ABSTRACT
Ralstonia solanacearum is a notorious pathogen of bacterial wilt on Solanum lycopersicum. Most isolates from diseased tomato tissues are biovar 3, and their genomes are publicly available; however, information on biovar 4 strains is limited. Here, the complete genome sequence of R. solanacearum Bs715, a biovar 4 strain, is presented.
ABSTRACT
Pseudomonas syringae pv. phaseolicola, the causative agent of halo blight, is the most important bacterial pathogen of bean. Both nontoxigenic (Tox(-)) and toxigenic (Tox+) strains of this pathogen cause halo blight in beans. However, nontoxigenic strains cannot be detected by currently available molecular and serological tools. In this study, a TaqMan probe and primer set were designed based on the phage integrase family site-specific recombinase of P. s. pv. phaseolicola 1448A because it is known that most site-specific recombinases are structurally and functionally diverse. The specificity of the probe and primers was evaluated using purified DNA from 29 isolates of 3 different pathovars of P. syringae. The probe and primer set were able to detect Tox(-) and Tox+ isolates of P. s. pv. Phaseolicola, but no other phytopathogenic bacteria. The assay was also able to detect at least 5 genome equivalents of cloned amplified target DNA, using purified DNA, or 7 colony forming unit (CFU) per reaction when using calibrated cell suspensions. Thus, the TaqMan real-time PCR-based method can be used for the rapid detection of both types of P. s. pv. Phaseolicola, and will potentially simplify and facilitate the diagnosis and monitoring of this pathogen, and guide plant disease management.
Subject(s)
Bacterial Proteins/genetics , DNA Primers/genetics , Fabaceae/microbiology , Polymerase Chain Reaction/methods , Pseudomonas syringae/isolation & purification , Recombinases/genetics , Bacterial Proteins/metabolism , Base Sequence , Ornithine/analogs & derivatives , Ornithine/genetics , Ornithine/metabolism , Plant Diseases/microbiology , Pseudomonas syringae/enzymology , Pseudomonas syringae/genetics , Pseudomonas syringae/metabolism , Recombinases/metabolism , Sensitivity and Specificity , Taq Polymerase/chemistryABSTRACT
To determine the pathogenicity domain and to apply cross protection, Pepper mild mottle virus (PMMoV) point-mutations in the replicase (REP) gene between the methyl-transferase and helicase domains, and deletions truncating pseudoknots in the 3' non-coding region (NCR), were constructed. Some mutants substituting a single amino acid in REP residue 348 exhibited mild symptoms in Nicotiana benthamiana or pepper plants. Accumulation of these mutants was higher than that of other REP mutants or wild-type PMMoV. Deletion mutants in the 3' NCR pseudoknot showed the lowest virus replication and accumulation among the mutants tested. Six attenuated mutants, which combined 3' NCR deletions and single or double REP substitution mutations were constructed to investigate cross protection effects on pepper plants. All six of the attenuated mutants showed milder symptom development than wild-type virus. These results suggest that REP and the pseudoknot in the 3' NCR are major pathogenicity determinants of the virus, and engineered PMMoV attenuated mutants can be useful for protection against the virus in pepper plants.
Subject(s)
Capsicum/virology , Plant Diseases/virology , Tobamovirus/pathogenicity , 3' Untranslated Regions/genetics , Amino Acid Substitution , Mutagenesis , RNA, Untranslated , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics , Nicotiana/virology , Tobamovirus/genetics , Viral Interference , Virulence/genetics , Virus Replication/geneticsABSTRACT
Five isolates of Cucumber mosaic virus (CMV) from Lilium sp. (lily), which were isolated from specimens in Japan, Korea and Taiwan, were unable to support satellite RNA (satRNA) accumulation. In order to map the CMV sequences that are involved in satRNA support, HL-CMV (Japanese lily isolate), Y-CMV (ordinary strain) and Y-satellite RNA (Y-sat) were used as the source material. The pseudorecombinants between Y-CMV and HL-CMV revealed that RNA1 was essential for satRNA replication in lily. The results of chimeric constructs and various mutations showed that two amino acid residues (at positions 876 and 891) in the 1a protein were the determinants for the inability of HL-CMV to support a satRNA. Specifically, Thr at position 876 had a more pronounced effect than Met at position 891. Specific changes in RNA sequence were also detected in the 3' terminus of Y-sat and these particular alterations allowed it to be supported by HL-CMV. It is believed that, through evolution, the adaptation of CMV to lily resulted in the introduction of amino acid changes in the 1a protein, changes that coincidentally affected the ability of lily CMV to support satRNAs.
