Serological detection of yam viruses in farmers' fields in Ogun State,Nigeria

A field survey was conducted in eight local government areas (LGA) of Ogun state, Nigeria to assess the incidence of viral diseases of yams in the areas. Leaf samples were collected from 90 yam plants which were either symptomatic or asymptomatic. These were bulked into 45 during serological tests and the viruses indexed include yam mosaic virus (YMV); Dioscorea alata bacilliform virus (DaBV) and cucumber mosaic virus (CMV). DaBV was the most prevalent virus on the field with incidence of 48.9% (22/45) followed by YMV which occurred in 42.2% (19/45). CMV had the lowest percentage of incidence; 2.2% (1/45). Of all the LGAs visited, Abeokuta north and Abeokuta south had the highest incidence of YMV and DaBV, respectively. Mixed virus infections were also detected.     

Isolation, characterisation and identification of a potyvirus from Dioscorea alata L. (water yam) in Nigeria

A yam potyvirus was isolated from Dioscorea alata samples collected in Nigeria. The virus was not transmissible mechanically but was transmitted by Aphis craccivora to four cowpea cultivars (Ife Brown, IT84S-2114, IT82E-10 and TVu2657), and from which it could be mechanically transmitted between the cowpea cultivars. In infectivity- tests using cowpea extracts, the virus had a dilution end point of 10^-4, a thermal inactivation point of 60–65°C and longevity in vitro of 2 days at room temperature. The virus coat protein had an estimated molecular weight of 32 100 daltons. The virus was identified as an isolate of Dioscorea alata virus (DAV; syn. yam virus 1) due to its biological characteristics and its serological reaction with antiserum raised against DAV. The virus is not related to yam mosaic virus, but distantly related to blackeye cowpea mosaic virus and cowpea aphid-borne mosaic virus.     

Development of DNA microsatellite markers in tropical yam (Dioscorea sp.)

We developed new simple sequence repeat (SSR) markers in different species of yam (Dioscorea sp.). A microsatellite‐enriched bank was created from Dioscorea alata, Dioscorea abyssinica and Dioscorea praehensilis. Sixteen polymorphic loci were characterized. Several of these markers are transferable to species of other Dioscorea sections.     

Comparison of Three Plant Rhabdovirus Isolates by Two Different Serological Techniques

Three different rhabdovirus isolates, eggplant mottled dwarf virus (EMDV), tomato veinyellowing virus (TVYV) and a hitherto undefined isolate from tomato in Portugal (Tom-P) were compared by two different serological techniques, immunosorbent electron microscopy (ISEM) and electro-blot-immunoassay (EBIA). Antisera were prepared either against purified virus or against partially purified virus preparations extracted by a one-step procedure. Even the partially purified virus preparations yielded antisera that allowed unequivocal identification of two of the five structural virus proteins, G and N, in EBIA and were suitable for specific trapping of complete virus particles on electron microscope grids. With both serological techniques identical results were obtained indicating a close serological relationship between the three isolates tested. Cross-reactions between the G proteins could be deduced from heterologous trapping of complete virus particles in ISEM. The cross-reactions for the G proteins were substantiated by the EBIA-results which indicated in addition cross-reactions between the remaining three major structural virus proteins, N, M₁, and M₂, but also revealed significant differences in the molecular weights of the G and M₁ proteins between TVYV and the other two isolates. The results obtained indicate that Tom-P is serologically closely related and in respect of protein molecular weights identical to EMDV, and that TVYV is not a separate virus but rather a strain of EMDV.     

The Partial Characterization of a Badnavirus Infecting the Greater Asiatic or Water Yam (Dioscorea alata)

A bacilliform virus from Dioscorea alata, designated Dioscorea alata bacilliform virus (DaBV), from Barbados and West Africa and from other Dioscorea spp. from West African, Carribean, Asian and South American countries, has been characterized. The virus was transmitted by the mealybug, Planococcus citri and by mechanical transmission of partially purified preparations to several Dioscorea spp. DaBV was serologically related to a distinct bacilliform virus from Dioscorea bulbifera, to one isolate of sugarcane bacilliform badnavirus and two isolates of banana streak badnavirus (BSV) but was not related to another isolate of BSV or to Kalanchoe top spotting or cacao swollen shoot badnaviruses. The coat protein of DaBV was about 56 kDa and the nucleic acid was double-stranded DNA of about 7.5 kbp, part of which showed distant homology with other badnaviruses. Thus, DaBV is a distinct hitherto uncharacterized badnavirus.     

