Maize bushy stunt phytoplasma affects native corn at high elevations in Southeast Mexico

Abstract

In the 2013-2014 growing season, field surveys were conducted in native corn fields located in high altitude agricultural communities in the 'Sierra Norte de Puebla' in Mexico. Symptoms typical of maize bushy stunt (MBS) disease were observed and DNA extracted from symptomatic native corn plants was used as template to confirm the presence of phytoplasmas.Amplification and sequencing of 16S rRNA-encoding sequences and chaperonin 60 universal target (cpn60 UT) sequences followed by in vitro restriction fragment length polymorphism and phylogenetic analyses revealed that the phytoplasma detected belongs to the subgroup 16SrI-B, 'Candidatus Phytoplasma asteris'. Based on 16S rRNA-encoding gene sequence analysis and on a single nucleotide polymorphism within the cpn60 UT sequence, two MBS strains, MBS-Puebla and MBS-Veracruz, were identified. This is the first detection of MBS phytoplasma (MBSP) affecting native corn and the first molecular survey made in corn fields in Mexico to detect and characterize MBSP. We discuss these results in light of the potential evolutionary relationship between corn and MBSP.

Phytoplasma classification and phylogeny based on in silico and in vitro RFLP analysis of cpn60 universal target sequences

“…The tree topology corresponded with the topology previously obtained by Wei and colleagues in 2007 using 16S rRNA gene sequences (Wei et al , 2007). This result confirms the ability of cpn60 UT sequences to identify phytoplasmas through cladistics analysis, as previously suggested (Dumonceaux et al , 2014; Pérez-López et al , 2016b). …”

“…So far, phytoplasma cpn60 sequences have been reported from members of the groups 16SrI, 16SrII, 16SrV, 16SrVII, 16SrIX, 16SrX, 16SrXII, 16SrXIII and 16SrXIV (Dumonceaux et al , 2014; Pérez-López et al , 2016b). Altogether, after trimming the cpn60 UT sequence from the five completely sequenced phytoplasma genomes (Andersen et al , 2013; Bai et al , 2006; Kube et al , 2008; Oshima et al , 2013; Tran-Nguyen et al , 2008), from the draft genome belonging to the group 16SrII-A, strain PnWB (Chung et al , 2013) and 16SrIX-B strain SA213 (Quaglino et al , 2015), from the cpn60 sequences reported by Mitrović et al (2011) for members of the group 16SrI, and members of the group 16SrXIV (Mitrović et al , 2015), from the 3.6 kb DNA fragments obtained by Kakizawa et al (2006), and the sequences previously obtained by our group, we had 96 cpn60 UT sequences in this study.…”

“…However, draft genomes for phytoplasma strains from the 16SrIII group suggest that this subgroup may lack this gene (Saccardo et al , 2012), which would limit the utility of cpn60 -based classification tools for this subgroup. Nevertheless, the recent development of methods to access cpn60 UT sequences from phytoplasmas (Dumonceaux et al , 2014), has enabled the use of these sequences to develop diagnostic methods, and facilitates phytoplasma characterization based on polymorphisms detected among the different phytoplasma groups and subgroups (Dumonceaux et al , 2014; Pérez-López et al , 2016b). This primer cocktail has been shown to amplify the cpn60 UT from a diverse array of phytoplasmas (sharing as little as 61 % identity at the nucleotide level) from the major groups of phytoplasmas (Chung et al , 2013; Dumonceaux et al , 2014), although it is acknowledged that this amplification strategy may need to be modified as new sequences accrue, particularly from genomic sequencing efforts.…”

First report of ‘CandidatusLiberibacter asiaticus’ affecting sour orange in urban areas of Mayabeque, Cuba

“…L. asiaticus’ (CLas) genome (Duan et al 2009), diversity studies of CLas have been restricted to the 16S-23S rRNA intergenic spacer region, the omp gene, the β -operon sequence, or bacteriophage-type DNA polymerase region (DNA pol) (Subandiyah et al 2000; Bastianel et al 2005; Ding et al 2009; Furuya et al 2010). The cpn60 universal target ( cpn60 UT) (Goh et al 1996), a fragment of approximately 550 bp, has been extensively used in the study of microbial communities, in the development of molecular diagnostic methods, and has been demonstrated to be a suitable molecular barcode for the domain Bacteria (Links et al 2012; Pérez-López et al 2016b). Partial cpn60 gene sequences (500 to 550 bp) have been also useful to identify new species, and recently identified as an additional marker to improve the differentiation of plant pathogens within the genera ‘ Candidatus Phytoplasma’ (Pérez-López et al 2016a) and Xanthomonas spp.…”

Periwinkle proliferation disease associated with 16SrI-B phytoplasma in Mexico

“…Collected samples were placed in plastic bags in a cooler during transportation to the lab and stored at 4°C until DNA extraction. DNA was extracted from 0.3 g of leaf midribs using a CTAB-based method (Pérez-López et al 2016b).…”