Characterization of Phytoplasmas Related to Aster Yellows Group Infecting Annual Plants in Iran, Based on the Studies of 16S rRNA and RP Genes

Several annual field crops, vegetables, ornamentals, oilseed crops, and weeds showing phytoplasma diseases symptoms were collected to detect phytoplasmas related to ‘Candidatus Phytoplasma asteris’. The collecting was done in the central regions of Iran. For general detection of phytoplasmas, 16S rRNA gene fragments were amplified using phytoplasma universal primer pair P1/P7 in polymerase chain reaction (PCR) followed by primer pair R16F2n/R16R2 in nested PCR. Then, for finer detection of phytoplasmas related to ‘Ca. P. asteris’, DNA samples were used to extend the rp and tuf gene fragments by PCR using aster yellows group specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy, respectively. Restriction fragment lenght polymorphism (RFLP) analysis of rp gene fragments using digestion with AluI, MseI, and Tsp509I restriction enzymes indicated that aster yellows group related phytoplasmas in these Iranian regions, belong to rpI-B subgroups. Sequence analysis of partial 16S rRNA and rp genes from representative phytoplasma isolates confirmed the RFLP results. This research is the first report of annual plants infected with phytoplasmas related to subgroup rpI-B in Iran.

First Report of Streptocarpus flower break virus in Streptocarpus in the United States

Streptocarpus flower break virus (SFBV) belongs to the genus Tobamovirus and was described from naturally infected streptocarpus plants in 1995 (2). The complete genomic sequence was recently reported (1). Prominent symptoms include flower breaking while foliar symptoms are often lacking. In March 2007, four streptocarpus plants (cv. Indigo Dream) from San Diego County, CA were tested for the presence of SFBV by ELISA and reverse transcription (RT)-PCR. Symptoms suggestive of a virus infection were not present on these mother plants at the time of sampling. ELISA with SFBV-specific antiserum showed that all samples were infected with SFBV. The ELISA results were verified by RT-PCR followed by cloning and sequencing. Two sets of primer pairs were used in separate RT-PCR tests. One was a degenerate tobamovirus group-specific primer pair and the second primer pair was specific to SFBV (1). The tobamovirus group-specific primer pair consisted of Tob Uni1, 5′-ATT TAA GTG GAG GGA AAA CCA CT-3′ and Tob Uni2, 5′-GTY GTT GAT GAG TTC GTG GA-3′. The SFBV-specific primers were SFBVcpF: 5′-AAA ATG TCG TAC GTG GTG GT and SFBVcpR: 5′-ACC CAC AGA ACT TCC TTC AA-3′ (1). PCR amplicons of the expected size (686 bp for the tobamovirus group-specific primer pair and 562 bp for the SFBV-specific primer pair) were obtained for each primer pair. The positive PCR test using the SFBV-specific primer pair confirmed the presence of SFBV. To further verify the identity of the virus, the amplicons obtained with each primer pair were separately cloned and sequenced. At least two clones for each amplicon were sequenced in both directions. Sequence comparisons with those available in GenBank showed 98% sequence identity with the corresponding genomic region (GenBank Accession No. NC_008365) of SFBV (1). To our knowledge, this is the first report of SFBV in the United States and it highlights the need for testing for this virus to ensure propagation and distribution of virus-free material. References: (1) C. Heinze et al. Arch. Virol. 151:763, 2006. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 101:311, 1995.

Molecular Profiling of the Clostridium leptum Subgroup in Human Fecal Microflora by PCR-Denaturing Gradient Gel Electrophoresis and Clone Library Analysis

ABSTRACT A group-specific PCR-based denaturing gradient gel electrophoresis (DGGE) method was developed and combined with group-specific clone library analysis to investigate the diversity of the Clostridium leptum subgroup in human feces. PCR products (length, 239 bp) were amplified using C. leptum cluster-specific primers and were well separated by DGGE. The DGGE patterns of fecal amplicons from 11 human individuals revealed host-specific profiles; the patterns for fecal samples collected from a child for 3 years demonstrated the structural succession of the population in the first 2 years and its stability in the third year. A clone library was constructed with 100 clones consisting of 1,143-bp inserts of 16S rRNA gene fragments that were amplified from one adult fecal DNA with one forward universal bacterial primer and one reverse group-specific primer. Eighty-six of the clones produced the 239-bp C. leptum cluster-specific amplicons, and the remaining 14 clones did not produce these amplicons but still phylogenetically belong to the subgroup. Sixty-four percent of the clones were related to Faecalibacterium prausnitzii (similarity, 97 to 99%), 6% were related to Subdoligranulum variabile (similarity, ∼99%), 2% were related to butyrate-producing bacterium A2-207 (similarity, 99%), and 28% were not identified at the species level. The identities of most bands in the DGGE profiles for the same adult were determined by comigration analysis with the 86 clones that harbored the 239-bp group-specific fragments. Our results suggest that DGGE combined with clone library analysis is an effective technique for monitoring and analyzing the composition of this important population in the human gut flora.