Production and some characteristics of monoclonal antibodies against beet necrotic yellow vein virus

Four rat monoclonal antibodies (MAbs) specific for beet necrotic yellow vein virus (BNYVV) were produced. In indirect ELISA, all four MAbs reacted strongly with BNYVV infected plant leaf extracts (19 isolates from eight countries) but they did not react with beet soil-borne virus (BSBV), an unnamed rod-shaped soil-borne beet virus isolate (86 - 109) from Sweden or barley stripe mosaic virus (BSMV). However, two of the MAbs, MAFF 6 and MAFF 7 did not detect BNYVV in ELISA of infected sugar beet roots whereas MAbs MAFF 8 and MAFF 9 did detect virus in root extracts. In electro-blot immunoassay (EBIA), MAFF 6 and MAFF 7 readily detected BNYVV coat protein from leaf extracts whereas MAFF 8 and MAFF 9 reacted only weakly. None of the MAbs reacted with BSBV, 86 - 109, BSMV or plum pox virus in EBIA. MAFF 6 coated BNYVV particles which were trapped from infected leaf or root sap on to electron microscope grids by polyclonal antibodies. MAFF 6 was partially purified from tissue culture supernatant fluid by cation exchange chromatography and the preparation used to coat microtitre plates and successfully trap BNYVV in ELISA of leaf sap extracts.     

Rapid assessment of resistance of tissue-cultured water yam (Dioscorea alata) and white guinea yam (Dioscorea rotundata) to anthracnose (Colletotrichum gloeosporioides Penz.)

Yam anthracnose is caused by the pathogen Colletotrichum gloeosporioides Penz. and has been identified as the most important biotic constraint to yam production worldwide. Rapid assessment of the disease is vital to its effective diagnosis and management. In this study, tissue-cultured yam plantlets of five lines of Dioscorea alata and nine of D. rotundata were rapidly assessed for their reactions to two isolates of yam anthracnose. The plantlets, obtained from meristem of the nodal cuttings, were grown for 8?weeks on Murashige and Skoog (MS) basal medium, acclimatised for 3?weeks, hardened for an additional 3?weeks, arranged in screen house in completely randomised design and sprayed with spore inocula prepared from 7?day-old culture of the two strains of Colletotrichum gloeosporioidies Penz. The relative resistance of the different Dioscorea spp. was evaluated using three disease indices – severity at seventh day after inoculation, SD7; area under disease progress curve, AUDPC; and disease severity rate, Rd. A modified rank-sum classification method put TDa 1425 and TDr 2040, with rank sum of 2.0 each, as resistant. TDr 2121, TDr 2287 and TDr 2048 were susceptible with rank sum of 27.50, 25.50 and 24.50, respectively. Dioscorea alata TDa 1425 and Dioscorea rotundata TDr 2040 were recommended in areas endemic with yam anthracnose, and also as parent lines while breeding for resistance to anthracnose.     

Identification and potential use of RAPD markers linked to Yam mosaic virus resistance in white yam (Dioscorea rotundatd)

Resistance to Yam mosaic virus (YMV) in tetraploid white yam (Dioscorea rotundatd) is inherited differentially as a dominant and recessive character. Elite D. rotundata breeding lines with durable resistance to YMV can be developed by pyramiding major dominant and recessive genes using marker‐assisted selection (MAS). The tetraploid breeding line, TDr 89/01444, is a source of dominant genetic resistance to yam mosaic disease. Bulked segregant analysis was used to search for random amplified polymorphic DNA (RAPD) markers linked to YMV resistance in F₁ progeny derived from a cross between TDr 89/01444 and the susceptible female parent, TDr 87/00571. The F₁ progeny segregated 1:1 (resistantsusceptible) when inoculated with a Nigerian isolate of YMV, confirming that resistance to YMV in TDr 89/01444 was dominantly inherited. A single locus that contributes to YMV resistance in TDr 89/01444 was identified and tentatively named Ymv‐1. Two RAPD markers closely linked in coupling phase with Ymv‐1 were identified, both of which were mapped on the same linkage group: OPW18₈₅₀ (3.0 centiMorgans [cM]) and OPX15₈₅₀ (2.0 cM). Both markers successfully identified Ymv‐1 in resistant genotypes among 12 D. rotundata varieties and in resistant F1 individuals from the cross TDr 93–1 × TDr 877 00211, indicating their potential for use in marker‐assisted selection. OPW18₈₅₀ and OPX15₈₅₀ are the first DNA markers for YMV resistance and represent a starting point in the use of molecular markers to assist breeding for resistance to YMV.     

