Mitochondrial Genomics of Six Cacao Pathogens From the Basidiomycete Family Marasmiaceae

Abstract

Thread blight disease has recently been described as an emerging disease on cacao (Theobroma cacao) in Ghana. In Ghana, thread blight disease is caused by multiple species of the Marasmiaceae family: Marasmius tenuissimus, M. crinis-equi, M. palmivorus, and Marasmiellus scandens. Interestingly, two additional members of the Marasmiaceae; Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches' broom disease), are major pathogens of cacao in the Western hemisphere. It is important to accurately characterize the genetic relationships among these economically important species in support of their disease management. We used data from Illumina NGS-based genome sequencing efforts to study the mitochondrial genomes (mitogenomes) of the four cacao thread blight associated pathogens from Ghana and compared them with published mitogenomes of Mon. roreri and Mon. perniciosa. There is a remarkable interspecies variation in mitogenome size within the six cacao-associated Marasmiaceae species, ranging from 43,121 to 109,103 bp. The differences in genome lengths are primarily due to the number and lengths of introns, differences in intergenic space, and differences in the size and numbers of unidentified ORFs (uORF). Among seven M. tenuissimus mitogenomes sequenced, there is variation in size and sequence pointing to divergent evolution patterns within the species. The intronic regions show a high degree of sequence variation compared to the conserved sequences of the 14 core genes. The intronic ORFs identified, regardless of species, encode GIY-YIG or LAGLIDADG domain-containing homing endonuclease genes. Phylogenetic relationships using the 14 core proteins largely mimic the phylogenetic relationships observed in gene order patterns, grouping M. tenuissimus with M. crinis-equi, and M. palmivorus with Mon. roreri and Mon. perniciosa, leaving Mar. scandens as an outlier. The results from this study provide evidence of independent expansion/contraction events and sequence diversification in each species and establish a foundation for further exploration of the evolutionary trajectory of the fungi in Marasmiaceae family.

Keywords: Marasmiaceae; basidiomycete; cacao (Theobroma cacao); mitochondrial genome (mitogenome); thread blight disease.

FIGURE 1

Conservation of gene order and interspecies variation in the mitogenome structure of four cacao thread blight associated pathogens from Ghana and the Western hemisphere frosty pod rot and witches’ broom pathogens of cacao, all basidiomycetes within the Marasmiaceae family. Whole mitochondrial genome sequence involved are Marasmius tenuissimus (GHA07), Marasmiellus scandens (GHA19), Marasmius crinis-equi (GHA76); Marasmius palmivorus (GHA12), Moniliophthora roreri (Genbank no. HQ259115.1), Moniliophthora perniciosa (Genbank no. AY376688.1). Mitochondrial gene annotation was performed with MFannot using the NCBI translation code 4, and the physical map of the mitogenomes were created with OrganellarGenome-DRAW (OGDRAW) v 1.2.

FIGURE 2

Circos plot mapping the block rearrangements, synteny and identity level of (A) Marasmius tenuissimus and (B) Marasmiellus scandens against Marasmius palmivorus mitogenomes. Green and red block in the beginning and end of the sequences indicate the sequence orientation. Colors blocks and ribbons represent the local alignments produced by BLAST the score/max bits core ratio, with blue ≤ 0.25, green ≤ 0.50, orange ≤ 0.75, and red > 0.75 being the best quartile. The figure was produced using Circoletto (Darzentas, 2010).

FIGURE 3

Conservation of gene order and Intraspecies variation in the mitogenome structure of seven isolates of the cacao thread blight associated pathogen Marasmius tenuissimus. Mitochondrial gene annotation was performed with MFannot using the NCBI translation code 4, and the physical map of the mitogenomes were created with OrganellarGenome-DRAW (OGDRAW) v 1.2.

FIGURE 4

Molecular phylogenetic analysis of four cacao thread blight associated pathogens from Ghana (M. tenuissimus, M. palmivorus, M. crinis-equi, Mar. scandens) and the Western hemisphere frosty pod rot (Mon. roreri) and witches’ broom (Mon. perniciosa) pathogens of cacao along with some other members of Marasmiaceae and Agaricales. The Rhizoctonia solani was used as an outgroup. The analysis was based on the amino-acid sequences of 14 conserved mitochondrial proteins with 3,154 distinct alignment positions and 1,000 rapid bootstrap inferences. Sequences were combined and aligned using ClustalW2 tool under default setting and the phylogenetic tree was reconstructed using the Maximum Likelihood method. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Analyses were conducted in MEGA6 (Tamura et al., 2013). The analysis was re-run with seven M. tenuissimus and M. crinis-equi as an outgroup.

FIGURE 5

Molecular phylogenetic analysis of four cacao thread blight associated pathogens from Ghana (M. tenuissimus, M. palmivorus, M. crinis-equi, Mar. scandens) and the Western hemisphere frosty pod rot (Mon. roreri) and witches’ broom (Mon. perniciosa) pathogens of cacao based on the rps3 gene. The analysis was based on amino-acid sequences of rps3 homologs from each isolate with 81 distinct alignment positions and 1,000 rapid bootstrap inferences. (A) Three M. tenuissimus isolates with other five species. (B) Seven isolates of M. tenuissimus. Sequences were combined and aligned using ClustalW2 tool under default setting and the phylogenetic tree was reconstructed using the Maximum Likelihood method. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Analyses were conducted in MEGA6 (Tamura et al., 2013). (C) Visualization of rps3 gene region sequence alignment between M. tenuissimus isolate GHA64 and GHA63. The mVISTA program (http://genome.lbl.gov/vista/mvista/submit.shtml) was used to compare the section of mitochondrial genomes with default parameters in Shuffle-LAGAN mode and a sequence conservation profile was visualized in an mVISTA plot (Frazer et al., 2004).

FIGURE 6

Molecular phylogenetic analysis of cox1 introns of four cacao thread blight associated pathogens from Ghana (M. tenuissimus, M. palmivorus, M. crinis-equi, Mar. scandens) and the Western hemisphere frosty pod rot (Mon. roreri) and witches’ broom (Mon. perniciosa) pathogens of cacao. The analysis was based on nucleotide sequences of cox 1 introns from each isolate with 1,566 distinct alignment positions and 1,000 rapid bootstrap inferences. (A) Two M. tenuissimus isolates with other five species. (B) Seven isolates of M. tenuissimus. Sequences were combined and aligned using ClustalW2 tool under default setting and the phylogenetic tree was reconstructed using the Maximum Likelihood method. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Analyses were conducted in MEGA6 (Tamura et al., 2013).