The virobacterial agglutination test as a rapid means of detecting cocoa swollen shoot virus

The virobacterial agglutination (VBA) test was developed as a means of detection of cocoa swollen shoot virus (CSSV). Identification of CSSV-infected Theobroma cacao in the field has only been possible by visual examination of symptoms, by virus transmission using mealybugs and by grafting to induce symptom expression in Amelonado cocoa seedlings. Detection of latent infection has not been possible even using enzyme-linked immunosorbent assays (ELISA). The VBA test successfully detected CSSV in infected sap diluted to 1/2560. Antisera to a range of mild and severe CSSV isolates were tested, and the results suggest a close relationship between seven isolates (1A, Bosomtwi, Bosomuoso, Nkrankwanta, Nsaba, Seidi-Nkawie and SS365B) while the mild isolate N1 appears to be less closely related. The VBA test was compared with both direct and indirect ELISA in the field. Only VBA detected all the cocoa trees which were known to be infected and additionally identified infection in many symp-tomless trees.     

Typing Primus necrotic ringspot virus isolates by serology and restriction endonuclease analysis of PCR products

Primus necrotic ringspot virus (PNRSV) isolates were characterised by bioassays, serotyping and restriction fragment length polymorphism (RFLP) analysis of PCR products. Based on symptoms in host trees and bioassays it was concluded that only one of the 16 tested isolates is severe. The serotyping results demonstrated that by using four different MAbs in TAS-ELISA the tested isolates could be divided into four subgroups, however, the severe isolate could not be singled out. RFLP analysis of PCR products supported the serotyping data but did not differentiate between isolates of the two main serological subgroups. A restriction map, derived from sequence analysis of the PCR products obtained from selected isolates, allowed exact location of the restriction sites within the PCR products of each isolate. A mild isolate with a unique genome structure was identified by both serological and RFLP assays. As far as we are aware, this is the first report on sub-grouping of PNRSV isolates by bioassays, serotyping with MAbs and RFLP analysis.     

Nucleotide sequence heterogeneity of the RNA from a natural population of foot-and-mouth-disease virus

The genomic RNA from isolates of foot-and-mouth-disease virus (FMDV) of serological types O or C obtained during epizootic outbreaks have been analysed by two-dimensional gel electrophoresis of the T1 RNase-generated oligonucleotides (T1 fingerprinting). Among virus isolates that are closely related serologically, 4-12 oligonucleotide changes were detected constitute the genome, the variations affect 0.7%-2.2% positions in FMDV RNA. Higher nucleotide-sequence divergence exists between the genomic RNAs from serologically unrelated viruses, while a 100-fold lower RNA sequence heterogeneity has been detected by analysis of individual clones derived from one viral isolate. Oligonucleotide mapping indicates that the variant oligonucleotides are scattered throughout the FMDV genome. We suggest that extensive genetic variability at many RNA sites is the basis for the antigenic diversity of FMDV.     

Numerical analysis of isozyme variation in Glycine tomentella

Isozyme variation among 114 accessions of the Glycine tomentella Hayata was analysed by single linkage cluster analysis and the unweighted pair group centroid method (UPGMC). The diploid accessions fell into six distinct, well defined groups, which conformed with differences in chromosome number (2n − 38 or 2n − 40) or in geographic origin. The majority of the tetraploid accessions belonged to a large, geographically widespread group, predominantly aneuploid (2n − 78) group. The remaining four tetraploid groups were distinct on the basis of morphology or geographic distribution. The validity of tetraploid isozyme groupings for reflecting subspecific differentiation was supported by the published reports of hybrid fertility. All of the nineteen crosses between isozyme groups have yielded sterile hybrids, whereas five crosses within groups have yielded fertile hybrids. The relationship between diploids and tetraploids was examined either as the similarity between individual accessions, or that between isozyme groups. These analyses indicated that each tetraploid group is closely related to only one or two of the diploid groups or subgroups.     