Analysis of community structures in anaerobic processes using a quantitative real-time PCR method

The methanogenic community structures of four different anaerobic processes were characterized using a quantitative real-time PCR with group-specific primer and probe sets targeting the 16S rRNA gene (rDNA). The group specific primer and probe sets were developed and used to detect the orders Methanosarcinales, and the families Methanosarcinaceae and Methanosaetaceae. Two separate sets targeting the domains Archaea and Bacteria were also used. Each microbial population in different anaerobic processes was determined and the relative abundance in the system was compared with each other. Dominant methanogenic populations and the community structures in the processes were varied by hydraulic retention time and acetate concentration. This indicates that the real-time PCR method with the primer and probe sets is a promising tool to analyze community structures in anaerobic processes.

First Report of Phytoplasma Infection in Hop Plants

Disease symptoms of severe shoot proliferation resembling phytoplasmal disease symptoms were observed in early spring of 2003 in hop (Humulus lupulus L.) plant cvs. Magnum and Marynka that were grown in a commercial farm in Poland. Proliferation of shoots has not been previously reported in hop plants. To detect the presence of phytoplasmas in hops, young shoots from four symptomatic (two cultivars) and two symptomless (‘Magnum’) plants were assayed for phytoplasma 16S rDNA using polymerase chain reaction (PCR). In addition, leaf samples from healthy Catharanthus roseus plants and plants experimentally infected with the reference strains of aster yellows phytoplasma (AY1, group 16SrI-B) or apple proliferation phytoplasma (AP, group 16SrX-A) were included for comparison. Amplifications were performed using the universal phytoplasma primer pair P1/P7 in an initial assay, and universal primer pairs fA/rA (1), Pc399/P1694, or R16F2n/R16R2 (2) and group specific primer pair R16(I)F1/R16(I)R1 (3) in a nested reaction. Specific products were obtained in direct PCR with the universal primer pairs P1/P7 only for the control samples of the reference strains AY and AP. No visible product was amplified by the direct PCR from samples obtained from hops and healthy periwinkle plants. However, in nested PCR with primer pairs P1/P7 followed by primer pairs fA/rA, R16F2n/R16R2, Pc399/P1694, or R16(I)F1/R16(I)R1, specific DNA bands were observed from naturally infected hop plants (both four symptomatic and two symptomless) tested. No amplification products were observed from healthy periwinkle plants. The specificity of PCR products (obtained with universal R16F2n/R16R2 primer pair) was confirmed by restriction fragment length polymorphism (RFLP) analysis using AluI, MseI, HhaI, and RsaI for enzymatic digestion. RFLP patterns of these rDNA fragments for samples of naturally infected hops and for AY1 reference strain were similar and were characteristic of phytoplasma 16SrI-B subgroup. To our knowledge, this is the first evidence that hop shoot proliferation disease is associated with natural infection by phytoplasmas. Furthermore, detection of phytoplasma in asymptomatic hops underscores the need to fully elucidate the etiological role of this pathogen in the disease. References: (1) U. Ahrens and E. Seemüller. Phytopathology 82:828, 1992. (2) I.-M. Lee et al. Phytopathology 83:834, 1993. (3) I.-M. Lee et al. Phytopathology 84:559, 1994.

Rapid PCR-based Detection of Phytoplasmas from Infected Plants

Five simplified DNA preparation procedures for polymerase chain reaction (PCR) amplification were tested for detection of phytoplasmas from infected herbaceous and woody plants. Thin freehand cross-sections made from infected plant tissues and stored in acetone were used as sources for DNA preparation. The tissue sections were treated by: 1) grinding in sodium hydroxide; 2) sonicating in water; 3) microwaving in water; 4) boiling in sodium hydroxide; or 5) placing directly in PCR tube. PCR amplification was performed with a universal phytoplasma-specific primer pair in a reaction buffer containing 0.5% (v/v) Triton X-100, 1.5 mm magnesium chloride, and 10 mm Tris-HCl. All five procedures provided phytoplasmal template DNA for successful PCR amplification from infected herbaceous plants {periwinkle [Catharanthus roseus (L.) G. Don (periwinkle)], carrot (Daucus carota L.), maize (Zea mays L.)}, while the grinding, microwaving, and boiling procedures also allowed positive amplification from a woody plant [green ash (Fraxinus pennsylvanica Marsh.)]. The quality of the resulting DNA was adequate for subsequent identification of the aster yellows and ash yellows phytoplasmas through nested-PCR using phytoplasma group-specific primer pairs. These methods provide remarkable savings in labor and materials, making disease testing and indexing of plant materials much more attractive.