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.     

Molecular characterization of distinct YMV (Yellow mosaic virus) isolates affecting pulses in India with the aid of coat protein gene as a marker for identification

The present study was carried out to find out the variations present in different isolates of yellow mosaic virus (YMV) causing yellow mosaic disease of pulses in southern parts of India. The coat protein gene of YMV was amplified using gene specific and deng universal primers with DNA isolated from YMV infected samples. Further, cloning and DNA sequencing of CP gene was carried out. CP gene decrypt sequences revealed that YMV infected samples of Black gram, Cowpea and Green gram were similar to the MYMV-Tamil Nadu isolates. Whereas the YMV infected sample of Horse gram was found to be similar with HYMV. Hence, in the present study, two distinct YMV infecting pulses in Tamil Nadu (MYMV and HYMV species) were identified and it was observed that there exists considerable genetic variation among these species. In addition, Cowpea crop which was earlier supposed not to be susceptible for YMV infection also showed the presence of this virus similar to the MYMV. Overall, the findings of the present study indicate that the CP region is efficient enough to provide a simple, rapid, and reliable method for early detection of YMV infections in pulses, which would help to develop proper management strategies to control these viruses.     

Infection of yam tubers by Aspergillus niger in relation to isolate, yam species, and temperatures

Infection of yam (Dioscorea spp.) tubers by a range of geographical isolates of Aspergillus niger from yam and other hosts was tested on tubers imported into the UK, using a standard inoculation procedure. A yam isolate of A. niger from Nigeria was generally more aggressive than one from Barbados or from isolates from different hosts. There were inconsistent differences in infection between different portions of the tubers. Infection and weight loss of yam tubers were evaluated at temperatures ranging from 18 to 36°C, using the Nigerian and Barbados isolates. Mean percentage infection increased across the temperature range for the Nigerian isolate; for the Barbados isolate, infection increased with temperatures only from 18 to 30°C. Comparison of different yam species in relation to A. niger infection is difficult due to lack of information on cultivar and provenance.     

Book Review

To study the variability and to identify the species of Begomovirus associated with yellow mosaic disease of blackgram in Andhra Pradesh, India, infected blackgram samples were collected from six districts belonging to three regions of Andhra Pradesh. The total DNA was isolated by modified CTAB method and amplified with coat protein gene-specific primers (RHA-F and AC abut) resulting in 900?bp gene product. The PCR products were cloned, sequenced and deposited in GenBank. The sequence analysis of six clones showed that the size of amplified CP gene of YMV was 920?bp. Based on nucleotide sequence identity of six isolates representing three regions of Andhra Pradesh, the isolates from Rayalaseema and Telangana region are the same variant of YMV (>99.5% identity) and isolate from coastal Andhra is another variant of YMV (>95.4%) when compared with other region isolates. Comparison of CP gene sequence of YMV-TPT isolate with 27 other isolates in database revealed more than 93.2 and 86.2% identity with MYMIV isolates and less than 80 and 64% identity with MYMY isolates that originate from Indian sub-continent and South-East Asia at nucleotide and amino acid level, respectively. Phylogenetic tree based on CP gene sequences of six isolates with other isolates from GenBank formed unique cluster with MYMIV. Hence the YMV infecting blackgram in Andhra Pradesh is caused by MYMIV rather than MYMY as reported in Tamil Nadu which is adjoining state in southern India.     

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     

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.     

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.     

家养动物体表蜱中发热伴血小板减少综合征布尼亚病毒分离及鉴定

为了解发热伴血小板减少综合征布尼亚病毒(SFTSV)的传播机制,采集了山东疫区家养牛、羊和狗等动物体表蜱,分类鉴定后,通过Real-time PCR筛查、病毒分离培养和基因组序列分析等方法分离鉴定蜱中的病毒。所采集的蜱,以长角血蜱为主,占91.4%。其中3头SFTSV核酸检测阳性,阳性率为2.14%,并在其中一份羊体表蜱标本中分离到SFTSV病毒,命名为SDLZTick12。序列分析显示与我国在不同省份患者标本中分离的病毒全基因序列具有高度同源性,且病毒的抗原性和生长特性与人源病毒相同。本研究首次在山东疫区蜱中分离到新型布尼亚病毒,并与人源病毒进行了系统比较研究,提示蜱可能为该新病原体的传播媒介,对疾病的防控具有重要的指导意义。     

