of purified DGMV and YV-N all migrated as a single band with an apparent molecular weight of 36 kd. Coat protein of purified DaV showed up to 5 bands with molecular weights of 36 to, 32 kd. Polypeptides of purified BtMV-Y had an estimated molecular weight of 35 kd but those from infected plant extracts had a molecular weight of 36 kd. DGMV, YV-N, and BtMV-Y particles contained a single nucleic acid with an apparent molecular weightof 3.2, 3.2, and 3.1 Md, respectively. Using λ-DNA digested with Hind III as a marker, the molecular weight of DGMV and BtMV-Y nucleic acid was calculated to be 3.6 Md ± 10%. The nucleic acid was determined to be single-stranded RNA by enzymatic digestion and by staining with acridine orange. In serological studies using immunoelectron microscopy (IEM), electro-blot immunoassay (EBIA), and enzyme-linked immunosorbent assay (ELISA), DGMV and YV-N were closely related. Strong serological reactions were also obtained in IEM and EBIA when DGMV and YV-N were tested with antiserum to yam mosaic virus (YMV). Antisera against DGMV, YV-N, and YMV also reacted strongly with DaV antigen. Serological reactions between these viruses and BtMV-Y were usually not found or were weak. A very close serological relationship could be detected between BtMV-Y and beet mosaic virus isolated from beet (BtMV); both isolates were also very similar in host range, symptomatology, and cytopathology.     

Detection of potato virus Y in the aphid Myzus persicae by enzyme-linked immunosorbent assay (ELISA)

Potato virus Y was detected by enzyme linked immunosorbent assay (ELISA) in at least 50% of groups of five Myzus persicae. The mean A₄₀₅ value for groups of viruliferous aphids was 2–3 times greater than that for virus-free ones. PVY was not detected in Aphis craccivora, A. citricola or A. gossypii, three other species which transmitted the virus to peppers, and it was detected in only a small proportion of groups of Acyrthosiphon pisum. In a series of trials, success in detection of PVY by ELISA was not correlated with the ability of other aphids from the same source plant to transmit the virus to test plants. The limitation of ELISA for quantitative assay of PVY in aphids and for epidemiological work is discussed.     

A single rep protein initiates replication of multiple genome components of faba bean necrotic yellows virus, a single-stranded DNA virus of plants

Faba bean necrotic yellows virus (FBNYV) belongs to the nanoviruses, plant viruses whose genome consists of multiple circular single-stranded DNA components. Eleven distinct DNAs, 5 of which encode different replication initiator (Rep) proteins, have been identified in two FBNYV isolates. Origin-specific DNA cleavage and nucleotidyl transfer activities were shown for Rep1 and Rep2 proteins in vitro, and their essential tyrosine residues that catalyze these reactions were identified by site-directed mutagenesis. In addition, we showed that Rep1 and Rep2 proteins hydrolyze ATP, and by changing the key lysine residue in the proteins' nucleoside triphosphate binding sites, demonstrated that this ATPase activity is essential for multiplication of virus DNA in vivo. Each of the five FBNYV Rep proteins initiated replication of the DNA molecule by which it was encoded, but only Rep2 was able to initiate replication of all the six other genome components. Furthermore, of the five rep components, only the Rep2-encoding DNA was always detected in 55 FBNYV samples from eight countries. These data provide experimental evidence for a master replication protein encoded by a multicomponent single-stranded DNA virus.     

Differentiation Among Bean Leafroll Virus Susceptible and Resistant Lentil and Faba Bean Genotypes on the Basis of Virus Movement and Multiplication

