Complete genome sequence of cherry virus T, a novel cherry-infecting tepovirus.

Double-stranded RNA and total RNA purified from sour cherry leaves (Prunus cerasus, cv. Amarelka Chvalkovicka) was analyzed by high-throughput sequencing. BLAST annotation identified contigs with homology to several already known cherry-infecting viruses (prune dwarf virus, prunus necrotic ringspot virus, prunus virus F, little cherry virus 1) as well as contigs with sequences more distantly related to those of members of the family Betaflexiviridae and in particular to prunus virus T of the genus Tepovirus. The full genome sequence of a putative virus (6,847 nucleotides [nt]; GenBank no. MT090966) was assembled and completed at the genome ends. The genome has a typical tepovirus organization, containing three overlapping open reading frames (ORFs), encoding a replication-associated protein, a movement protein and a capsid protein, respectively. Both its genome organization and its phylogenetic relationships show that the virus belongs to the genus Tepovirus, but considering the species demarcation criteria for the family Betaflexiviridae, it appears to represent a novel virus species, and we propose the name "cherry virus T" (ChVT) for this virus.

Identification and complete genome analysis of a virus variant or putative new foveavirus associated with apple green crinkle disease.

A virus identified as "apple green crinkle associated virus" (AGCaV) was isolated from Aurora Golden Gala apple showing severe symptoms of green crinkle disease. Evidence was obtained of a potential causal relationship to the disease. The viral genome consists of 9266 nucleotides, excluding the poly(A) tail at the 3'-terminus. It has a genome organization similar to that of members of the species Apple stem pitting virus (ASPV), the type species of the genus Foveavirus, family Betaflexiviridae. ORF1 of AGCaV encodes a replicase-complex polyprotein with a molecular mass of 247 kDa; the proteins of ORFs 2, 3, and 4 (TGB proteins) are estimated to be 25.1 kDa, 12.8 kDa, and 7.4 kDa, respectively; and ORF5 encodes the CP, with an estimated molecular mass of 43.3 kDa. Interestingly, AGCaV utilizes different stop codons for ORF1, ORF3, and ORF5 compared to the ASPV type isolate PA66, and between the two viruses, six distinct indel events were observed within ORF5. AGCaV has four non-coding regions (NCRs), including a 5'-NCR (60 nt), a 3'-NCR (134 nt), and two intergenic (IG) NCRs: IG-NCR1 (69 nt) and IG-NCR2 (91 nt). A conserved stable hairpin structure was identified in the variable 5'-NCR of members of the genus Foveavirus. AGCaV may be a variant or strain of ASPV with unique biological properties, but there is evidence that it may be a distinct putative foveavirus.

First Report of Apricot pseudo-chlorotic leaf spot virus Infecting Plum (Prunus domestica) in the Czech Republic.

Apricot pseudo-chlorotic leaf spot virus (APCLSV) is a novel, still poorly known Trichovirus in the family Betaflexiviridae. It is most closely related to Apple chlorotic leaf spot virus (ACLSV) (2,4) and infects stone fruit trees of the Prunus genus. Its presence has so far been detected in apricot, plum, Japanese plum, and peach trees in Italy, Spain, France, Hungary, Turkey, Jordan, and Australia (1,2,4). During the summers of 2008 and 2010, leaf samples of old Czech local plum cultivars were obtained from the Holovousy collection and assessed for the presence of viruses belonging to the Capillovirus, Trichovirus, and Foveavirus genera using the polyvalent degenerate oligonucleotides (PDO) nested reverse transcription (RT)-PCR test (3). Following amplification from total RNAs extracts, the amplicons were cloned and several clones were sequenced for each plant sample. In plum (Prunus domestica) cv. Babce, a mixture of amplicons was observed and BlastN and BlastX analyses of the obtained sequences revealed the presence of ACLSV and APCLSV. The 310-bp APCLSV amplicon (GenBank Accession No. JN790294) showed highest identity (82.9% in nucleotide sequence and 97.1% in amino acid sequence) with the Sus2 isolate of APCLSV (4) and clustered with APCLSV isolates in a phylogenetic analysis. APCLSV infection was further confirmed with an APCLSV-specific RT-PCR assay (4), which yielded a product of the expected 205-bp size (GenBank Accession No. JN653070) with closest homology again to the Sus2 APCLSV isolate (83.4 and 94.3% nucleotide and amino acid identity, respectively). To our knowledge, this finding represents the first detection of APCLSV in domestic plums in the Czech Republic, extending our vision of APCLSV diversity and its geographic distribution. For unknown reasons, APCLSV has almost always been reported in mixed infection with ACLSV (1,2,4) and the situation in cv. Babce does not deviate from this trend. This has greatly hindered the analysis of the pathogenicity of APCLSV, a situation further complicated in the current case because the Babce cultivar was also infected by Plum pox virus. References: (1) M. Barone et al. Acta Hortic. 781:53, 2008. (2) T. Candresse et al. Virus and Virus-Like Diseases of Pome and Stone Fruit Trees. A. Hadidi et al., eds. The American Phytopathological Society, St. Paul, MN, 2011. (3) X. Foissac et al. Phytopathology 95:617, 2005. (4) D. Liberti et al. Phytopathology 95:420, 2005.