Diagnostic and molecular evaluation of three iridovirus-associated salamander mortality events

In 1998 viruses were isolated from tiger salamander larvae (Ambystoma tigrinum diaboli and A. tigrinum melanostictum) involved in North Dakota and Utah (USA) mortality events and spotted salamander (A. maculatum) larvae in a third event in Maine (USA). Although sympatric caudates and anurans were present at all three sites only ambystomid larvae appeared to be affected. Mortality at the North Dakota site was in the thousands while at the Utah and Maine sites mortality was in the hundreds. Sick larvae were lethargic and slow moving. They swam in circles with obvious buoyancy problems and were unable to remain upright. On the ventral surface, near the gills and hind limbs, red spots or swollen areas were noted. Necropsy findings included: hemorrhages and ulceration of the skin, subcutaneous and intramuscular edema, swollen and pale livers with multifocal hemorrhage, and distended fluid-filled intestines with areas of hemorrhage. Light microscopy revealed intracytoplasmic inclusions, suggestive of a viral infection, in a variety of organs. Electron microscopy of ultra thin sections of the same tissues revealed iridovirus-like particles within the inclusions. These viruses were isolated from a variety of organs, indicating a systemic infection. Representative viral isolates from the three mortality events were characterized using molecular assays. Characterization confirmed that the viral isolates were iridoviruses and that the two tiger salamander isolates were similar and could be distinguished from the spotted salamander isolate. The spotted salamander isolate was similar to frog virus 3, the type species of the genus Ranavirus, while the tiger salamander isolates were not. These data indicate that different species of salamanders can become infected and die in association with different iridoviruses. Challenge assays are required to determine the fish and amphibian host range of these isolates and to assess the susceptibility of tiger and spotted salamanders to heterologous virus isolates.     

RNA/cDNA Hybridization and Infectivity Tests Suggest that Barley Yellow Mosaic Virus Isolate M has a Bipartite Genome

Northern blot analysis with random-primed cDNAs revealed that RNAs 1 and 2 of barley yellow mosaic virus (Ba YMV) isolate M have no extensive base sequence homologies. Both RNAs are needed for infection. RNA 2 is therefore neither a subgenomic nor a, satellite RNA, but rather an essential part of the viral genome. Ba YMV isolate M has thus a bipartite genome.     

Vegetative Compatibility Among Isolates of Colletotrichum gloeosporioides from Yam (Dioscorea spp.) in Nigeria

Isolates of Colletotrichum gloeosporioides obtained from yam‐based cropping systems in Nigeria, previously characterized on the basis of morphology, virulence and rDNA internal transcribed spacer (ITS) sequence variation were further compared for vegetative compatibility (VC). Chlorate‐resistant nitrate non‐utilizing (nit) mutants were generated from the isolates and used in complementation (heterokaryon) tests. Tests of VC between complementary mutants from different isolates indicated the presence of several genotypes within a single field, suggesting limited clonal spread. In some cases, isolates obtained from the same lesion were observed to belong to different vegetative compatibility groups (VCGs). No compatibility was observed between isolates of the highly virulent slow‐growing grey (SGG), the moderately virulent fast‐growing salmon (FGS) and the avirulent/weakly virulent fast‐growing grey (FGG) strains. Forty‐one C. gloeosporioides isolates belonged to 28 VCGs, giving a genotype diversity estimate of 0.68. This diversity confirmed the high variability of the pathogen population as revealed by previous characterization studies, however, a correlation between VCGs and isolate groupings based on morphology and virulence was not found. The finding that an isolate from weed was compatible with yam isolates indicated that transfer of important traits, such as virulence, may take place between isolates from yam and non‐yam hosts. The VCG diversity revealed by this study suggests that in addition to asexual reproduction, sexual reproduction may play an important role in the epidemiology of anthracnose on yam.     