我国水稻条纹病毒RNA3片段序列分析—纤细病毒属重配的又一证据

测定了来源于我国水稻条纹叶枯病常年流行区的辽宁盘锦 (PJ)、云南昆明 (KM)、云南宜良 (YL)及病害暴发区的江苏洪泽 (HZ)的水稻条纹病毒 (RSV) 4个分离物RNA3全长序列 ,其长度分别为 2 480bp、2 5 0 9bp、2 489bp和 2 497bp。与已报道的RSV云南Y、日本T和M分离物RNA3序列进行比较的结果表明 ,这 7个分离物可分为两组 ,其中 ,KM、YL分离物为一组 ,PJ、HZ、Y、T和M分离物为另一组。组与组之间 ,RNA3的毒义链 (vRNA3)及RNA3的毒义互补链 (vcRNA3)上的ORF的核苷酸同源性分别为 97%～ 98%和 93%～ 94% ,但在氨基酸水平上则没有明显差异。结合上述RSV分离物RNA4的核苷酸全序列比较结果 ,推测认为RSV自然种群中存在两个与地理因素相关的不同类型的亚群 ,Y分离物不同片段具有不同来源可能是由重配引起的。     

我国水稻条纹病毒一个强致病性分离物的RNA4序列测定与分析

对我国水稻条纹病毒(Rice Stripe Virus,RSV)一个强致病性分离物(辽宁PJ分离物)的RNA4区段进行扩增、克隆和测序,其核苷酸序列全长2157bp。与已报道的日本T和M分离物及我国云南CX分离物的RNA4序列进行比较分析,结果表明,这4个分离物可分为两组,其中,PJ、T和M分离物为一组,组内分离物之间,RNA4的毒义链(vRNA4)及RNA4的毒义互补链(vcRNA4)上的ORF的核苷酸一致性分别为970%和970%～975%,5′末端和3′末端非编码区的序列则完全一致。但PJ分离物与T分离物的亲缘关系更为密切,其基因间隔区(IR)与T分离物的等长,核苷酸一致性为930%,比M分离物的IR多了一段长19bp的插入序列,核苷酸一致性仅为850%。另一组为我国CX分离物,组与组之间,vRNA4及vcRNA4上的ORF的核苷酸一致性分别为940%和925%～935%,但在氨基酸水平上则没有明显的差异。CX分离物的IR与PJ分离物相比有一段长84bp的插入序列,组间,IR的核苷酸一致性仅为720%～750%,5′末端非编码区的序列完全一致,但3′末端非编码区有两个碱基的差异。这些结果表明,RSV在自然界的分子变异与其地理分布具有密切的关系。此外,非编码区序列的高度保守性暗示着它们在病毒基因转录和复制的调控方面具有重要的功能。本文还讨论了RSV的分子流行学。     

Biological and Molecular Characterization of Polish Isolates of Tomato torrado virus*

Three isolates of Tomato torrado virus (ToTV) were found in Poland. The isolates were characterized on the basis of their symptomatology on plant species, serological reactions, electron microscopy, and nucleotide and amino acid sequence analyses of coat protein subunit genes. In comparative tests, the Polish ToTV isolates were shown to be closely related to each other and also to the isolate from Spain.     

Detection and Comparison of some Ghanaian Isolates of Cacao Swollen Shoot Virus (CSSV) by Enzyme-Linked Immunosorbent Assay (ELISA) and Immunoelectron Microscopy (IEM) Using an Antiserum to CSSV Strain 1A

Various isolates of Cacao Swollen Shoot Virus (CSSV) were detected without difficulty in leaves of Theobroma cacao L. by ELISA and immunosorbent electron microscopy (ISEM) using an antiserum to severe strain 1A. Many isolates were detected with relatively high values at dilutions of 1:30, whereas some other isolates were hardly or not at all detected at this dilution. Strain 1A was detected at dilutions of up to 1: 2560 of crude leaf extracts. All isolates yielding high reactions seem to be serologically closely related to strain 1A. Strains of the mottle-leaf type (AD 191, AD 196, AD 7, AD 36, AD 135, Kpeve) and others were poorly detected; their relationship to strain 1A is discussed. A close correlation was found between results obtained by ELISA and ISEM.     

Diverse rhizobia associated with woody legumes Wisteria sinensis, Cercis racemosa and Amorpha fruticosa grown in the temperate zone of China

Fifty-nine bacterial isolates from root nodules of the woody legumes Wisteria sinensis, Cercis racemosa and Amorpha fruticosa grown in the central and eastern regions of China were characterized with phenotypic analysis, PCR-based 16S and 23S rRNA gene RFLP, Box PCR and 16S rRNA gene sequencing. Seven main phena were defined in numerical taxonomy, which corresponded to distinct groups within the genera Agrobacterium, Bradyrhizobium, Mesorhizobium and Rhizobium in 16S and 23S rRNA gene PCR-RFLP. The phylogenetic relationships of the 16S rRNA genes supported the grouping results of PCR-RFLP. Most of the isolates from Amorpha fruticosa were classified into two groups closely related to Mesorhizobium amorphae. Seventeen of the 21 isolates from Wisteria sinensis were identified as two groups related to Rhizobium and Agrobacterium. Six out of 10 isolates from Cercis racemosa were identified as a group related to Bradyrhizobium. Our results indicated that each of the investigated legumes nodulated mainly with one or two rhizobial groups, although isolates from different plants intermingled in some small bacterial groups. In addition, correlation between geographic origin and grouping results was found in the isolates from Amorpha fruticosa. These results revealed that the symbiotic bacteria might have been selected by both the legume hosts and the geographic factors.     