Differentiation of Plum pox virus isolates by single-strand conformation polymorphism and low-stringency single specific primer PCR analysis of HC-Pro genome region.

Single-strand conformation polymorphism (SSCP) and low-stringency single specific primer (LSSP)-PCR were assessed for suitability and reliability in genotyping of Plum pox virus (PPV) isolates. Examined PPV isolates included 16 PPV-D, 12 PPV-M, and 14 PPV-Rec isolates collected in Czech Republic. The analysis was performed on the helper component protease (HC-Pro) region of the PPV genome. SSCP and LSSP-PCR allowed the differentiation of PPV strain, but SSCP was not able to distinguish isolates within the same strain. The individual genotyping of each PPV isolate was obtained by LSSP-PCR. Nevertheless, both SSCP and LSSP-PCR techniques are suitable for preliminary screening of genetic variability of plant RNA viruses.

Phytoplasma associated with witches'-broom disease of Ulmus minor MILL . in the Czech Republic: Electron microscopy and molecular characterization.

Visual inspections of elm trees in south Moravia in 1997-2007 revealed a rare occurrence of plants with smaller and cowl-forming leaves on some twigs, i.e. a feature resembling witches'-broom disease observed on the end of twigs. The presence of phytoplasma-like bodies was observed by transmission electron microscopy of phloem tissue. On the other hand, no phytoplasmas were found in asymptomatic trees. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rRNA sequences specific for phytoplasmas. Sequence analyses of the 16S-23S ribosomal operon (1852 bp) allowed for the classification of the detected phytoplasmas in the elm yellows group, but its position remained on the boundary of the 16SrV-A and 16SrV-C ribosomal subgroups. Sequence analyses of the ribosomal protein of the rpl22-rps3 and secY genes lead to further classification and revealed the phytoplasmas' affiliations to the 'Candidates Phytoplasma ulmi'. Some exceptions in unique oligonucleotide sequences defined for 'Ca. Phytoplasma ulmi' were found in the Czech isolate. This is the northernmost confirmed occurrence of phytoplasma on elm trees within Europe.

Genetic and biological diversity of the Pea seed-borne mosaic virus isolates occurring in Czech Republic.

Eight isolates of the Pea seed-borne mosaic virus (PSbMV) from the Czech Republic were studied regarding their biological and molecular characteristics. Molecular characterization using RT-PCR was done on the 5'(Nter)NIb-CP-UTR3' region amplified using universal CPUP/P9502 primer pair and the newly designed PSB8812/PSB944, and PSB8800/PSB9440 primer pairs, respectively. Sequential and phylogenetic analysis of CP-UTR3' region from all isolates showed that the available Czech and GenBank PSbMV isolates were distributed into 4 clusters in agreement with their diversification and according to their biological characteristics (i.e. pathotype). The molecular data were confirmed by biological testing on different pea cultivars. The Czech isolates were distributed into two pathotypes, the P-1 (7 isolates) and P-4 (1 isolate).

First Report of Brown Rot Caused by Monilinia fructicola on Various Stone and Pome Fruits in the Czech Republic.