Transmissibility of Broad bean wilt virus 1 by aphids: influence of virus accumulation in plants,virus genotype and aphid species

Broad bean wilt virus 1 (BBWV‐1) is transmitted by several aphid species in a non‐persistent manner. Transmission efficiency by vectors is a key factor for understanding virus epidemiology and applying disease control measures based on limiting virus spread. We evaluated the transmission rates of two genetically divergent BBWV‐1 isolates (PV‐132 from USA and Ben from Spain) infecting broad bean (Vicia faba L.) by isofemale lines of nine aphid species from eight different genera collected in Spain. Our analyses showed that: (a) the virus concentration in the source plant was a key factor in BBWV‐1 transmissibility; (b) The Spanish isolate Ben was transmitted more efficiently than the American isolate PV‐132 by most aphid species, but this was only due to the higher accumulation of Ben in plants, as both isolates had similar transmissibility after adjusting virus concentration and (c) The transmission rate varied greatly between the different aphid species.     

Determination of the races of potato virus X and its host rang in Karaj, Damavand and Ardabil, Iran

Potato virus x (PVX) is found commonly in potato-growing areas, worldwide. It is an economically important virus which causes losses in tuber yield of approximate 5 to 15 percent. In a 2 year survey, potato leaf and tuber samples were collected from various fields in Damavand and Karaj. The initial isolations from potato were made by mechanical inoculation first to Gomphrena globosa L. and later to Dartura stramonium L. It was not transmitted by 2 species of Cuscuta but transmitted mechamically. The isolates were inoculated to Nicotiana glutinosa L. in which they were maintained throughout the work. Sap from infected N. glutinosa was ineffective after dilution to 10-6, after 10 minutes at 70 degrees C and after 10 weeks at room temperature. The virus had filamentous and slightly flexuous particles with a normal length of about 490-500 nm and 12 nm width. According to the symptoms, TIP results and serological comparisons, the compared isolates showed no difference and they belong to XN group. In order to estimate disease incidence, 773 tubers from Damavand area were tested and compared with that in Ardabil area. Disease incidence in Damavand ranged from 1.1-20.9 percent and was lower than disease incidence in Ardabil. In 8 genera of collected weeds from fields of potato and tomato samples by using test plants and serological methods, they didn't show existence of the potato virus x.     

Specific Insect-Virus Interactions Are Responsible for Variation in Competency of Different Thrips tabaci Isolines to Transmit Different Tomato Spotted Wilt Virus Isolates

Local adaptation between sympatric host and parasite populations driven by vector genetics appears to be a factor that influences dynamics of disease epidemics and evolution of insect-vectored viruses. Although T. tabaci is the primary vector of Tomato spotted wilt virus (TSWV) in some areas of the world, it is not an important vector of this economically important plant virus in many areas where it occurs. Previous studies suggest that genetic variation of thrips populations, virus isolates, or both are important factors underlying the localized importance of this species as a vector of TSWV. This study was undertaken to quantify variation in transmissibility of TSWV isolates by T. tabaci, in the ability of T. tabaci to transmit isolates of TSWV, and to examine the possibility that genetic interactions and local adaptation contribute to the localized nature of this species as a vector of TSWV. Isofemale lines of Thrips tabaci from multiple locations were tested for their ability to transmit multiple TSWV isolates collected at the same and different locations as the thrips. Results revealed that the probability of an isofemale line transmitting TSWV varied among virus isolates, and the probability of an isolate being transmitted varied among isofemale lines. These results indicate that the interaction of T. tabaci and TSWV isolate genetic determinants underlie successful transmission of TSWV by T. tabaci. Further analysis revealed sympatric vector-virus pairing resulted in higher transmission than allopatric pairing, which suggests that local adaptation is occurring between T. tabaci and TSWV isolates.     

Genetic diversity of enzootic isolates of vesicular stomatitis virus New Jersey.