Disease-induced changes in carotenoid content of edible yams (Dioscorea spp.) infected by Botryodiplodia theobromae and Aspergillus niger

When pieces of Dioscorea dumentorum, Dioscorea alata and Dioscorea cayenensis were inoculated with suspensions of Botryodiplodia theobromae and Aspergillus niger and incubated at room temperature, the quantities of carotenes and xanthophylls were decreased by infection. The quantities of carotenes and xanthophylls were highest in D. dumentorum followed by D. cayenensis; D. alata had the lowest amounts.     

Some hosts, properties and possible affinities of a labile virus from Hypochoeris radicata (Compositae)

Hypochoeris mosaic virus (HMV) is common in Hypochoeris radicata (‘cat's ear’) in western Canada. It infected 10 of 53 mechanically inoculated species in five of twelve families, but was not transmitted by aphids or through seed or soil. Sap from infected Nicotiana clevelandii was sometimes infective after dilution to 10^-1 and occasionally 10², after 10 min at 45 but not 50°C, and after 1 but not 2 days at 20°C. Infectivity of crude nucleic acid extracts from infected leaves was rapidly abolished by RNase but not by DNase. Host sap contained very few rod-shaped particles or particle fragments mostly 21.0–22.5 nm in diameter, and up to 420 nm long but with predominant lengths of 120–140 and 240–260 nm. Many rods in purified virus preparations were less than 240 nm long, and the majority were c. 140 nm or shorter. The particles had a helical substructure with a pitch of 2.58 nm and contained a single type of protein of estimated mol. wt 24.5 × 10³. HMV showed no serological relationship to eight morphologically similar viruses (beet necrotic yellow vein, broad bean necrosis, barley stripe mosaic, peanut clump, potato mop-top, Nicotiana velutina mosaic, wheat soil-borne mosaic and defective strains of tobacco mosaic). It is probably a hitherto undescribed tobamovirus.     

Serological studies on cassava latent virus

Particles of cassava latent virus (CLV) were purified by a method that yielded up to 3 mg per 100 g of systemically infected Nicotiana benthamiana leaf. Specific antiserum was prepared and used for enzyme-linked immunosorbent assay (ELISA), which detected purified virus at 5 ng/ml. As estimated by ELISA, CLV antigen reached a greater concentration in leaves of N. benthamiana plants kept at 20–25 °C than in those at 15 °C or 30 °C. CLV was also detected in leaf extracts of naturally infected cassava plants kept at 25 C but its concentration was only 1–7% of that in comparable extracts from N. benthamiana. Staining sections of N. benthamiana leaves with fluorescent antibody indicated that CLV particle antigen accumulates in the nuclei of many phloem cells and of some cells in other tissues. In tests on mosaic-affected cassava plants of Angolan origin, three plants were found in which CLV could not be detected by either ELISA or immunosorbent electron microscopy, or by transmission to indicator plants. This suggests that the mosaic symptoms were caused by a pathogen other than CLV, but no such agent was detected by electron microscopy of leaf extracts. Three kinds of serological test indicated that CLV is related to bean golden mosaic virus. Evidence was also obtained of a distant relationship to beet curly top virus but none was detected to four other geminiviruses.     

