The role of internal transcribed spacer 2 secondary structures in classifying mycoparasitic Ampelomyces

Abstract

Many fungi require specific growth conditions before they can be identified. Direct environmental DNA sequencing is advantageous, although for some taxa, specific primers need to be used for successful amplification of molecular markers. The internal transcribed spacer region is the preferred DNA barcode for fungi. However, inter- and intra-specific distances in ITS sequences highly vary among some fungal groups; consequently, it is not a solely reliable tool for species delineation. Ampelomyces, mycoparasites of the fungal phytopathogen order Erysiphales, can have ITS genetic differences up to 15%; this may lead to misidentification with other closely related unknown fungi. Indeed, Ampelomyces were initially misidentified as other pycnidial mycoparasites, but subsequent research showed that they differ in pycnidia morphology and culture characteristics. We investigated whether the ITS2 nucleotide content and secondary structure was different between Ampelomyces ITS2 sequences and those unrelated to this genus. To this end, we retrieved all ITS sequences referred to as Ampelomyces from the GenBank database. This analysis revealed that fungal ITS environmental DNA sequences are still being deposited in the database under the name Ampelomyces, but they do not belong to this genus. We also detected variations in the conserved hybridization model of the ITS2 proximal 5.8S and 28S stem from two Ampelomyces strains. Moreover, we suggested for the first time that pseudogenes form in the ITS region of this mycoparasite. A phylogenetic analysis based on ITS2 sequences-structures grouped the environmental sequences of putative Ampelomyces into a different clade from the Ampelomyces-containing clades. Indeed, when conducting ITS2 analysis, resolution of genetic distances between Ampelomyces and those putative Ampelomyces improved. Each clade represented a distinct consensus ITS2 S2, which suggested that different pre-ribosomal RNA (pre-rRNA) processes occur across different lineages. This study recommends the use of ITS2 S2s as an important tool to analyse environmental sequencing and unveiling the underlying evolutionary processes.

Fig 1. Normalized nrDNA ITS sequence lengths from environmental fungi are similar to those from the outgroup.

The graph shows the normalized sequence length (%) of the ITS region from Ampelomyces strains (n = 376), putative Ampelomyces Groups 1 (n = 5) and 2 (n = 5) as well as the outgroup (n = 5). The normalized ITS sequence length of the Ampelomyces strains was significantly higher than those from putative Ampelomyces Groups 1 and 2 as well as from fungi belonging to the outgroup taxa, with a Kruskal-Wallis chi-squared statistic value of 53.27, a p-value of 1.59e-11 and 3 degrees of freedom. The error bars indicate the S.E.M. and the * indicates the statistical significance at the 5% confidence level.

Fig 2. Differences in ITS nucleotide contents help in distinguishing putative Ampelomyces from the ‘true’ Ampelomyces.

(A) The A/T and (B) C/G content values around the median for ITS sequences from Ampelomyces spp. (1; n = 376), putative Ampelomyces Groups 1 (2; n = 5) and 2 (3; n = 5) as well as the outgroup (4; n = 5). For each box plot, the central line as well as the top and bottom of each box represent the median, the third and first quartile, respectively. The whiskers indicate the maximum and minimum values, while circles above each box plot represent the outliers of each dataset. The statistical significance of the results was assessed using the Kruskal-Wallis test with Dunn’s post-hoc test for multiple comparisons (p-values< 0.05) with 3 degrees of freedom. The differences were statistically significant at the 5% confidence level.

Fig 3. Two variations in the hybridization model of the proximal stem region were found in Ampelomyces.

(A) The typical 5.8S and 28S hybridization model was obtained from an ITS2 sequence extracted from Ampelomyces conidia infecting P. xanthii. (B) A variation in the 5.8S and 28S hybridization model detected in Ampelomyces sp. infecting P. xanthii. (C) Another variation in the 5.8S and 28S hybridization model obtained from Ampelomyces sp. infecting E. necator (Section Uncinula) chasmothecia. The blue nucleotides denote the 5.8S strand, the red nucleotides indicate the 28S strand and the black nucleotides comprise the flanking regions of the ITS2 spacer. The formation of an internal loop is indicated by the two black arrows. The typical free nucleotides found in the 5.8S and 28S strands are indicated with blue and red arrows, respectively. The GenBank accession number is shown below each structure. The Gibbs free energy (dG) and enthalpy (dH) values are shown after the taxon name.

