Experimental Host Species Research Articles

Characterisation and diagnosis of frangipani mosaic virus from India

Frangipani mosaic virus (FrMV) is known to infect frangipani tree (Plumeria rubra f. acutifolia) in India but the virus has not been characterized at genomic level and diagnosis is not available. In the present study, an isolate of FrMV (FrMV-Ind-1) showing greenish mosaic and vein-banding symptoms in P. rubra f. acutifolia in New Delhi was characterized based on host reactions, serology and genome sequence. The virus isolate induced local symptoms on several new experimental host species: Capsicum annuum (chilli), Nicotiana benthamiana, Solanum lycopersicum and S. melongena. N. benthamiana could be used as an efficient propagation host as it developed systemic mottle mosaic symptoms all round the year. The genome of FrMV-Ind-1 was 6643 (JN555602) nucleotides long with genome organization similar to tobamoviruses. The Indian isolate of FrMV shared a very close genome sequence identity (98.3 %) with the lone isolate of FrMV-P from Australia. FrMV-Ind-1 together with FrMV-P formed a new phylogenetic group i.e. Apocynaceae-infecting tobamovirus. The polyclonal antiserum generated through the purified virus preparation was successfully utilized to detect the virus in field samples of frangipani by ELISA. Of the eight different tobamoviruses tested, FrMV-Ind-1 shared distant serological relationships with only cucumber green mottle mosaic virus, tobacco mosaic virus, bell pepper mottle virus and kyuri green mottle mosaic virus. RT-PCR based on coat protein gene primer successfully detected the virus in frangipani plants. This study is the first comprehensive description of FrMV occurring in India.

Transcriptome analyses of Anguillicola crassus from native and novel hosts.

Anguillicola crassus is a swim bladder nematode of eels. The parasite is native to the Asian eel Anguilla japonica, but was introduced to Europe and the European eel Anguilla anguilla in the early 1980s. A Taiwanese source has been proposed for this introduction. In the new host in the recipient area, the parasite appears to be more pathogenic. As a reason for these differences, genetically fixed differences in infectivity and development between Taiwanese and European A.crassus have been described and disentangled from plasticity induced by different host environments. To explore whether transcriptional regulation is involved in these lifecycle differences, we have analysed a “common garden”, cross infection experiment, using deep-sequencing transcriptomics. Surprisingly, in the face of clear phenotypic differences in life history traits, we identified no significant differences in gene expression between parasite populations or between experimental host species. From 120,000 SNPs identified in the transcriptome data we found that European A. crassus were not a genetic subset of the Taiwanese nematodes sampled. The loci that have the major contribution to the European-Taiwanese population differentiation show an enrichment of synonymous and non-coding polymorphism. This argues against positive selection in population differentiation. However, genes involved in protein processing in the endoplasmatic reticulum membrane and genes bearing secretion signal sequences were enriched in the set of genes most differentiated between European and Taiwanese A. crassus. These genes could be a source for the phenotypically visible genetically fixed differences between European and Taiwanese A. crassus.

Complete genome analysis of three isolates of narcissus late season yellows virus and two of narcissus yellow stripe virus: three species or one?

Complete genome sequences of two new isolates of narcissus late season yellows virus (NLSYV) from Australia were compared with the other NLSYV genome from China and with two complete genomes of isolates designated narcissus yellow stripe virus (NYSV), one from Australia and the other from China. On the basis of symptoms on natural and experimental host species, and genome sequence identity, the isolates could either be classified as closely related members of three different species or placed together in one taxon. Options for classification of these potyvirus isolates are discussed.

Experimental cross-infections by Perkinsus marinus and P. chesapeaki in three sympatric species of Chesapeake Bay oysters and clams

In controlled laboratory transmission experiments, uniform doses of axenic in vitro isolate cultures of Perkinsus marinus from a Crassostrea virginica oyster, and of independent P. chesapeaki isolates from Chesapeake Bay Mya arenaria and Macoma balthica clams, were used to reciprocally challenge Perkinsus sp.-free C. virginica, M. arenaria, and M. balthica experimental hosts. Following mantle cavity inoculations, all 3 experimental hosts acquired high incidences (30 to 100%) of infections by each of the 3 Perkinsus sp. isolates, based on PCR assays of DNAs from experimental host tissues that were collected through 60 d post-inoculation. Lesions containing proliferating pathogen cells were documented histologically in tissues of all experimental host species challenged with all isolates of both Perkinsus species. Experimental Perkinsus sp. challenge isolates were re-isolated and propagated in vitro from infected tissues of host molluscs from most (5 of 9) experimental treatment groups. Koch's postulates were generally satisfied to confirm experimental infections in all bivalve molluscs that were challenged with 3 isolates of 2 Perkinsus spp. These results suggest potential broad and overlapping host specificities for the 2 current Chesapeake Bay-endemic Perkinsus species: P. marinus and P. chesapeaki.

