Intra and extragenomic variation between 16S rRNA genes found in 16SrI‐B‐related phytopathogenic phytoplasma strains

Abstract

Phytoplasmas are bacterial pathogens that pose a threat to cereal and vegetable production worldwide. However, there is limited information regarding the occurrences of phytoplasmas in Central Europe. In this study we examined samples taken from crop and ornamental plants showing symptoms indicative of phytoplasma infection. Phytoplasmas related to 'Candidatus Phytoplasma asteris' were detected in eight plant species (carrot, lupine, pea, cauliflower, mustard, maize, juniper and marigold). Based on the restriction fragment length polymorphism analysis of the 16S rDNA, the phytoplasmas were classified as subgroup 16SrI-B, 16SrI-L or a complex strain, which harboured the I-B and I-L-type 16S rRNA genes. Individual 16S rDNA sequences were analysed from those heterogeneous strains and as many as five genetic variants were distinguished. By operon-specific amplification of the 16S rRNA genes with primers complementary to RP166 phytoplasma genome, we detected 16SrI-L/B phytoplasma strains in the multivariant samples. The I-L-type sequences were amplified from rrnA and the I-B-type sequences were amplified from rrnB.Molecular phylogeny based on the 16S rRNA showed that all the detected phytoplasma variants were clustered with a 'Ca. Phytoplasma asteris' subclade and were closely related to the 16SrI-B subgroup. Despite the variability of 16S rDNA, the strains from all the eight crops shared identical tuf gene sequences. We report, for the first time, 16SrI-B phytoplasma infecting lupine, field pea, cauliflower and mustard, 16SrI-L phytoplasma infecting maize and field pea and 16SrI-L/B phytoplasma infecting carrot, lupine and marigold. We further demonstrate variability in the 16S rRNA genes found in the 16SrI-B-related phytoplasma genomes worldwide. For clearer and more unambiguous nomenclature, we propose that in the future, the more intuitive subgroup 16SrI-L/B be used for heterogenic phytoplasma strains instead of 16SrI-(B/L)L.

Limited variation in microbial communities across populations ofMacrostelesleafhoppers (Hemiptera: Cicadellidae)

“…The plant-pathogenic bacterium Phytoplasma was detected in 57 individuals, but was generally found at relatively low abundance: in one individual, it was 44.8% and in a few others ∼10%, but typically, it was well below 1% (Figure 4a, Supplementary table S5-6). This large variation among individual insects in Phytoplasma relative abundance may explain the limited overlap between its detection based on amplicons as opposed to diagnostic nested PCR (Zwolińska and Borodynko□Filas, 2021). When looking at the 156 M. laevis individuals from Szczecinek and Sośnicowice (Poland) that were pre-selected for microbiome characterization based on the results of Phytoplasma screen, we found a discrepancy between Phytoplasma detection based on nested PCR and amplicon data (Figure 4A).…”

“…For further steps, the manufacturer protocol was followed. For amplicon sequencing, we selected samples that tested positive and negative for Phytoplasma infections in the earlier nested-PCR screen, which followed the standard method for phytoplasma detection as previously described (Zwolińska and Borodynko□Filas, 2021). Our goal was to include 20 samples from each of the two categories — positive and negative — for each site-date combination, although there were instances where lower numbers were available.…”

Identification of phytoplasmas in stone fruit (Prunus sp.) and persimmon (Diospyros kaki L.) trees exhibiting leaf alterations and witches'‐broom in Jordan

“…16S rRNA‐encoding gene amplicon‐derived chromatograms showed no evidence of double peaks, indicating the absence of intra‐genomic heterogeneity or mixed infections (Zwolińska & Borodynko‐Filas, 2021). According to 16S rRNA gene sequence identity versus the reference strains of ‘ Ca .…”

Molecular detection and identification of phytoplasmas associated with Catharanthus roseus, Pinus eldarica, and Petunia hybrida in southeastern Iran's urban green spaces

“…Inter‐ and intragenomic 16S rRNA gene variability in bacterial populations, including phytoplasmas with two rRNA operon copies, causes prokaryotic diversity (Johnson et al, 2019; Sun et al, 2013; Zwolińska & Borodynko‐Filas, 2021). Differences between phytoplasmas have been found in just one of the two 16S rRNA genes in some cases (Lee et al, 1998).…”