Fig 4. Common features of the ITS2 S2 from Ampelomyces.

(A) The ITS2 S2 from Ampelomyces extracted from seven PM genera. The ITS2 contained a core structure with four helices (I–IV), a U-U mismatch on helix II and a large helix III containing a UGG motif. (B) This ITS2 S2 was found in Ampelomyces sampled in China (GI: DQ490752). The UGGU and UU motifs were located in helix III. The minimum free energy structures were calculated with RNAfold and visualized using a force directed graph layout with the Vienna RNA Web Services [64]. The helices are indicated with Roman numerals (I–IV). The U-U mismatch on helix II and the UGG, UGGU and UU motifs are indicated with arrows.

Fig 5. The most common ITS2 S2 (Model 1) found in the Ampelomyces population.

(A) The ITS2 S2 Model 1 derived from Ampelomyces extracted from seven PM genera. (B) The ITS2 S2 Model 7 derived from Ampelomyces extracted from A. mougeotii. Compared to Model 1, Model 7 contains several nucleotide substitutions. Specifically, nucleotides cytosine and uracil in helices III and IV, respectively of Model 1 are present as uracil and thymine in the helices III and IV, respectively of Model 7; (red arrows indicate nucleotide substitutions). The helices are indicated by Roman numerals (I–IV). The following shared ITS2 features are shown in black arrows and circles: (1) a U-U mismatch on helix II and (2) the UGG motif on helix III. A single-stranded ring between helices I and IV is indicated with red left right arrows. It comprises the sequence GTACCC or *UACCC when T is substituted with U. The structures were directly folded and modelled using the ITS2-DB.

Fig 6. The second most common ITS2 S2 (Model 2) found in the Ampelomyces population.

(A) The ITS2 S2 Model 2 derived from mycoparasites extracted from the three PM genera Arthrocladiella, Golovinomyces and Podosphaera. (B) The ITS2 S2 Model 3 was only found in one ITS2 sequence (GenBank accession number DQ490754) from Ampelomyces sp. extracted from P. fusca. The helices are indicated by Roman numerals (I–IV). Single-stranded rings (between helices I and IV) for Models 2 and 3 with the sequence GTGTCC are indicated with blue left right arrows. The following shared ITS2 features are indicated by black arrows and circles: (1) a U-U mismatch on helix II and (2) the UGGU and UU motifs on helix III. The structures were directly folded and modelled with the ITS2-DB.

Fig 7. Predicted ITS2 S2 found in the Ampelomyces population isolated from two PM genera, Podosphaera and Erysiphe.

(A) The ITS2 S2 Model 4 predicted from Ampelomyces spp. strains extracted from P. leucotricha on Malus domestica. (B) The ITS2 S2 Model 5 from P. ferruginea on Sanguisorba officinalis. (C) The ITS2 S2 Model 6 from E. necator infecting Vitis sp.. M. domestica and S. officinalis are plants from the family Rosaceae, whereas Vitis sp. is a plant from the family Vitaceae. The U-U mismatch motifs, a single-stranded ring rich in adenine nucleotides between helices II and III and a large helix III with a UGGU motif are indicated with black arrows and circles. The structures were directly folded and modelled with the ITS2-DB. The helices are indicated with Roman numerals (I–IV). The single-stranded ring between helices I and IV is indicated with green left right arrows. However, the one from Model 4 is not complete and the green arrow is not shown.

Fig 8. Predicted putative Ampelomyces ITS2 S2s were modelled to those from Didymella, Epicoccum and Phoma.