Systemic movement of Bean leafroll virus (BLRV) in susceptible and resistant lentil and faba bean genotypes was studied using plants grown in a plastic house. All the plants studied were inoculated with BLRV by viruliferous pea aphids (Acyrthosiphon pisum). Five plants/genotype of lentil and faba bean were harvested, respectively, at 3, 6, 9, 12 and 18 days and 1, 2, 3, 4 and 5 weeks after inoculation. Each plant was split into growing point, stem, stem base and root, and each was tested using tissue blot immunoassays (TBIA). Virus concentration in each section was estimated using a 0–3 score and a relative TBIA value was estimated accordingly for each genotype. In susceptible lentil genotypes (ILL 8063 and ILL 2581), BLRV was present in low concentrations in the growing point 3 days after inoculation and in high concentrations in all parts of the plant after 6 days. By contrast, the virus was not detected in the highly resistant genotype (ILL 74) until 18 days after inoculation. In the faba bean genotypes studied, BLRV was detected in high concentrations in all parts of the highly susceptible genotype (Fiord) 1 week after inoculation but only after 3 weeks in resistant genotypes (e.g. BPL 5274), but was not detected in the highly resistant genotypes (BPL 5278 and BPL 5279) 5 weeks after inoculation. The replication and systemic movement of BLRV was thus slower in resistant genotypes than in susceptible genotypes. Moreover, the use of TBIA scores clearly and easily differentiated resistant and susceptible genotypes. Our results suggest that BLRV movement and multiplication can be useful criteria when differentiating resistant from susceptible genotypes. In addition, undertaking the preliminary screening in a plastic house requires less space than direct planting in the field.     

Comparison of ELISA and immunoblotting techniques for the detection of cherry mottle leaf virus

Formaldehyde treated cherry mottle leaf virus (ChMLV) and the isolated coat protein were used successfully for the production of polyclonal and monoclonal antibodies. The monoclonal antibodies had a titre of 1:51 200 and consisted of IgG1 and IgG2. The antibodies reacted with all 11 isolates of ChMLV, from five locations in Canada and the USA, included in this study. Several serological procedures were assessed to compare their sensitivity for detecting ChMLV. Plate-trapped antigen ELISA (PTA-ELISA) and dot-blot immunobinding assay (DBIA), using virus specific MAbs, were the most sensitive tests in this study. Triple antibody sandwich ELISA (TAS-ELISA) and Western blot were found to be less sensitive. Dilution of the samples appeared to increase the sensitivity of both PTA-ELISA and Western blot detection. Young leaves and flowers of Prunus avium were the best tissue for detecting the virus which could also be detected in the fruit and leaves of P. tomentosa. April and May were optimal for detection of the virus in the field, whereas both April to May and August to September were optimal for screenhouse-grown plants.     

侵染浙贝母3种病毒CP基因的原核表达、抗血清制备及其病毒检测

根据GenBank报道的浙贝母花叶病毒(Thunberg fritillary mosaic virus,TFMV)、浙贝母Y病毒(Fritillary virus Y,FVY)和百合斑驳病毒(Lily mottle virus,LMoV)序列设计引物,扩增其CP基因。将CP基因插入表达载体pSBET,转化大肠杆菌BL21(DE3)Plys E菌株,IPTG诱导表达。经12% SDS-PAGE和5%~20%梯度SDS-PAGE两次纯化CP,分别免疫小鼠获得抗CP血清。采用Western blot分析确定抗体的特异性及其之间的血亲学关系;采用ELISA分析确定抗体是否能与天然病毒粒子结合。采用Western blot、间接ELISA法和Dot-ELISA 法检测侵染浙贝母的3种病毒。结果表明,制备的抗体对CP有高度特异性,相互之间无交叉反应,且能与天然病毒离子结合。制备的抗血清可以用于检测3种病毒,其中间接ELISA法和Dot-ELISA法检测效果较好。     

Antixenosis resistance to Aphis fabae Scopoli (Hemiptera:Aphididae) in bean cultivars

Aphis fabae Scopoli (Hemiptera: Aphididae) is heteroecious and polyphagous that is harmful on secondary hosts such as many important agricultural products like beet, common bean, faba bean, potato and other products. This aphid is the cause of more than 33 viral transition. One of the mechanisms of plant resistance is antixenosis. This mechanism influences on placement and nutrition of pests that result in less damage. In this study, antixenosis resistance mechanism of 12 varieties of bean was tested. Experiment was on completely randomised design with 12 treatments and 6 replications. Bean varieties include of white bean, kidney bean and wax bean, and each replication includes one pot, and then, pots were placed under the isolated room that were filled with winged adult aphids in circular form. After 24 and 48?h, aphids and level of nymph production were counted. The lowest number of adult aphids was observed on Sayad variety among 12 varieties (during 24?h). The least number of produced nymphs was in Daneshkade variety. In Sayad variety, the frequency of matured insects and produced nymphs was minimum.     

