Silvija Černi

Stem pitting and seedling yellows symptoms of Citrus tristeza virus infection may be determined by minor sequence variants

The isolates of Citrus tristeza virus (CTV), the most destructive viral pathogen of citrus, display a high level of variability. As a result of genetic bottleneck induced by the bud-inoculation of CTV-infected material, inoculated seedlings of Citrus wilsonii Tanaka displayed different symptoms. All successfully grafted plants showed severe symptoms of stem pitting and seedling yellows, while plants in which inoculated buds died displayed mild symptoms. Since complex CTV population structure was detected in the parental host, the aim of this work was to investigate how it changed after the virus transmission, and to correlate it with observed symptoms. The coat protein gene sequence of the predominant genotype was identical in parental and grafted plants and clustered to the phylogenetic group 5 encompassing severe reference isolates. In seedlings displaying severe symptoms, the low-frequency variants clustering to other phylogenetic groups were detected, as well. Indicator plants were inoculated with buds taken from unsuccessfully grafted C. wilsonii seedlings. Surprisingly, they displayed no severe symptoms despite the presence of phylogenetic group 5 genomic variants. The results suggest that the appearance of severe symptoms in this case is probably induced by a complex CTV population structure found in seedlings displaying severe symptoms, and not directly by the predominant genomic variant.

East Adriatic—a reservoir region of severe Citrus tristeza virus strains

Citrus tristeza virus (CTV) represents one of the major threats to citrus production worldwide. In the East Adriatic region, CTV symptoms are mostly absent due to traditional citrus grafting on trifoliate orange (Poncirus trifoliata), a CTV-tolerant rootstock. Therefore, the virus has been continuously spreading by the propagation of infected material. The genetic variability of CTV was studied on nineteen citrus samples, collected from orchards in the coastal region of Croatia, Montenegro and Albania, that previously tested positive by ELISA and immunocapture RT-PCR. Single-strand conformation polymorphism of the amplified coat protein gene demonstrated the presence of different CTV variants in each amplicon, while sequence analysis of cloned CP gene variants confirmed their clustering into six out of the seven phylogenetic groups so far delineated. Four of these groups include sequences of severe quick decline, seedling yellows and stem-pitting (SP) isolates, thought to be found only rarely in the Mediterranean region. Regardless of the lack of symptoms in the field, CTV isolates from the East Adriatic displayed high genetic variability and pathogenic potential, additionally confirmed by biological characterisation. The high percentage of mixed infections suggest the potential for further diversification and a greater risk of severe variants spreading into new areas.

Multilocus sequence analysis of ‘Candidatus Phytoplasma asteris’ strain and the genome analysis of Turnip mosaic virus co-infecting oilseed rape

Molecular characterization of a pathogenic complex infecting winter oilseed rape (Brassica napus ssp. oleifera (DC.) Metzg.) plants showing typical rape phyllody symptoms along with some atypical changes.

The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity.

Genetic heterogeneity of RNA populations influences virus pathogenesis, epidemiology and evolution. Therefore, accurate information regarding virus genetic structure is highly important for both diagnostic and scientific purposes. For the Hepatitis E virus (HEV), the causal agent of hepatitis in humans, the intra-host population structure has been poorly investigated, mainly using the less sensitive RFLP-based approach. The objective of this study was to assess the suitability and the accuracy of single strand conformation polymorphism (SSCP) analysis, a well-established tool in genetic variation research, for the characterization of HEV quasispecies. The analysis was conducted on 50 clones of five swine isolates and 30 clones of three human HEV isolates. To identify and quantify the sequence variants present in each HEV isolate, 348bp long fragments of the amplified conserved ORF2 region were separated by cloning. Ten clones per isolate were subjected to SSCP and sequenced in a parallel experiment. The results show a high correlation of SSCP haplotype profiling with the sequencing results, confirming the sensitivity and reliability of this simple, rapid and low cost approach in the characterization of HEV quasispecies.

Comparison of different methods for Citrus tristeza virus detection in Satsuma mandarins

Citrus tristeza virus (CTV) infection in 13 Satsuma (Citrus unshiu (Mak.) Marc.) symptomless clones was investigated by different diagnostic methods, namely ELISA (Enzyme Linked Immunosorbent Assay), DTBIA (Direct Tissue Blot Immunoassay), and biological testing. The objective was to correlate the outcomes of the two serological tests mutually and with the severity of tristeza disease symptoms on indicator plants. ELISA and DTBIA were performed on the clones and on inoculated tissue samples of Mexican lime, while biological testing was performed on four citrus indicators. An additional goal of this study was to evaluate the efficiency of CTV DTBIA testing using different tissue types, including Satsuma immature ovaries and flower petioles. ELISA performed on spring shoots tissue of Mexican lime and DTBIA performed on mature ovaries of Satsuma field samples, showed high intensity of reactions and detection efficiency. Both tests were equally reliable and would make a good choice for fast CTV detection.

Citrus wilsonii: biological response to infection with different Citrus tristeza virus genotypes.

SUMMARY Citrus wilsonii Tanaka is cold-resistant, rarely grown chance hybrid between Citrus ichangensis and Citrus grandis with a potential to be used as a rootstock in colder climates. In order to evaluate its reaction to Citrus tristeza virus (CTV), C. wilsonii seedlings were graft-inoculated with previously characterized CTV isolates monophyletic with respect to the p25 gene and clustering to phylogenetic groups Gp 1, Gp 2, Gp 3a, Gp 4, Gp 5 and Gp M. The evaluation of C. wilsonii symptoms and comparison with the reaction of standard citrus indicators infected with the same CTV isolates revealed that C. wilsonii infected with isolates Gp 2 and Gp 4 developed stem pitting (SP). This is in accordance with the SP-inducing potential of these CTV isolates in the standard sweet orange SP indicator. The obvious and numerous pits shown by C. wilsonii stems suggests that it reacts strongly to severe SP CTV isolates.

In vitro system for studying interactions between Citrus exocortis viroid and Gynura aurantiaca (Blume) DC. metabolism and growing conditions

Citrus exocortis viroid (CEVd) is an economically important plant pathogen, which infects a broad range of hosts. In order to investigate complex interactions among viroid, host-growing conditions, and plant secondary metabolism, we setup an in vitro system for the cultivation of CEVd-infected and viroid-free Gynura aurantiaca (Blume) DC. shoots. Both basal Murashige and Skoog (MS) medium and MS medium supplemented with cytokinines supported the growth of shoots, but viroid-free shoots performed better than infected ones. The addition of cytokinines induced an increase in the concentration of purple-reddish pigment, cyanidin tetra-glucoside (GAA), which did not depend on the presence of viroid. The differences in single strand conformation polymorphism patterns between CEVd sequence variants obtained from plants grown on medium supplemented with cytokinines and those obtained from plants grown on basal MS medium or in the greenhouse, indicated that cytokinines may influence not only the plant metabolism, but also the viroid population structure.