(A) The ITS2 S2 from A. quercinus was homology modelled based on the ITS2 S2 from D. pomorum (FJ839851). (B) The ITS2 S2 from A. humuli were modelled according to the ITS2 S2 from Epiccocum sp. (GU973791) and (C) from the ITS2 S2 from D. glomerata (FJ839851). (D) The ITS2 S2 from putative Ampelomyces sp. (Group 1) was homology modelled based on the structure of P. labilis (GU237868). (E) The ITS2 S2 from putative Ampelomyces sp. (Group 2) extracted from crosstie waste was directly folded and modelled. The helices are indicated with Roman numerals (I–IV). The single-stranded ring between helices I and IV is indicated by orange left right arrows. The U-U mismatch on helix II is indicated with red arrows and circles. The S2s were obtained with the ITS2-DB.

Fig 9. Phylogram based on ITS2 S2s enhance the discrimination between fungal DNA environmental samples and the ‘true’ Ampelomyces.

(A) The phylogram with the highest log likelihood (-866.33) was based on the ITS2 S2s and inferred via the maximum likelihood (ML) method and the Kimura two-parameter model. An evolutionary rate among sites was modelled with a discrete gamma distribution (+G) parameter = 0.99. (B) The phylogenetic tree with the highest log likelihood (-2040.24) was based on the ITS region and estimated with the maximum likelihood method and the Tamura-Nei model with a discrete gamma distribution (+G) parameter = 0.48. The ML bootstrap values >60% are indicated over the branches and are expressed as percentages. The scale bar represents the nucleotide substitutions per site. The tree was edited with FigTree v1.4.4 software. The GenBank accession numbers are indicated before the taxa names. Abbreviations: ITS2 S2 variations of main Models 1 (M1, M1-2 and M1-3); Models 2 (M2, M2-1, M2-2 and M2-3); Model 3 (M3); Model 4 (M4); Model 5 (M5); Models 6 (M6-1 and M6-2); and Model 7 (M7).

Fig 10. Phylogram based on 26 Ampelomyces ITS2 S2s shows that the S2s are not associated with PM hosts.

The phylogram with the highest log likelihood (-829.62) is shown based on the Kimura two-parameter DNA model and an evolutionary rate among sites modelled with a discrete gamma distribution (+G) parameter = 0.90. The ML bootstrap values >60% are indicated over the branches and are expressed as percentages. The scale bar represents the nucleotide substitutions per site. The tree was edited with FigTree v1.4.4 software. The GenBank accession numbers are indicated before the taxa names. Abbreviations: ITS2 S2 variations of main Models 1 (M1, and M1-3); Models 2 (M2, M2-1 and M2-3); Model 3 (M3); Model 4 (M4); Model 5 (M5); Models 6 (M6-1 and M6-2); and Model 7 (M7).

Fig 11. Four major consensus ITS2 S2s from Ampelomyces are represented in three main clades.

(A) Consensus ITS2 S2 of Models 2 and 3, which are grouped in clade 1a. (B) Consensus ITS2 S2 belonging to clade 1b are represented by Model 6. (C) Consensus ITS2 S2 of Models 4 and 5 that belong to clade 2. (D) Consensus ITS2 S2 of Models 1 and 7 that belong to clade 3. All consensus ITS2 S2s have gaps and were obtained with the 4SALE v1.7.1 software. Yellow nucleotides indicate conserved motifs (U-U mismatch and AAA motifs) known to be found in yeasts and vertebrates [73]. UGG, UGGU and GGUU motifs on helix III are indicated with black circles. The bar on the upper right side indicates the level of nucleotide conservation, with the most conserved nucleotides in green. The gaps are depicted in red. The GenBank IDs used to build each consensus are indicated below each S2.

Fig 12. Highly diverse consensus ITS2 S2s from the outgroup taxa.

(A) The consensus ITS2 S2 was elaborated without gaps. The GenBank ID numbers of four ITS2 sequences used to predict the ITS2 S2 are indicated. (B) The consensus ITS2 S2 build with gaps. The consensus ITS2 S2s were obtained using the 4SALE v1.7.1 software. The helices are indicated by Roman numerals (I–IV). Nucleotides in yellow indicate the U-U mismatch motifs in helix II. The bar on the upper right side indicates the nucleotide conservation across the four ITS2 sequences used to obtain the consensus structure. Gaps in the structure are indicated in red.