Effect of sowing date and straw mulch on virus incidence and aphid infestation in organically grown faba beans (Vicia faba)

The effect of sowing date on aphid infestation and the incidence of aphid-transmitted viruses were investigated in organically managed, small-scale field experiments with two faba bean cultivars over 3 years (2002–04). As an additional factor, straw mulch was applied in 2 of the 3 years shortly before the start of vector activity in May. Virus incidence was determined using enzyme-linked immunosorbent assay and immunoelectron microscopy. Aphid flight activity was monitored using standard yellow water traps. Bean colonising aphids were assessed throughout the vegetation period by counting the number of plants infested with Acyrthosiphon pisum , Megoura viciae and Aphis fabae . Pea enation mosaic virus and bean yellow mosaic virus were the most abundant aphid-transmitted viruses, being detected in 22–54% and 9–69%, respectively, of the total number of virus-infected plants analysed per year. Further aphid-transmitted viruses found in faba bean were bean leaf roll virus, beet western yellows virus, clover yellow vein virus (in 2002) and soybean dwarf virus (in 2004). A. pisum was the predominant aphid species colonising faba bean plants. Early sowing compared with late sowing led to a significant reduction of the total virus incidence in faba bean in all 3 years. However, significantly decreased levels of A. pisum colonisation as a result of early sowing were observed only in 1 year and one cultivar. Irrespective of sowing date, straw mulching had no significant effects on virus incidence and aphid colonisation. Compared with late sowing, early sowing significantly increased bean yield in all 3 years and kernel weight in 2 years, whereas straw mulching had no effect on yield.     

Vectoring ability of aphid clones of Rhopalosiphum padi (L.) and Sitobion avenae (Fabr.) and their capacity to retain barley yellow dwarf virus

Vectoring ability of four aphid clones, Rp-M and Rp-R26 of Rhopalosiphum padi and Sa-R1 and Sa-V of Sitobion avenae, to transmit barley yellow dwarf (PAV, MAV and RPV) luteoviruses (BYDV) was compared in controlled conditions. Significant differences between highly efficient vectors (HEV), Rp-M and Sa-Rl, and poorly efficient vectors (PEV), Rp-R26 and Sa-V, were found in transmission of their specific viruses with acquisition and inoculation access periods (AAP, IAP) of 5 days. BYD-RPV was occasionally transmitted by both clones of S. avenae. None of 150 tested apterous adults of the Rp-R26 transmitted BYD-MAV, while 10% of transmission was observed from those of the Rp-M in a parallel test. An improved ELISA and immuno-PCR were adapted to test for viruses in aphids. The results obtained by the improved ELISA indicated there was a good correlation between virus detection in single aphids of HEV clones after a 5 day AAP and virus transmission by them. In contrast, the percentages of virus-carrying aphids of PEV clones were generally higher than those of their transmission rates. BYD-MAV and BYD-RPV were also detected by the improved ELISA in single aphids of their PEV clones, with the exception of BYD-RPV in those of Sa-V. However, after a 2-day IAP, the improved ELISA in most cases failed to detect these viruses in single aphids of PEV clones. Detection by immuno-PCR demonstrated that all three viruses could be acquired and retained by the aphids of both HEV and PEV clones. But, as visualised from electrophoretic bands, after the 2-day IAP the amplified products from aphid extracts of PEV clones were reduced. The detection in a batch of nine aphids by the improved ELISA revealed that virus content in PEV clones decreased more rapidly than that in HEV clones during transmission. Thus, the difference in transmission efficiency of the aphid clones within species was not caused by an inability to acquire virus, but was determined by variation in vectoring ability between them. This was due to differences in ability to prevent the passage of virions from haemocoel to salivary duct and/or different capacities for the retention of BYDV.