Properties of Johnsongrass Chlorotic Stripe Mosaic Virus

Physicochemical, biological, and cytopathological properties of Johnsongrass chlorotic stripe mosaic virus (JCSMV) found in Iran were investigated. Virus particles were polyhedral, showed a knobbed surface structure, were c. 30 nm in diameter and had a buoyant density of 1. 359 g/ml in cesium chloride. Virions contained one major protein with a molecular weight of 41 kd and a single species of ssRNA with a molecular weight of 1. 43 × 10⁶ d. Acid hydrolysis of the virus followed by thin-layer electrophoresis gave the following molar percentages of the bases: A: 23. 5, G: 27. 5, C: 26 and U: 23. Separation of nucleotides of the viral RNA using alkaline hydrolysis was not successful. Mechanical inoculation of freshly purified virus or isolated RNA failed to infect Johnsongrass or maize plants. The virus was readily detected by ELISA in seeds from infected plants and young seedlings raised from such seeds, but not in later stages of growth. Ultrathin sections of infected cells showed high concentrations of virus particles in the cytoplasm and vacuoles. Virus-like particles also occurred in the stroma of chloroplasts and mitochondria. Cisternae of endoplasmic reticulum (ER) were often extremely inflated and filled by a fine fibrillar material. Small membrane-associated vesicles were frequently found in ER elements and occurred also in the permuclear space. Based on particle structure, properties of the nucleic acid, molecular weight of the coat protein and cytopathology, the virus resembles carmoviruses. However, lack of mechanical transmissibility is not known for any virus classified with this group. No serological reaction was detected with a total of 30 antisera to carmoviruses and other isometric viruses.     

Yellow mosaic virus affects the ion leakage fromVigna aconitifolia

Yellow mosaic virus (YMV) causes a greater loss of electrolytes from infected leaf tissues ofVigna aconitifolia (mothbean). Four highly susceptible entries of mothbean were examined for the pattern of electrolyte loss after virus has invaded the tissues. It was observed that YMV triggered a heavy loss of ions at initial stages of disease development, but the loss receded at advanced stages of infection. Maximal damage to electrolytes occurred at the second stage, showing about 50% infection. The findings are interesting as the present observations on viral disease differ from other plant diseases.     

Characterisation and serology of virus-like particles associated with faba bean necrotic yellows

A disease showing chlorosis, leaf rolling and stunting in Vicia faba and other legumes was observed in West Asia and North Africa during 1987–1988. The putative causal agent could not be transmitted mechanically, but could be transmitted by aphids, most efficiently by Acyrthosiphon pisum, in the persistent manner. Further studies revealed isometric virus-like particles (VLPs) closely associated with the disease, although their infectivity could not be demonstrated by membrane feeding. These particles, measuring c. 18 nm in diameter and containing a capsid protein of about 22 kDa and ssDNA of about 1 kb, are hereafter designated faba bean necrotic yellows virus (FBNYV). A high proportion of circular nucleic acid molecules of about 0.9 kb were visualised by electron microscopy. Hybridisation analysis of cloned viral DNA suggests that the circular genome is larger than 1 kb and consists of several components of similar size. An antiserum produced against FBNYV was used in ELISA, immunoelectron microscopy (IEM) and Western blot experiments for virus detection in aphids and field samples and for serological comparison with other viruses. Weak heterologous reactions between FBNYV and subterranean clover stunt virus (SCSV) were detected in IEM, but could not be confirmed in ELISA or Western blots. No serological relationship to banana bunchy top virus (BBTV) was detected. Using a direct tissue blot immunoassay (TBIA), FBNYV was detected in vascular tissue of infected faba bean leaves and stems.     

Purification and some properties of oat golden stripe virus

Oat golden stripe virus (OGSV) was maintained in oats by mechanical inoculation and purified by extraction of leaves in borate buffer, two cycles of centrifugation through sucrose cushions and isopycnic centrifugation in CsCl. An antiserum with a titre of 1/1024 in precipitin tests was prepared. Particle length distribution was bimodal with median values, respectively, of 150 and 300 nm from dip preparations. Measurements from immunosorbent electron microscopy (ISEM) and purified preparations showed that the particles had partially degraded during these procedures. The virus sedimented as two components of 168 S and 218 S and had a buoyant density of 1321 g cm^-3. Four isolates of OGSV reacted with the antiserum. Antiserum to members and possible members of the furovirus group were tested in ISEM decoration tests and in ELISA. OGSV was related to soil-borne wheat mosaic virus but not to beet necrotic yellow vein virus, hypochoeris mosaic virus or potato mop-top virus.     