The RNA genomes of 43 vesicular stomatitis virus (VSV) isolates of the New Jersey (NJ) serotype were T1-ribonuclease fingerprinted to compare the extent of genetic diversity of virus from regions of epizootic and enzootic disease activity. Forty of these viruses were obtained from Central America during 1982 to 1985. The other three were older isolates, including a 1970 isolate from Culex nigripalpus mosquitos in Guatemala, a 1960 bovine isolate from Panama, and a 1976 isolate from mosquitos (Mansonia indubitans) in Ecuador. The data indicate that extensive genetic diversity exists among virus isolates from this predominantly enzootic disease zone. Six distinct T1 fingerprint groups were identified for the Central American VSV NJ isolates from 1982 to 1985. The 1960 VSV NJ isolate from Panama and the 1976 isolate from Ecuador formed two additional distinct fingerprint groups. This finding is in sharp contrast to the relatively close genetic relationship existing among VSV NJ isolates obtained from predominantly epizootic disease areas of the United States and Mexico during the same period (S. T. Nichol, J. Virol. 61:1029-1036, 1987). In this previous study, RNA genome T1 fingerprint differences were observed among isolates from different epizootics; however, the isolates were all clearly members of one large T1 fingerprint group. The eight T1 fingerprint groups described here for Central American and Ecuadorian viruses are distinct from those characterized earlier for virus isolates from the United States and Mexico and for the common laboratory virus strains Ogden and Hazelhurst. Despite being isolated 14 years earlier, the 1970 insect isolate from Guatemala is clearly a member of one of the 1982 to 1985 Central American virus fingerprint groups. This indicates that although virus genetic diversity in the region is extensive, under certain natural conditions particular virus genotypes can be relatively stably maintained for an extended period. The implications of these findings for the evolution of VSV NJ and epizootiology of the disease are discussed.     

Species delimitation in the African Armillaria complex by analysis of the ribosomal DNA spacers

Thirteen Armillaria isolates, collected from various geographical areas in tropical Africa and previously characterized by cultural morphology, pairing tests and isozyme analysis, were evaluated using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). DNA regions corresponding to the intergenic spacer (IGS) and internal transcribed spacer (ITS) were amplified and analyzed by restriction enzyme digestion. The IGS amplification products were about 875 bp long and uniform in length among the isolates. The amplified-ITS region showed two different lengths corresponding to two groups. The first group included the isolates believed to belong to A. mellea ssp. africana and two Kenyan isolates (K11 and K12) belonging to a yet unnamed biological species. The second group included isolates identified as A. heimii and a Tanzanian isolate (T7). Each length variant of the ITS showed distinct RFLP banding patterns. Digestion with EcoRI confirmed the two polymorphic groups while the endonucleases AluI and NdeII discriminated the A. mellea isolates from the Kenyan isolates K11 and K12. In addition, the latter enzyme showed a slight dissimilarity between the A. heimii isolates from Western and Eastern Africa (C1 and Z1). Digestion with HinfI cleaved the isolates of A. heimii into two sub-groups corresponding to the heterothallic and homothallic forms. This endonuclease also indicated that the isolate T7, originating from Tanzania, was clearly similar to the heterothallic species A. heimii. Data presented support the maintenance of three distinct species of Armillaria in tropical Africa with A. heimii as a variable species, the isolates of which were separated in accordance with their sexual system. The results indicate that PCR-RFLP can be used as a simple and speedy taxonomical tool for the ecological studies of Armillaria species.     

Strain variation, based on the hemagglutinin gene, in Norwegian ISA virus isolates collected from 1987 to 2001: indications of recombination.

Infectious salmon anemia (ISA) is caused by a virus that probably belongs to the Orthomyxoviridae and was first recorded in Norway in 1984. The disease has since spread along the Norwegian coast and has later been found in Canada, Scotland, the Faroe Islands, Chile, and the USA. This study presents sequence variation of the hemagglutinin gene from 37 ISA virus isolates, viz. one isolate from Scotland, one from Canada and 35 from Norway. The hemagglutinin gene contains a highly polymorphic region (HPR), which together with the rest of the gene sequence provides a good tool for studies of epizootics. The gene shows temporal and geographical sequence variation, where certain areas are dominated by distinct groups of isolates. Evidence of transmission of ISA virus isolates within and between regions is given. It is suggested that the hemagglutinin gene from different isolates may recombine. Possible recombination sites are found within the HPR and in the 5'-end flanking region close to the HPR.