Genetic diversity of Candidatus phytoplasma aurantifolia using single-strand conformational polymorphism analysis and sequence analyses of the groupII intron gene

Background and Aim: Forty-one citrus trees infected by ‘Candidatus Phytoplasma aurantifolia’ were examined using single-strand conformation polymorphism (SSCP) and sequence analysis of a variable groupII intron gene fragment. SCP analysis of polymerase chain reaction-amplified groupII intron gene fragments revealed diverse profiles, differing in number and position of the bands. PCR fragments were sequenced to corroborate SSCP results. The phylogenetic tree derived from the nucleic acid sequences revealed three different sequevar and agreed with the SSCP typing. The polymorphism of the groupII intron fragment predestine this gene as a molecular marker for tracking strains in epidemiological studies Methods: Plant material Leaf midribs from ۴۱ phytoplasma-infected (Candidatus phytoplasma aurantifolia) citrus plants grown in commercial orchards or private gardens in Hormozgan and Kerman Provinces, were sampled and lyophilized. Table ۱ Candidatus phytoplasma aurantifolia Phytoplasmas isolates used in this study Isolate code Host plant genus/species common name Region Elevation GenBank accession numbers ۱۶S-۲۳SrDNA BWB۱ Citrus hybrid Bakraee Hormozgan ۱۱۷۶ MK۴۴۳۳۱۸ MK۴۴۳۳۱۹ MK۴۴۳۳۲۲ MK۴۴۳۳۲۳ MK۴۴۳۳۲۴ MK۴۴۳۳۲۵ MK۴۴۳۳۲۶ MK۴۴۳۳۲۷ MK۴۴۳۳۲۸ MK۴۴۳۳۲۹ MK۴۴۳۳۳۰ MK۴۴۳۳۳۱ MK۴۴۳۳۳۲ MK۴۴۳۳۳۳ MK۴۴۳۳۳۴ MK۴۴۳۳۳۵ MK۴۴۳۳۳۶ MK۴۴۳۳۳۷ MK۴۴۳۳۳۸ MK۴۴۳۳۳۹ MK۴۴۳۳۴۰ MK۴۴۳۳۴۱ MK۴۴۳۳۴۲ MK۴۴۳۳۴۳ MK۴۴۳۳۴۴ MK۴۴۳۳۴۵ MK۴۴۳۳۴۶ MK۴۴۳۳۴۷ MK۴۴۳۳۴۸ MK۴۴۳۳۴۹ MK۴۴۳۳۵۰ MK۴۴۳۳۵۲ MK۴۴۳۳۵۳ MK۴۴۳۳۵۵ MK۴۴۳۳۵۶ MK۴۴۳۳۵۷ MK۴۴۳۳۵۸ MK۴۴۳۳۵۹ MK۴۴۳۳۶۰ MK۴۴۳۳۶۱ MK۴۴۳۳۶۲ BWB۲ Citrus hybrid Bakraee Hormozgan ۱۱۷۶ DA۱ Citrus aurantifolia Mexican lime Hormozgan ۱۸۱ DA۲ Citrus aurantifolia Mexican lime Hormozgan ۱۶۹ DE۱ Citrus aurantifolia Mexican lime Kerman ۸۲۳ DE۳ Citrus aurantifolia Mexican lime Kerman ۷۹۰ DE۵ Citrus aurantifolia Mexican lime Kerman ۷۹۰ DE۷ Citrus aurantifolia Mexican lime Kerman ۷۹۰ DE۸ Citrus aurantifolia Mexican lime Kerman ۷۹۱ FA۱ Citrus aurantifolia Mexican lime Hormozgan ۳۳۵ FA۳ Citrus aurantifolia Mexican lime Hormozgan ۳۳۲ FA۶ Citrus aurantifolia Mexican lime Hormozgan ۳۳۰ FA۷ Citrus aurantifolia Mexican lime Hormozgan ۳۱۷ FA۸ Citrus aurantifolia Mexican lime Hormozgan ۳۱۹ GWB۱ Citrus paradisi Grapefruit Hormozgan ۲۱۲ GWB۴ Citrus paradisi Grapefruit Hormozgan ۲۱۲ HA۶ Citrus aurantifolia Mexican lime Hormozgan ۲۲۸ HA۷ Citrus aurantifolia Mexican lime Hormozgan ۲۳۴ JD۲ Citrus aurantifolia Mexican lime Kerman ۶۴۹ JI۱ Citrus aurantifolia Mexican lime Kerman ۶۴۳ JI۲ Citrus aurantifolia Mexican lime Kerman ۶۳۸ KAH۱ Citrus aurantifolia Mexican lime Kerman ۵۲۳ KAH۲ Citrus aurantifolia Mexican lime Kerman ۵۲۳ KAH۳ Citrus aurantifolia Mexican lime Kerman ۵۲۴ ME۱ Citrus aurantifolia Mexican lime Kerman ۳۶۱ ME۲ Citrus aurantifolia Mexican lime Kerman ۳۴۹ ME۳ Citrus aurantifolia Mexican lime Kerman ۳۵۶ ME۴ Citrus aurantifolia Mexican lime Hormozgan ۳۶۰ MIN۱ Citrus aurantifolia Mexican lime Hormozgan ۲۱ MIN۳ Citrus aurantifolia Mexican lime Hormozgan ۲۱ NO۱ Citrus aurantifolia Mexican lime Kerman ۴۷۴ RO۷ Citrus aurantifolia Mexican lime Hormozgan ۲۶۹ RO۸ Citrus aurantifolia Mexican lime Hormozgan ۲۶۷ RO۱۴ Citrus aurantifolia Mexican lime Hormozgan ۱۹۲ RO۱۷ Citrus aurantifolia Mexican lime Hormozgan ۲۴۹ RO۱۸ Citrus aurantifolia Mexican lime Hormozgan ۲۶۱ RO۱۹ Citrus aurantifolia Mexican lime Hormozgan ۲۰۹ SA۱ Citrus aurantifolia Mexican lime Hormozgan ۱۸۲ SA۲ Citrus aurantifolia Mexican lime Hormozgan ۲۶۰ TO۱ Citrus aurantifolia Mexican lime Hormozgan ۲۹۷ ZR۱ Citrus aurantifolia Mexican lime Hormozgan ۹۹ Total DNA extraction and polymerase chain reaction The total genomic DNA extraction was performed following the protocol described by Murray and Thompson (۱۹۸۰). The DNA extracted were subjected to PCR with phytoplasma universal primer pairs P۱A and P۷A (Lee et al., ۲۰۰۴) to amplify A major portion of ribosomal DNA. The following primers (Siampour et al., ۲۰۱۵) were used were used to amplify a ~۹۰۰-bp fragment of retroelement gene homologues from all phytoplasma strains: IntF۱ (۵’-ATAACACGTTGAAGAATCGCT -۳’) / IntR۱ (۵’-TATACGAGTTTTATTGTGGATTC -۳’). Amplifications of Phytoplasma ribosomal DNA and retroelements were performed with PCR conditions as described (Lee et al., ۲۰۰۴, Siampour et al., ۲۰۱۵) Single-strand conformation polymorphism (SSCP) and gel analysis The SSCP analysis of PCR product from retroelement gene was carried out following the protocol described in more details elsewhere (Music´ & Škoric´, ۲۰۱۳). Samples were separated on ۸% acrylamide-bisacrylamide non denaturing gels and electrophoresed in ۱× TBE buffer at ۲۰۰ V for ۴ h at ۴℃. The SSCP profiles were visualized by ethidium bromide staining (Yap & McGee, ۱۹۹۲). Sequence analysis Phytoplasma identification was performed by sequence analysis of ribosomal DNA (۱۶SrDNA) amplicons using the primers P۱A/P۷A. nucleotide sequence data of ۱۶SrDNA and retroelement gene were submitted to the GenBank (Table ۱). The sequence data of retroelement gene fragment has been submitted to the GeneBank database with accession numbers MH۶۲۲۸۷۰ (FA۱), MH۶۲۲۹۰۳ (ZR۱), MH۶۲۲۸۷۷ (HA۶), MH۶۲۲۸۷۹ (JD۲), MH۶۲۲۸۸۵ (ME۱), MH۶۲۲۸۸۳ (KAH۲), MH۶۲۲۸۶۷ (DE۵) and MH۶۲۲۸۸۹ (MIN۱).Results: A total of ۴۱ field sites were analyzed in this study, ۲۶ from the Hormozgan province and ۱۵ field from Kerman province. The P۱A/P۷A amplicons were sequenced and species identified by comparison with ۱۶S rDNA sequences of reference phytoplasmas in GenBank/EMBL databases. The analysis confirmed that citrus trees were infected by ‘Ca. P. aurantifolia’ (Table ۱). SSCP analysis was performed on all amplified phytoplasma retroelement fragments. The SSCP analysis of the citrus samples yielded three different banding patterns. This analysis showed that each infected sample carries mainly one sequence sequevar of Pat. Examples of the various profile Fig. ۱ Three different SSCP banding patterns obtained from analysis of ۹۰۰ bp PCR products from the retroelement genes of ۴۱ Pat are depicted in Fig. ۱. The single stranded DNA molecules typically formed ۲ to ۳ bands after denaturation in formamide buffer. The PCR products from the selected representative samples showing different SSCP profiles were