Molecular Characterization Of Phytoplasma Research Articles

Molecular Characterization of 'Candidatus Phytoplasma prunorum' in the Czech Republic and Susceptibility of Apricot Rootstocks to the Two Most Abundant Haplotypes.

'Candidatus Phytoplasma prunorum' is one of the most destructive pathogens of Prunus species, where susceptible species render unproductive several years after infection. In epidemiology, the molecular characterization of phytoplasmas is based on sequence analysis of variable nonribosomal genes. In this study aceF, pnp, imp and secY genes were used for characterization of the 'Ca. P. prunorum' genotypes present in the Czech Republic. In total, 56 plant and 33 vector (Cacopsylla pruni) samples positive to 'Ca. P. prunorum' collected in seven localities were used in the study. Based on sequence analysis, four aceF, two pnp, six imp, and three secY genotypes were identified in analyzed samples. The most abundant in both plant and insect samples were the A6, P2, I4, and S2 genotypes. Most of the Czech 'Ca. P. prunorum' haplotypes clustered together in the haplotype network analysis. Next, two isolates representing the two most abundant Czech haplotypes (A6-P2-I4-S2 and A5-P2-I4-S2) were used in the susceptibility test of three apricot rootstock types (St. Julien A, M-VA-1, GF-305). Susceptibility was analyzed by phytoplasma quantification using quantitative real-time PCR and evaluation of symptom manifestation. Based on the results, the influence of the rootstock type on the phytoplasma titer and symptom manifestation was greater than of the phytoplasma isolate, while the year of analysis had no influence on the results. The results also showed that the phytoplasma titer is increasing in plant tissues during the vegetation period.

Detection and Molecular Characterization of Phytoplasma and its Effect on the Vitamin and Fibers in Corchorus olitorius Crops in Egypt

Detection and Molecular Characterization of Phytoplasma and its Effect on the Vitamin and Fibers in Corchorus olitorius Crops in Egypt

Detection and molecular characterization of phytoplasmas in mango seedlings

Detection and molecular characterization of phytoplasmas in mango seedlings

First detection and molecular characterization of grapevine phytoplasmas in Kosovo

AbstractA search for phytoplasma‐associated diseases was conducted for the first time in the main grapevine‐growing localities of the Dukagjini plain in Kosovo. A total of 144 samples were collected from grapevine cultivars displaying leaf yellowing, reddening, discolouration and irregular wood ripening, and analysed using nested and quantitative PCR assays. These assays showed that 35.4% of samples belonging to eight cultivars were positive to the presence of phytoplasmas in the 16SrXII group. The 16S rDNA phytoplasma sequences obtained from 15 samples shared identity greater than 99.5% with ‘Candidatus Phytoplasma solani’. Sequence analysis of the tuf gene showed that the strains found in Kosovar grapevines are in the tuf‐type b1 group, sharing 99.6% to 99.8% identity with ‘Ca. P. solani'‐related strains associated with the “bois noir” grapevine disease in many European countries; the secY gene sequences, on the other hand, shared 100% identity with ‘Ca. P. solani' strains from Bosnia and Herzegovina, Serbia, Croatia and Turkey. This study constitutes the first report on the presence and molecular characterization of phytoplasmas in Kosovar vineyards. Based on these results, it is recommended that testing for phytoplasma be included in the certification program for grapevine in Kosovo.

Identification and molecular characterization of phytoplasmas and rickettsia pathogens associated with ‘Bunchy Top Symptom’ (BTS) and ‘Papaya Bunchy Top’ (PBT) of papaya in Cuba

Two different papaya diseases have been previously reported in Cuba, Bunchy Top Symptom (BTS) associated with a phytoplasma of group 16SrII ‘Candidatus Phytoplasma aurantifolia’ and Papaya Bunchy Top (PBT), associated with a rickettsia. Regarding the regional phytosanitary impact of both diseases for the papaya crop, the present study investigated the occurrence of BTS and PBT in papaya fields in Cuba, and the possible mixed infection of phytoplasma and rickettsia pathogens associated. Papaya plants showing symptoms of BTS or PBT or both, were collected in Las Tunas and Havana provinces from January 2009 to February 2010, and evaluated for phytoplasma and rickettsia by PCR with primers targeting the 16S ribosomal RNA and the rickettsial succinate deshydrogenase (sdhA) genes, respectively. Phytoplasmas and rickettsia were individually detected in 76/86 BTS-symptomatic and 22/22 PBT-symptomatic papaya plants, and simultaneously detected in 5/86 (5.81%) of the BTS-symptomatic and 17/22 (77.27%) of the PBT-symptomatic plants. Conventional and virtual RFLP analyses of the 16S rDNA sequences revealed the presence of phytoplasmas of group 16SrI ‘Candidatus Phytoplasma asteris’ and 16SrII in papaya plants affected by BTS and PBT, and identified two new phytoplasma subgroups, 16SrI-X and 16SrII-N in papayas fields of Las Tunas, which was confirmed by phylogenetic analysis. The partial rickettsia sdhA gene sequences were 100% identical to that of the rickettsia associated with PBT in Puerto Rico. Results confirm that phytoplasmas are consistently associated with both BTS and PBT symptoms, and that mixed infections of phytoplasma and rickettsia pathogens can occur in either BTS or PBT-affected papaya fields, which implies new epidemiological constraints for the disease control.