Capparis spinosa- a new host of Cucumber mosaic virus in Italy

In spring 2004, a survey was conducted to investigate the phytosanitary status of caper ( Capparis spinosa ) crops in Sicily, the Eolie Islands (Salina and Lipari) and Pantelleria. Two viruses have previously been isolated from caper in Salina (Campo et al ., 2000): Caper latent virus (CapLV, genus Carlaviruses , family Flexiviridae ) (Gallitelli & Di Franco, 1987) and Pittosporum vein yellowing virus (PVYV, genus Nucleorhabdovirus , family Rhabdoviridae ) (Nuzzaci et al ., 1993) . Crude sap taken from collected leaf samples was used to mechanically inoculate the experimental host species Chenopodium quinoa , C. amaranticolor , Nicotiana benthamiana and N. glutinosa . Some samples from the Eolie crops produced chlorotic local lesions on Chenopodium spp. a few days post-inoculation and severe systemic mosaic leaf malformations and shoestringing on Nicotiana spp., resembling symptoms caused by C ucumber mosaic virus (CMV, genus Cucumovirus , family Bromoviridae ). ELISA, using a CMV-specific rabbit polyclonal antiserum (ISPaVe-CMV22), detected the presence of the virus in samples from Salina and Lipari. To characterize the virus isolates, total RNA was extracted from caper leaves (Qiagen, RNeasy Plant Mini Kit) and was analysed in a one-step RT-PCR using primer sets CMV-CPI or CMVCPII (Lumia et al ., 2001), specific to I and II subgroups of CMV, respectively. An amplified product of the expected size was mostly commonly obtained when CMV-CPI primers were used (672 bp), and less frequently with CMV-CPII primers (674 bp). In a few samples both subgroups were detected. During the spring, CMV was detected in plants showing reddish mottle, vein yellowing and yellow rings or spots, and in caper plants without symptoms that showed interveinal leaf chlorosis on a few basal shoots later in summer. Plants with yellowing disorder were found to be infected with both CMV and PVYV. No CMV-infected plants were detected in Pantelleria. This is the first report of Capparis spinosa as a natural host of CMV and adds a new plant family, Capparaceae, to the already wide virus host range.

Turnip yellow mosaic virus: transfer RNA mimicry, chloroplasts and a C‐rich genome

SUMMARY Taxonomy: Turnip yellow mosaic virus (TYMV) is the type species of the genus Tymovirus, family Tymoviridae. TYMV is a positive strand RNA virus of the alphavirus-like supergroup. Physical properties: Virions are non-enveloped 28-nm T = 3 icosahedrons composed of a single 20-kDa coat protein that is clustered in 20 hexameric and 12 pentameric subunits. Infectious particles and empty capsids coexist in infected tissue. The genomic RNA is 6.3 kb long, with a 5'(m7)GpppG cap and a 3' untranslated region ending in a tRNA-like structure to which valine can be covalently added. The genome has a distinctive skewed C-rich, G-poor composition (39% C, 17% G). Viral proteins: Two proteins, whose open reading frames extensively overlap, are translated from the genomic RNA. p206, which contains sequences indicative of RNA capping, NTPase/helicase and polymerase activities, is the only viral protein that is necessary for genome replication in single cells. It is produced as a polyprotein and self-cleaved to yield 141- and 66-kDa proteins. p69 is required for virus movement within the plant and is also a suppressor of gene silencing. The coat protein is expressed from the single subgenomic RNA. Hosts and symptoms: TYMV has a narrow host range almost completely restricted to the Cruciferae. Experimental host species are Brassica pekinensis (Chinese cabbage) or B. rapa (turnip), in which diffuse chlorotic local lesions and systemic yellow mosaic symptoms appear. Arabidopsis thaliana can also be used. Clumping of chloroplasts and the accumulation of vesicular invaginations of the chloroplast outer membranes are distinctive cytopathological symptoms. High yields of virus are produced in all leaf tissues, and the virus is readily transmissible by mechanical inoculation. Localized transmission by flea beetles may occur in the field.

Suitability of the maize weevil and angoumois grain moth as hosts for the parasitoids Anisopteromalus calandrae and Pteromalus cerealellae

AbstractTwo parasitoids, Pteromalus cerealellae (Ashmead) and Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae), were compared for their ability to parasitize two important internally‐developing insect pests of stored maize (Zea mays L.). Parasitism by P. cerealellae was greater on Angoumois grain moth, Sitotroga cerealella (Olivier), than on maize weevil, Sitophilus zeamais Motschulsky, in no‐choice experiments. Anisopteromalus calandrae parasitized more maize weevils than did P. cerealellae. The former parasitoid parasitized only a few Angoumois grain moths successfully in maize, but parasitized many in wheat if the hosts were younger than 3 weeks old. Thus, both host age and type of grain affect suitability for parasitism. The effects of parental host (species on which the female developed) and experimental host (species exposed to parasitism) on parasitism rate of P. cerealellae were tested in a host‐switching experiment. Parasitism by parasitoids reared on maize weevils was 23% lower than that of parasitoids reared on Angoumois grain moth. This effect was independent of which host the filial generation of parasitoids was tested on. However, the experimental host species had a much greater effect on parasitoid fecundity than the parental host species. Female progeny had smaller body sizes when emerging from maize weevil than from Angoumois grain moth, which may explain the parental host effect on fecundity. There was also a slight intergenerational effect of host species on parasitoid body size.

Host specificity of Salmonella infection in chickens and mice is expressed in vivo primarily at the level of the reticuloendothelial system.

By experimental infection, host-specific Salmonella serotypes were shown to demonstrate specificities for chickens, mice, and other laboratory animals. Following oral inoculation, four strains of Salmonella gallinarum and two S. pullorum strains, isolated from diseased poultry, were more virulent for chickens than for mice. By contrast, four strains each of S. choleraesuis and S. dublin, isolated from diseased pigs and cattle, respectively, were more virulent for mice than for chickens. These results were also reflected in the degree of virulence expressed after parenteral inoculation. In addition, S. choleraesuis, but not other serotypes, killed rats, guinea pigs, and rabbits. S. typhimurium strains varied widely in their virulence, and some strains were virulent for both mice and chickens. Four other serotypes isolated from poultry or human food poisoning cases and a nonpathogenic Escherichia coli strain were much less virulent for both experimental host species. Most of the host-specific Salmonella serotypes studied were able to colonize the distal alimentary tract and invade the tissues in both mice and chickens to various degrees. There was, however, a greater difference in the ability to survive and multiply in the visceral organs, particularly the spleen and the liver, once invasion had occurred which correlated with the virulence for the host species involved.