sequenced on both strands using the same primers employed for their amplification with direct Sanger sequencing by Microsynth, Switzerland. Sequencing of retroelement area (groupII intron reverse transcriptase/mature) allowed the assembly of ۸۸۹ nucleotides fragment, including the partial N-terminal reverse transcriptase (RT) domain. Multiple alignments of chosen sequenced fragments showed that the ones having the same SSCP profiles also showed sequence variation and three sequevars (seq) including: seq۱ (FA۱, ZR۱, HA۶, JD۲, ME۱ and MIN۱), seq۲ (DE۵) and seq۳ (KAH۲) were detected among phytoplasmas infecting citrus host.The nucleic acid homology amongst the ‘Ca. P. aurantifolia strain. ranged between ۹۶.۱-۱۰۰%. In a multiple alignment of the protein sequences (Fig. ۲) substitutions occurred in less than ۴ positions (Fig. ۲) in sequevar۳ (KAH۲). Phylogenetic analyses (Fig. ۳) of phytoplasma sequences obtained in this study corroborated the SSCP results. Phylogeny based on groupII intron reverse transcriptase/matures distinguished four separate clusters supported by high bootstrap value and revealing much higher diversity among Iranian strains. The nucleotide sequences of fragments having similar SSCP profiles clustered together on the phylogenetic tree, indicating their close relatedness. Fig. ۲ Sequence alignment and characteristics of group II intron reverse transcriptase/maturase proteins C. P. aurantifolia. Deduced amino acid sequence highlited in gray and are underlined Fig. ۳ Phylogenetic trees for the representative phytoplasma retroelement (groupII intron reverse transcriptase/maturase) sequences. The trees were constructed by the UPGMA method with the Tamura ۳-parameter method by using the MEGA v۷ program package. Bootstrap values are given above the branches. Trees for the were rooted by using corresponding Peanut withes’ broom, Vaccinium withes’ broom phytoplasma and Bacillus cereus sequences as an outgroup.Conclusion: The SSCP analysis results of the phytoplasma retroelement genes revealed the presence of molecular variability among Iranian C. P. aurantifolia strains. Based on the present study, molecular markers present in the ۱۶S-۲۳S rDNA locus showed (data not shown)close genetic relationship between citrus phytoplasma strains which is in agreement with previous study indicated the existence of a limited variation among ‘Ca. P. aurantifolia’ strains based on the ۱۶S rRNA gene.(Al-Ghaithi et al., ۲۰۱۸). Presence of SNPs in groupII intron reverse transcriptase/mature genes allowed grouping of the ‘Ca. P. aurantifolia’ into three sequevar, indicated that the groupII intron reverse transcriptase genes will greatly benefit as a molecular marker to identify strains and track their spread in the field. On other hand SSCP The technique provides an inexpensive, convenient, and sensitive method for determining sequence variation and to differentiate phytoplasma strains when dealing with a large number of field samples. Thus, SSCP analysis can also be considered as a successful fingerprinting tool in the analysis of phytoplasma genes.

Keywords:

Lime , Candidatus Phytoplasma aurantifolia , Diversity , SSCP