Nuclear ribosomal DNA secondary structures and statistical approach for the phylogeny of Ampelomyces

Nuclear ribosomal DNA secondary structures and statistical approach for the phylogeny of Ampelomyces

Fungi belonging to the genus Ampelomyces are intracellular mycoparasites of the Erysiphales, which cause powdery mildew diseases in economically important crops. As Ampelomyces spp. are parasites of other parasites, they are named hyperparasites. Several Ampelomyces-based biocontrol agents have been developed but its effectivity against different powdery mildew fungi can vary, causing to still rely on the use of harmful pesticides. Accurate identification of these hyperparasites is essential to discovery newly highly virulent isolates for using in plant protection. In addition, further research is needed in Ampelomyces to understand, for example, their underlying mechanisms of virulence.

One of the problems in studying Ampelomyces is their unresolved taxonomy, which has been mainly determined by phylogenetic analyses based on the nuclear ribosomal DNA internal transcribed spacer region (nrDNA ITS). The ITS region of the 18S-28S nuclear ribosomal DNA is the preferred fungal barcode and it consists of the ITS spacers 1 (ITS1) and 2 (ITS2), and the 5.8S gene.

Historically, Ampelomyces were confused with other closely related pycnidial fungi. Today, many nucleotide sequences are deposited in the GenBank database under the generic name of Ampelomyces, but it is unknown if these sequences belong to the type specimen of Ampelomyces, Ampelomyces quisqualis Ces.. As the hyperparasites asexual structures vary in dimensions and apparently are not linked to a particular mycoparasitic lineage, their identification rely on the use of molecular barcodes. At least four distinct lineages have been identified based on the ITS sequences and actin 1 gene fragments (actin1). These DNA markers are insufficient for species delimitation in this genus, and thus, other identifiers are sought to assist in this purpose.

This PhD thesis presents a comprehensive research and analysis conducted to optimise Ampelomyces identification. As the early works are showing the ITS spacers and secondary structures (S2s) are important molecular tools in phylogenetic studies. This PhD thesis has conducted an in-depth evaluation of the utility of ITS S2s in Ampelomyces phylogeny.

The results of this thesis showed that: 1) putative Ampelomyces ITS sequences derived from environmental DNA (eDNA) and deposited in the GenBank do not belong to this genus. Statistical tests revealed that there were significant differences in the sequence lengths of ITS region as well as A/T nucleotide content values between the 'true' Ampelomyces spp. and those from putative Ampelomyces; 2) nrDNA ITS S2s significantly improves likelihood-based estimates of phylogeny; 3) the statistical test, Kruskal-Wallis, supports that the ITS spacers are under functional constraints of their S2s and indicating are not evolving neutrally as usually assumed for the nrDNA gene repeats; 4) variations in the conserved ITS2proximal stem of two Ampelomyces ITS sequences reveals the presence of pseudogenes; 5) the simultaneous alignment of ITS spacers and S2s reflects the Ampelomyces phylogeny and reveals strong phylogenetic signal in structure variation across lineages; and 6) DNA repeats of Ampelomyces ITS1 sequences were different from those of putative Ampelomyces. Altogether, these findings are important for resolving the taxonomic classification of Ampelomyces and understanding their evolutionary history.

This computational work has also resolved (1) the previous difficulties in selecting a new DNA barcode with more phylogenetic resolution power than that of the ITS by using nrDNA ITS sequences and S2s based phylogeny and (2) some of the problems associated with selecting the 'true' Ampelomyces sequences without collecting and sequencing all previous ITS sequences deposited in the GenBank database. These findings will lead to accurately identify fungi in the future, improve DNA sequence data in databases as well as resolve the misidentification of Ampelomyces spp. sensu stricto.

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