Monilinia fructicola, a causal agent of brown rot, is one of the most important fungal pathogens of stone fruits. The disease causes major crop losses in peach, plum, prune, nectarine, and apricot. M. fructicola is commonly present in Asia, North and South America, and Australia. This is a quarantined pathogen in Europe; restricted occurrence has been observed in Austria and France. Recently, it was detected in Hungary and Switzerland on peach and nectarine fruits imported from Italy and Spain (1,4). During a survey in the summer of 2006, 56 samples were tested for the presence of Monilinia spp. M. fructicola was detected in 15 samples from 11 locations in the western area (Bohemia) of the Czech Republic, mainly on peaches (Prunus persica), apples (Malus × domestica), and sweet and sour cherries (Cerasus avium and C. vulgaris) and rarely on flowering plum (Prunus triloba) and Malus × moerlandsii cv. Liset. On the other hand, the pathogen was not detected on fruits of apricot (Prunus armeniaca) or pear (Pyrus communis). In all cases, M. fructicola was detected on fruits except for a single occurrence of the pathogen on a shoot of the Malus × domestica. The pathogen was always detected in mixed infections with M. fructigena and/or M. laxa. On both fruits and the shoot, symptoms appeared as brown, sunken lesions covered with grayish pustules. Many infected fruits became dry and mummified because rot progressed through the fruit surface. The infected shoot died back (3). M. fructicola was identified by means of colony and conidial morphology and molecular characteristics. The colonies cultivated on potato dextrose agar were entire and the colony surface was even. The color of the colony was gray, and sporulating colonies showed concentric rings that changed to a hazel color. Conidia were ellipsoid, hyaline, and 13.5 to 17.7 × 8.3 to 10.5 µm. Preliminary morphological identification was confirmed by PCR (2) on DNA isolated directly from mycelium on the examined fruits. A product that was 280 bp long was obtained in all cases. The BLAST analysis of our PCR product sequences showed 100% homology to sequences of M. fructicola (GenBank Accession Nos. DQ491506, AY2891185, Z73778, and AB125615). One sequence from our study was deposited in GenBank (Accession No. EF378628). To our knowledge, this is the first report of the quarantined fungus M. fructicola in the Czech Republic. References: (1) E. Bosshard et al. Plant Dis. 90:1554, 2006. (2) K. J. D. Hughes et al. EPPO Bull. 30:507, 2000. (3) J. M. Ogawa et al., eds. Compendium of Stone Fruit Diseases. The American Phytopathological Society, St. Paul, MN, 1995. (4) M. Petróczy and L. Palkovics. Plant Dis. 90:375, 2006.

Molecular characterization of Czech and Chinese leek yellow stripe virus isolates from garlic.

Using an RT-PCR specific for nuclear inclusion b (NIb) and coat protein (CP) genes of Leek yellow stripe virus (LYSV) we detected two Czech (LYSV-5CZ and LYSV-22CZ) and one Chinese (LYSV-16) isolate of LYSV in garlic plants. The RT-PCR products were cloned and sequenced. Phylogenetic analysis based on deduced amino acid sequence of NIb-CP region placed the Czech isolates in the group I and the Chinese isolate in the group II of LYSV.

First Incidence of Plum Pox Virus on Apricot Trees in China.

Plum pox disease, caused by Plum pox virus (PPV), is the most severe virus disease of plums, apricots, and peaches. The disease causes heavy losses for fruit growers and the international trade of propagation materials and fresh fruits. PPV was first reported in Bulgaria in 1917 (1). It is now widespread in Europe and has been reported from Cyprus, Syria, Egypt, India, Kazakhstan, Chile, the United States, and Canada. Leaves on symptomatic apricot trees (Prunus armeniaca cvs. Hong Mei and Bai Mei and a selected genotype) in the Hunan Province of China showed typical yellow rings and diffused chlorotic spots. Samples from three suspected trees were repeatedly analyzed using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) in the summers of 2001-2003. PPV was detected in leaves, bark, and leaf buds of all three trees using ELISA with polyclonal and monoclonal antibodies provided by M. Navratil, Palacky University, Olomouc, Czech Republic (3). The results were confirmed using RT-PCR amplification of a 243-bp of the coat protein gene with a PPV-specific primer pair (2). BLAST analysis of two RT-PCR product sequences (GenBank Accession Nos. AY750961 and AY795603) showed 100% homology to multiple sequences of the PPV-D strain (GenBank Accession Nos. X81080, AF440743, and AF401295). The third sequence (GenBank Accession No. AY795602) had a C at position 112 rather than the T found in the other sequences. The ELISA, RT-PCR, and sequence results indicate that PPV-D was present in the apricot trees. To our knowledge, this is the first indication of PPV occurrence in China. This sporadic incidence of PPV on apricot trees requires addressing problems with the occurrence and spread of plum pox diseases in China and starting an eradication program. References: (1) D. Atanasoff. Annu. Univ. Sofia Fac. Agron. et Sylvic. 11:49, 1932. (2) T. Candresse et al. Phytopathology. 88:198, 1998. (3) I. Hilgert et al. Hybridoma. 12:215, 1993.

[The relationship of antibody formation in immunization against typhoid fever to the acetylation phenotype]. / Sviaz' antiteloobrazovaniia pri immunizatsii protiv briushnogo tifa s fenotipom atsetilirovaniia.

The study of the dynamics of specific antibody formation in response to immunization against typhoid fever with adsorbed chemical vaccine T in its relationship to the phenotype of acetylation has yielded results characterizing a considerable variability of immune response simultaneously with the definite type of modification of parameter A.