Genetic relationships among a collection of Musa germplasm by fluorescent-labeled SRAP

Edible banana and plantains of the Musa genus are important staple food crops cultivated in humid tropical and subtropical climatic zones. These crops are important for subsistence farming in rural communities and also to generate significant employment and income. In an effort to increase the genetic variability of available cultivars, indexed accessions have been introduced into a regional collection in southeastern Mexico, through the Banana Bioversity International Program. The aim of this study was to use the fluorescently labeled sequence-related amplified polymorphism (SRAP) molecular marker system to characterize the genetic variability within 71 accessions of the existing collection and resolved uncertainties for the better management of the collection, as a preliminary step to establishing a breeding program. These accessions, which included wild species and cultivars of different subgroups, were consistently identified and separated by SRAP markers. A total of 330 polymorphic bands were detected using 12 primer combinations. The average number of polymorphic bands per primer pair was 27.5. The genetic similarity between accessions ranged between 0.44 and 0.97, as estimated using Jaccard's coefficient. Moreover, SRAP marker system probed to be useful to identify closely related accessions in the genus Musa and facilitated the recognition of duplicates to be eliminated and clarified uncertainties or mislabeled banana accessions introduced to the collection.     

Serological and immunoelectrophoretic relationships among viruses in the tombusvirus group

Serological cross-reactions among eighteen virus isolates of the tombusvirus group were compared in precipitin tube and immunodiffusion serological tests. The isolates were also compared by immunoelectrophoresis in agar gel. Although precipitin tube tests showed considerable and reproducible differences between the various isolates, the results were too greatly affected by other factors to be of value in assessing strain relationships. When pairs of isolates were compared for spur formation in gel-diffusion tests, the results suggested that most isolates could be placed in one of two groups; one group comprised isolates from pelargonium (leaf curl), the other consisted of petunia asteroid mosaic virus and artichoke mottled crinkle virus isolates from Italy and tomato bushy stunt isolates from soil around this Institute and from cherry. Four isolates did not fall into either of these groups; they nearly always formed spurs when compared among themselves, or with viruses in either of the two groups. Pairs of isolates that could be distinguished from each other in spur-formation tests using antiserum homologous to one of them could not always be differentiated when antiserum heterologous to both isolates was used. Immunoelectrophoresis gave consistent results with several methods of virus preparation; it indicated grouping and separation of the isolates in general agreement with the results of gel-diffusion tests: all pelargonium leaf curl isolates were grouped together with slow migration towards the cathode. The petunia asteroid mosaic isolate and the isolates from cherry and from soil from this Institute (GCRI) moved slowly towards the anode. Tomato bushy stunt virus type strain migrated rapidly to the cathode, differing greatly from all other isolates. The method offers a relatively simple means of typing isolates of the tombusvirus group.     

The Use of Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Cacao Swollen Shoot Virus (CSSV) in Theobroma cacao

Cacao swollen shoot virus (CSSV) was readily detected in different parts of Theobroma cacao using the ELISA technique. Different plant tissues contained varying amounts of CSSV; highest concentrations were found in leaf lamina. Methods to preserve the serological activity of CSSV were evaluated, and best results obtained with samples stored in a buffer or freeze-dried.     