Broad-bean stain and true broad-bean mosaic viruses

Autumn-sown crops of broad beans (Vicia faba L.) in England often contain plants with some leaves characteristically distorted and with a chlorotic mosaic. From some of these plants true broad-bean mosaic virus was isolated in 1959 and 1960 but not in 1965 and 1966. From other plants a similar but distinct virus, which caused staining of the seeds and we call broad-bean stain virus, was isolated in 1960, 1965 and 1966. The two viruses were readily distinguished in serological tests, and in some test plants. Both were seed-borne, and spread in crops, but were not transmitted by several animal species tested as vectors. Both viruses have isometric particles about 25 mμ in diameter. Some of these particles contain about 35% ribonucleic acid, some about 26% and some of those of broad-bean stain virus contain none; these three types of particles had sedimentation coefficients of about 120–130 S, 100 S and 60 S respectively. The ribonucleic acid of each virus had molar base content of G 23%, A 26%, C 18% and U 32%. These two viruses are members of the cowpea mosaic group of plant viruses; broad-bean strain virus was serologically related to cowpea mosaic, F I, red-clover mottle, and squash mosaic viruses. The particles of all these viruses and of true broad-bean mosaic virus were similar in appearance, sedimentation behaviour, and nucleic acid content and composition. The nucleic acid of red-clover mottle virus had a molar base content of G 20%, A 29%, C 20%, U 30%.     

Inheritance of resistance to Yam mosaic virus, genus Potyvirus, in white yam (Dioscorea rotundata)

Yam mosaic virus (YMV) causes the most-widespread and economically important viral disease affecting white yam (Dioscorea rotundata) in West Africa. The genetic basis of resistance in white yam to a Nigerian isolate of YMV was investigated in three tetraploid D. rotundata genotypes: TDr 93–1, TDr 93–2 and TDr 89/01444. F₁ progeny were produced using TDr 87/00571 and TDr 87/00211 as the susceptible parents. Segregation ratios indicated that a single dominant gene in a simplex condition governs the resistance in TDr 89/01444, while the resistance in TDr 93–2 is associated with the presence of a major recessive gene in duplex configuration. Segregation of progeny of the cross TDr 93–1×TDr 87/00211 fitted a genetic ratio of 2.48:1 resistant:susceptible, which can be expected when two simplex heterozygotes are crossed, indicating the possible modifying effect of the susceptible parent. A triple antibody immunosorbent assay (TAS-ELISA) was used for virus detection in inoculated plants. Slight mosaic symptoms appeared on most resistant individuals, while asymptomatic resistant genotypes with high ELISA (A₄₀₅) values were observed in all crosses. Such a heterogeneous response suggests the influence of additional modifier genes that segregate in the progeny. The finding that resistance can be inherited as a dominant or recessive character has important implications for YMV resistance breeding. Received: 15 August 2000 / Accepted: 12 April 2001     

Studies on Particle Components of Cacao Swollen Shoot Virus

Virions of the cacao swollen shoot virus (CSSV) strain 1A were purified and used for studies of its particle components. CSSV virions had a buoyant density of 1.365 g/cm³ in buffered CsCl. Following SDS polyacrylamide gel electrophoresis (PAGE), CSSV-specific proteins were identified in electroblot immunoassays (EBIA) with cross-absorbed polyclonal antibodies and especially well with monoclonal antibodies (MABs) to CSSV-1A. Based upon EBIA experiments with a selected MAB, CSSV virions appeared to have one capsid protein species with a relative molecular mass (M), of about 43 kd that was shown to be not glycosylated. However, this protein is sensitive to proteolytic degradation as degradation products ranging from 37 to 33 kd were found in addition to the 43 kd protein. Studies on the viral genome of CSSV revealed that CSSV virions contain a DNA of about 7. 5 kbp. Nucleic acid probes obtained by cloning parts of the viral genome yielded specific hybridization reactions with extracts and preparations from plants infected with strain 1A of CSSV but not with those from non-inoculated plants. One clone of 738 bp was sequenced and shown to contain a motif similar to the putative RNA binding domain of pararetroviruses. Based upon particle morphology and properties of the virion components, CSSV can be grouped with other nonenveloped bacilliform viruses for which the name badnaviruses has been proposed.     

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.