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Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of
Bernd Schneider1, Karen S Gibb1
1Faculty of Science, Northern Territory University, Darwin 0909, Australia.
Abstract:
Primers designed from sequences of the gene encoding the elongation factor Tu (tuf gene) of several culturable mollicutes amplified most of the tuf gene from phytoplasmas of the aster yellows, stolbur and X-disease groups. About 85% of the tuf gene from two aster yellows strains and a tomato stolbur phytoplasma was sequenced. The nucleotide sequence similarity between these related phytoplasmas was between 87.8 and 97.0%, whereas the homology with other mollicutes was 66.3-72.7%. The similarity of the deduced amino acid sequence was significantly higher, ranging from 96.0 to 99.4% among the phytoplasmas and 78.5% to 83.3% between phytoplasmas and the culturable mollicutes examined. From the nucleotide sequences of the phytoplasma strains, two pairs of primers were designed; one amplified the phytoplasmas of most phylogenetic groups that were established, the other was specific for the aster yellows and stolbur groups. The phytoplasmas of the various groups that were amplified could be distinguished by RFLP analysis using Sau3AI, Alul and HpaII. The aster yellows group could be divided into five Sau3AI RFLP groups. These results showed that the tuf gene has the potential to be used to differentiate and classify phytoplasmas. Southern blot analysis revealed that the tuf gene is present as a single copy.
Insights
The tuf gene can differentiate and classify phytoplasmas, which are plant pathogens. This gene
Area of Science:
- Phytopathology and Molecular Biology
- Bacteriology and Genomics
Background:
- Phytoplasmas are obligate intracellular plant pathogens causing significant agricultural losses.
- Accurate identification and classification of phytoplasmas are crucial for disease management.
Purpose of the Study:
- To evaluate the utility of the elongation factor Tu (tuf) gene for phytoplasma differentiation and classification.
- To develop molecular tools for identifying and distinguishing between different phytoplasma groups.
Main Methods:
- PCR amplification of the tuf gene from various phytoplasma strains.
- Nucleotide and amino acid sequence analysis of the amplified tuf gene.
- Restriction Fragment Length Polymorphism (RFLP) analysis using specific restriction enzymes (Sau3AI, Alul, HpaII).
- Southern blot analysis to determine tuf gene copy number.
Main Results:
- Primers targeting the tuf gene successfully amplified target DNA from multiple phytoplasma groups.
- High nucleotide (87.8–97.0%) and amino acid (96.0–99.4%) sequence similarity was observed among related phytoplasmas.
- RFLP analysis using Sau3AI, Alul, and HpaII allowed for the differentiation of phytoplasma groups, with five distinct RFLP types identified within the aster yellows group.
- The tuf gene was confirmed to be a single-copy gene.
Conclusions:
- The tuf gene is a valuable molecular marker for differentiating and classifying phytoplasmas.
- Developed tuf gene-based PCR primers and RFLP methods provide effective tools for phytoplasma identification.
- The findings contribute to a better understanding of phytoplasma diversity and evolution.