Predicting Salmonella enterica serotypes by repetitive sequence-based PCR

Repetitive extragenic palindromic sequence-based PCR (rep-PCR) utilizing a semi-automated system, was evaluated as a method to determine Salmonella serotypes. A group of 216 Salmonella isolates belonging to 13 frequently isolated serotypes and one rarer serotype from poultry were used to create a DNA fingerprint library with the DiversiLab System software. Subsequently, a blinded set of 44 poultry isolates were fingerprinted and queried against the library in an attempt to putatively assign a serotype designation to each Salmonella isolate. The query isolates were previously typed employing standard serological techniques. Utilizing pair-wise similarity percentages as calculated by the Pearson correlation coefficient, the predicted serotype of 28 isolates matched the serological typing result. For eight isolates, rep-PCR results were interpreted as one of two very closely-related serotypes, Hadar and the rarer Istanbul. Traditional serological assays have difficulty distinguishing between these groups, and sequencing interspacer regions of the rrfH gene was unable to differentiate among isolates of these two serovars. Six of the remaining isolates resulted in no match to the database (similarity values <95%) and these indeed proved to be serotypes not included in the original library. The two remaining samples proved discrepant at the 95% similarity threshold, however examination of electropherograms clearly indicated fingerprint variability between query and library samples, suggesting an expanded rep-PCR library will be necessary for increased utility. Since serological assays can take several days to weeks to provide information, the DiversiLab System holds promise for more rapid serotype classification for members of this group.     

Diversity of Mycobacterium species from marine sponges and their sensitivity to antagonism by sponge-derived rifamycin-synthesizing actinobacterium in the genus Salinispora

Eleven isolates of Mycobacterium species as well as an antimycobacterial Salinispora arenicola strain were cultured from the sponge Amphimedon queenslandica. The 16S rRNA, rpoB, and hsp65 genes from these Mycobacterium isolates were sequenced, and phylogenetic analysis of a concatenated alignment showed the formation of a large clade with Mycobacterium poriferae isolated previously from another sponge species. The separation of these Mycobacterium isolates into three species-level groups was evident from sequence similarity and phylogenetic analyses. In addition, an isolate that is phylogenetically related to Mycobacterium tuberculosis was recovered from the sponge Fascaplysinopsis sp. Several different mycobacteria thus appear to co-occur in the same sponge. An actinobacterium closely related to S. arenicola, a known producer of the antimycobacterial rifamycins, was coisolated from the same A. queenslandica specimen from which mycobacteria had been isolated. This Salinispora isolate was confirmed to synthesize rifamycin and displayed inhibitory effects against representatives from two of three Mycobacterium phylotype groups. Evidence for antagonism of sponge-derived Salinispora against sponge-derived Mycobacterium strains from the same sponge specimen and the production of antimycobacterial antibiotics by this Salinispora strain suggest that the synthesis of such antibiotics may have functions in competition between sponge microbial community members.     

Rabies emergence among foxes in Turkey

Sixteen rabies isolates recently collected from mainland Turkey and two isolates held within a British archive were used to form a representative cohort from a range of vectors, and were analyzed to identify potential causes for an increase of rabies within the fox (Vulpes vulpes) population in Turkey. Each isolate was characterized by sequence analysis of the nucleoprotein gene and compared phylogenetically to the cohort, to isolates from neighboring countries and to isolates from continental Europe and Russia. From this analysis the isolates could be divided into three groups associated with geographic location. This included a western group, an eastern group, and one isolate that did not group with any other Turkish isolate. This observation was also found using the heteroduplex mobility assay as an alternative method for typing rabies virus isolates. Further comparison with isolates from neighboring countries suggests that this isolate was related to viruses present in Georgia and could represent a recent import to Turkey from that country. Within the two larger groups, sequence data were obtained from both infected dogs and foxes suggesting that there has been transmission of virus between these two species. The direction of transmission could not be identified by the phylogenetic analysis, although absence of rabies within the fox population in previous years suggests that this could represent a recent spillover from the domestic dog to the fox.     

Infection of non‐host model plant species with the narrow‐host‐range Cacao swollen shoot virus

Cacao swollen shoot virus (CSSV) is a major pathogen of cacao (Theobroma cacao) in Africa, and long‐standing efforts to limit its spread by the culling of infected trees have had very limited success. CSSV is a particularly difficult virus to study, as it has a very narrow host range, limited to several tropical tree species. Furthermore, the virus is not mechanically transmissible, and its insect vector can only be used with difficulty. Thus, the only efficient means to infect cacao plants that have been experimentally described so far are by particle bombardment or the agroinoculation of cacao plants with an infectious clone. We have genetically transformed three non‐host species with an infectious form of the CSSV genome: two experimental hosts widely used in plant virology (Nicotiana tabacum and N. benthamiana) and the model species Arabidopsis thaliana. In transformed plants of all three species, the CSSV genome was able to replicate, and, in tobacco, CSSV particles could be observed by immunosorbent electron microscopy, demonstrating that the complete virus cycle could be completed in a non‐host plant. These results will greatly facilitate the preliminary testing of CSSV control strategies using plants that are easy to raise and to transform genetically.     

