Cross amplification of 16S rRNA bacterial primer 27F/1492R on horticultural crop chloroplast genome

Molecular techniques have been applied as fast and accurate detection methods of plant pathogenic bacteria. Bacteria-specific primer 27F paired with the universal primer 1492R was used in PCR to detect plant pathogenic bacteria in symptomatic leaves of tomato (Solanum lycopersicum L.), chili pepper (Capsicum annum L.), napa cabbage (Brassica rapa subsp. pekinensis L.), cabbage (Brassica oleracea var. capitata L.), and longan (Dimocarpus longan Lour.). The targeted single bands with length of 1400 bp were obtained but bidirectional sequencing of the PCR products recovered partial sequence of chloroplast instead of bacterial genomes. Thus, this result confirmed cross amplification of 27F/1492R primer pair against chloroplast genome of plants. Phylogenetic tree was constructed to separate the tested isolates according to their taxonomic relations: Order Sapindales, Solanales, and Brassicales. In-silico analysis on the new five and nineteen selected sequences in NCBI GenBank detected at least seven conserved regions with some single nucleotide polymorphisms. This report might be a cautionary advice for other researchers to avoid false positive results which could lead to misdiagnosis of bacterial plant diseases.


INTRODUCTION
Chloroplast is an important organelle in plant and algal cells where photosynthesis occurred.Just like mitochondria, chloroplast has its own DNA which was proposed in symbiognesis theory to be acquired by early eukaryotic cells from genome of photosynthesis performing endosymbiotic cyanobacterium (Ziehe et al., 2017).
Efforts then developed other primers to minimalized the chloroplast contamination in conventional PCR and also in Next generation sequencing, with different efficiencies (Hanshew et al., 2013;Reigel et al., 2020;Thomas et al., 2020).
Primers 27F (covering 8 -27 Escherichia coli rRNA positions) / 1492R (covering 1492 -1507 E. coli rRNA positions) (Heuer et al., 1997) are among the most commonly used for specieslevel determination due to its ability to amplify the full length of bacterial 16S gene (Frank et al., 2008).The primer pair is also compatible with chloroplast 16S thus prone to mismatch with the gene (Yu et al., 2013).However, this information seems to be not well known among researchers working with molecular identification of phytopathogenic bacteria and might also those with other types of bacteria (Ibrahim et al., 2023).
In this study, sequences of chloroplast 16S gene of five symptomatic plant species collected in Ngablak, Magelang Regency, Indonesia was inadvertently obtained using PCR with 27F/1492R primers during attempts to detect bacterial plant pathogen.Therefore, this report served as a cautionary advice for other researchers to avoid similar mistake that could be costly in financial term, and even may lead to misdiagnosis of plant diseases.Importance of sequencing to confirm PCR results (Pruvost et al., 2022) was also emphasized here.

Fields visit
Horticultural fields in Ngablak Subdistrict, Magelang Regency, Indonesia were surveyed on 16 September 2023 to observe bacterial diseases that may present and restrict production in the region.

DNA extraction and PCR
Total DNA was extracted from the five plant samples using Genomic DNA Mini Kit (Plant) (Geneaid Biotech Ltd., Taiwan) with standard protocols.PCRs were performed with 27F/1492R universal primer pair for amplification of ± 1400 bp bacterial 16S rRNA region (Heuer et al., 1997).PCR mix was each prepared in a volume of 40 μl: 20 μl MyTaq HS Red Mix (Bioline, Germany), 2 μl (10 pmol/μl) each of reverse primer and forward primer, 4 μl of DNA or cDNA, PCR products were loaded into 1% agarose gel stained with Florosafe DNA Staining (1st BASE, Malaysia) then electrophorized for 50 min at 50 V.Gel was then put on a UV transilluminator (Optima Inc., Japan) to observe band with the specific size.PCR products with single targeted band were send to 1st BASE biotechnological company in Malaysia to be sequenced bidirectionally using Sanger method.The recovered sequences were tested against NCBI GenBank database using nucleotide BLAST online software (https://blast.ncbi.nlm.nih.gov).
Nucleotide sequences of the novel isolates were registered to NCBI GenBank to obtain accession numbers.

Phylogeny and polymorphisms studies
Sequences of NCBI GenBank isolates with high percentage identities and coverage of at least 99% to the newly obtained isolates from Ngablak Subdistrict were aligned using ClustalW suits in MEGA X freeware (Kumar et al., 2018), and then trimmed according to the lengths of sequences of new isolates (Praptana et al., 2023).For additional comparison and conserved region analysis, sequences from different organisms including plant pathogenic bacteria, plant growth promoting rhizobacteria (PGPR), microalgae, cyanobacteria, and vascular plant were added to the alignment (Table 1).Construction of phylogenetic tree was conducted by MEGA X using Maximum Likelihood (ML) statistical method with Tamura-Nei parameter model (Tamura and Nei, 1993).Significancy of the constructed branches was statistically tested using 1000 bootstrap replicates.Nucleotide polymorphisms among aligned sequences were observed using BioEdit freeware v.7.2.5 (Hall, 1999;Santosa et al., 2023).
Clear single bands of around 1400 bp in size appeared on agarose after electrophoresis (Figure 1).This initial result indicated presence of bacteria, either plant pathogenic or nonpathogenic, in the tested samples.However, BLAST analysis showed that the recovered nucleotide sequences from those PCR amplification results were actually small subunit ribosomal RNA region of chloroplast of plants.The isolates were then listed in GenBank with acc.nos.OR939686-90.
The currently accepted symbiognesis theory thought that plants obtained their chloroplasts from ancient endosymbiotic cyanobacterium, and thus the nucleotide sequences of their chloroplasts shared high identity with those of bacteria.Despite its versality in amplification of    3).for amplification of bacterial 16S region (Hanshew et al., 2013;Reigel et al., 2020;Thomas et al., 2020).However, the new primers might not be efficient, as also was suggested by our in-silico study that the region amplified by 27F/1492R has only seven conserved regions (Figure 2).This could be one of reasons that 27F/1492R primers are still often used by many researchers to detect and identify bacteria for various purposes (Yuwantiningsih et al., 2015;Indraswari et al., 2021;Rodiansyah et al., 2021), including plant diseases diagnosis (Puspitasari et al., 2021).

CONCLUSIONS
Result of current study cautioned the application of 27F/1492R primers in molecular detection using total DNA extracted directly from plants as it may lead to misdetection of bacteria.
Researchers dealing with plant pathogenic bacteria were advised to obtain DNA from pure bacterial culture when using 27F/1492R.If DNA extraction from plant become essential, such as during dealing with obligate bacteria, it can be suggested to use other primers that are less likely to anneal to chloroplast sequences.Result of this study also strongly underlined the need of sequencing to confirm PCR amplification results.

Figure 2 .
Figure 2. A neighbor joining phylogenetic tree based on 1451 bp of 16S ribosomal RNA gene of plant chloroplasts (blue star) and chromosomal genome (green star) of eukaryote and bacteria.Dot signs highlighted the isolates of five plant species observed in this study, which were grouped into three subclusters according to their respective Order: Sapindales, Solanales, and Brassicales

Figure 3 .
Figure 3. Multiple alignment of five new and 19 partial sequences of 16SR DNA from chromosomal and chloroplast genomes listed in NCBI GenBank.Green boxes indicate the position of 27F and 1492R primer.The red boxes indicate conserved regions which showed relative high similarity among the sequences and few polymorphisms

Table 1 .
List of 16s rDNA and Chloroplast Sequences Used in This Study