Grapevine yellows diseases in Spain: eight year survey of disease spread and molecular characterization of phytoplasmas involved

Among grapevine yellows phytoplasma diseases in Europe, flavescence doree (FD) is the most devastating and in the last decade has reached Spanish vineyards, mainly in Catalonia. An eight-year survey was carried out in the areas where the disease has spread (Alt Emporda, Catalonia, Northern Spain) and in the remaining vine-growing areas of Catalonia. Sequence analyses of a portion of the 16S-23S ribosomal DNA cistron, from selected grapevine samples from Catalonia, showed that the phytoplasmas involved in grapevine yellows belong to 16S ribosomal subgroups V-D (flavescence doree, FD) and XII-A (bois noir, BN). A set of Spanish FD isolates collected during these years were further studied by RFLP analyses of the 16S-23S ribosomal DNA fragment, as well as the rpS3 and SecY genes. All the FD phytoplasma strains studied were related to phytoplasmas belonging to ribosomal protein subgroup rp-E.

Molecular characterization of phytoplasmas in lilies with fasciation in the Czech Republic

Lilium spp. with symptoms of severe fasciation were observed in Southern and central Bohemia during the period 1999–2003. Nucleic acids extracted from symptomatic and asymptomatic plants were used in nested-PCR assays with primers amplifying 16S-23S rRNA sequences specific for phytoplasmas. The subsequent nested-PCR with phytoplasma group-specific primers followed by RFLP analyses and the 16S ribosomal gene sequencing, allowed classification of the detected phytoplasmas in the aster yellows group, subgroups 16SrI-B and 16SrI-C alone, and in mixed infection. Samples infected by 16SrI-C phytoplasmas showed different overlapping RFLP profiles after TruI digestion of R16F2/R2 amplicons. Two of these amplicons were sequenced, one of them directly and the other after cloning; sequence analyses and blast alignment confirmed the presence of two different overlapping patterns in samples studied. The sequences obtained were closely related, respectively, to operon A and operon B ribosomal sequences of the clover phyllody phytoplasma. Direct PCR followed by RFLP analyses of the tuf gene with two restriction enzymes showed no differences from reference strain of subgroup 16SrI-C. Infection with aster yellows phytoplasmas of 16SrI-B subgroup in asymptomatic lilies cv. Sunray was also detected.

Electron microscopy and molecular characterization of phytoplasmas associated with strawflower yellows in the Czech Republic

Strawflower (Helichrysum bracteatum) with symptoms resembling those associated to phytoplasma infection were observed in several areas in the Czech Republic during the period 1994–2001. Plants with leaf bronzing, reddening and necrosis, proliferation of secondary shoots, flower abnormalities and dwarfing died in advanced stages of the disease. The disease incidence ranged from 2% to 70% and caused significant loss to the flower and seed production. Transmission electron microscopy showed phytoplasmas in sieve cells of affected plants, but not in healthy ones. Association of phytoplasmas with the disease was confirmed by polymerase chain reaction using phytoplasma universal ribosomal primers R16F2n/R16R2. An amplification product of the expected size (1.2 kb) was observed in all samples of the symptomatic strawflowers. The restriction profiles obtained following separate digestion with three endonucleases (AluI, HhaI, MseI) showed that phytoplasmas infecting strawflowers from different localities in the Czech Republic were uniform and undistinguishable from aster yellows (subgroup 16SrI-B). Sequence analysis of 1771 bp of the ribosomal operon amplified with primers P1/U3, R16F2n/R2 and 16R758/P7 indicated that the closest related phytoplasmas were those associated with 'Rehmannia glutinosa var. purpurea', both originating from Bohemia. This is the first report on the occurrence of a phytoplasma-associated disease of strawflower in the Czech Republic.