Serological identification of group D streptococci using commercial antisera

Three commercial group D streptococcal antisera were tested for the serological identification of 100 group D enterococci; 20 Streptococcus bovis; 5 isolated from each of the following streptococcal groups: A, B, C and G; and 3 isolates from serological group F. Antisera from Difco Laboratories, BBL, and Wellcome Reagents Limited were used in the classic capillary tube precipitin test on extracts prepared using the Rantz and Randall procedure. No false positive precipitin reactions were observed. Of the enterococcal isolates, all 100 reacted with the Wellcome, 99 reacted with the BBL, and 96 reacted with the Difco group D antisera. However, of the 20 S. bovis isolates, only 2 reacted with the BBL, and 1 reacted with both the Difco and the Wellcome antisera. Each antiserum was then used to prepare staphylococcal coagglutination (CoA) reagents and each isolate was subsequently tested. A simple extraction procedure was performed by suspending colonies of an isolate in a loopful of salin on a microscope slide and gently heating the slide directly in the flame of a Bunsen burner. All 100 enterococci and all 20 S. bovis gave positive results with the BBL and the Wellcome CoA reagents. Using the Difco reagent, 2 S. bovis isolates failed to produce postitive results. No false positive results were observed with the non-Group D isolates. Our results indicate that the CoA technique using commercial group D antisera may provide faster and sometimes more sensitive serological identification than the classic capillary tube precipitin test.     

A field experiment in Ghana on the tolerance of cocoa seedlings to cocoa swollen-shoot and cocoa mottle-leaf viruses

Seedings of Amelonado Cocoa and of two progenies obtained by crossing Iquitos (Upper Amazon) parents were infected with three strains of cocoa swollen-shoot virus (CSSV) and with cocoa mottle-leaf virus (CMLV). CSSV strain I A had the most severe effects on the growth and canopy condition of all varieties. All four virus isolates had more effect on the growth of Amelonado than on the Iquitos progenies, except that CSSV strain I W affected the growth of Amelonado and one of the Iquitos progenies equally, as did CMLV which caused the greatest decrease in the first crop of all varieties. The relative virulence of different CSSV strains in any one variety can be assessed from single criteria such as length of latent period or effect on growth; CMLV had a shorter latent period than CSSV Strain I A but less effect on growth.     

DNA sequence analysis of conserved genes reveals hybridization events that increase genetic diversity in Verticillium dahliae

The hybrid origin of a Verticillium dahliae isolate belonging to the vegetative compatibility group (VCG) 3 is reported in this work. Moreover, new data supporting the hybrid origin of two V. dahliae var. longisporum (VDLSP) isolates are provided as well as information about putative parentals. Thus, isolates of VDLSP and V. dahliae VCG3 were found harboring multiple sequences of actin (Act), β-tubulin (β-tub), calmodulin (Cal) and histone 3 (H3) genes. Phylogenetic analysis of these sequences, the internal transcribed sequences (ITS-1 and ITS-2) of the rRNA genes and of a V. dahliae-specific sequence provided molecular evidences for the interspecific hybrid origin of those isolates. Sequence analysis suggests that some of VDLSP isolates may have resulted from hybridization events between a V. dahliae isolate of VCG1 and/or VCG4A and, probably, a closely related taxon to Verticillium alboatrum but not this one. Similarly, phylogenetic analysis and PCR markers indicated that a V. dahliae VCG3 isolate might have arisen from a hybridization event between a V. dahliae VCG1B isolate and as yet unidentified parent. This second parental probably does not belong to the Verticillium genus according to the gene sequences dissimilarities found between the VCG3 isolate and Verticillium spp. These results suggest an important role of parasexuality in diversity and evolution in the genus Verticillium and show that interspecific hybrids within this genus may not be rare in nature.     

Limited degree of serological variability in pepper strains of potato virus Y as revealed by analysis with monoclonal antibodies

The degree of serological variability among pepper strains of potato virus Y (PVY) was assessed through the analysis of samples of infected pepper collected in three main pepper producing regions of Spain. Samples corresponding to the period 1980–1991 were analysed by ELISA with five different monoclonal antibodies (MAbs) produced against potato strains of the virus. The results obtained show a limited degree of epitope variability among pepper PVY-isolates, since only eight out of 32 possible serological profiles were found. Most isolates are not recognised by a MAb directed towards an epitope reported to be present in all potato-PVY isolates. The overall serological behaviour of pepper isolates with these MAbs places them as closer to the group O, of the three groups into which the potato isolates of PVY have been subdivided.