The complete mitochondrial genome of Clonostachys farinosa (Bionectriaceae, Hypocreales)

Abstract

The complete mitochondrial genome of Clonostachys farinosa was assembled using the strain CSC22A0184 that was isolated from the lichen Parmotrema clavuliferum. The total length of the mitogenome was 51,551 bp and contained 49 genes: 15 protein-coding genes, two ribosomal RNA genes, 26 transfer RNA genes, and six open reading frames. The GC content of the mitogenome was 28.5% and had a base composition of 36.4% A, 12.6% C, 15.9% G, and 35.1% T. Phylogenetic analysis based on 14 protein-coding genes indicates that C. farinosa is clustered in the family Bionectriaceae. This is the first study of the mitogenome of C. farinosa, which is helpful for understanding the evolution of species within the genus Clonostachys.

Keywords:

• Endolichenic fungi
• fungi
• lichens
• mitogenome
• phylogenetic analysis

Introduction

The genus Clonostachys Corda 1839 contains endophytic, parasitic, or saprotrophic fungi that primarily found in soils, insects, and plant-associated environments (Schroers 2001). Due to its mycoparasitic properties, Clonostachys has been extensively investigated as biocontrol agent against plant pathogens (Cota et al. 2009; Krauss et al. 2013; Sun et al. 2020). Additionally, the secondary metabolites produced by Clonostachys show various biological activities, including antibacterial, insecticidal, and cytotoxic activities (Han et al. 2020), thereby emphasizing their significant value in agricultural and medicinal applications.

Clonostachys farinosa (Henn.) Rossman 2014 is commonly found in plant-associated environments, such as on the bark or twigs of dead trees and the stems of living plants (Schroers 2001; Zhao et al. 2023). This species is recognized for its role as a biocontrol agent in managing plant pathogens by employing secondary metabolites with antimicrobial activities (Zheng et al. 2006; Krauss et al. 2013). While the ecology and taxonomy of Clonostachys species have been extensively studied, there exists a notable gap in genetic studies concerning the mitochondrial genome. Only mitochondrial genomes have been assembled for the following five species: C. compactiuscula, C. rogersoniana, C. rosea, C. solani, and Clonostachys sp. YFCC 8591 (Wang et al. 2017; Zhao et al. 2021). In this study, I have assembled the mitochondrial genome of C. farinosa and analyzed its phylogenetic relationship with other Clonostachys species. To the best of my knowledge, this is the first reported mitochondrial genome of C. farinosa, and it is expected to enhance our understanding of the Clonostachys mitochondrial genome characteristics.

Materials and methods

Clonostachys farinosa strain CSC22A0184 was isolated from the lichen Parmotrema clavuliferum (Figure 1), which was collected from Mt. Yeonhwa, Goseong, South Korea (35°04'24.1″N, 128°15'55.1″E, 385 m MSL). The fungal strain CSC22A0184 is deposited in the microbial symbiosis lab of Changwon National University, South Korea (Seung-Yoon Oh; [email protected]) under the name “Clonostachys byssicola”, which was its name before taxonomic review (Zhao et al. 2023). Genomic DNA was extracted from the strain which was cultured on potato dextrose agar for 2 weeks at 25 °C using the MG™ Genomic DNA Purification kit (Macrogen, Seoul, South Korea) following the manufacturer’s instructions. Paired-end libraries were constructed using the TruSeq Nano DNA Library Prep Kit (Illumina, San Diego, CA USA) with an insertion size of 350 bp and sequenced using the Illumina NovaSeq platform (Macrogen, South Korea).

Figure 1. Species reference photograph. (A) Colony morphology of Clonostachys farinosa strain CSC22A0184 on potato dextrose agar incubated at 25 °C in 7 days. The scale bar indicates 1 cm. (B) Lichen Parmotrema clavuliferum as an isolation source. The scale bar indicates 1 cm. Photograph by Seung-Yoon Oh.

The adapter sequence was trimmed from raw sequence reads, and the mitochondrial genome was assembled using NOVOPlasty v. 4.3.1 (Dierckxsens et al. 2017). The coverage depth map was generated using the method from the previous study (Ni et al. 2023) and showed the average depth 1013× (Figure S1). Annotation was performed using MFannot (https://megasun.bch.umontreal.ca/apps/mfannot) and RNAweasel (https://megasun.bch.umontreal.ca/apps/rnaweasel) with genetic code 4 and BLAST against a reference sequence for C. compactiuscula (MW030498), C. rogersoniana (MW030499), and C. rosea (KU668563). Transfer RNA (tRNA) genes were identified using the tRNAscan-SE program, v2.0.7 (Lowe and Eddy 1997). The genome map was visualized using the OGDRAW program (Greiner et al. 2019). Maximum likelihood phylogeny was constructed with the PROTGAMMA + MTZOA model and 1000 bootstrap replicates using raxmlGUI v. 2.0 (Edler et al. 2021) with amino acid sequences of 14 conserved protein-coding genes.

Results

The complete genome of C. farinosa strain CSC22A0184 (GenBank accession: OQ205181) consists of a circular molecule 51,551 bp in length, featuring a GC content of 28.5% and an overall nucleotide composition of 36.4%, 12.6%, 15.9%, and 35.1% for A, C, G, and T, respectively. The mitochondrial genome comprises 49 genes (Figure 2), including 15 protein-coding genes (atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, and rps3), two ribosomal RNA genes (rns and rnl), 26 tRNA genes, and 13 open reading frames (ORFs). Group I intron account for all introns, except for one Group II intron located within the nad2 gene (Table 1). Among the 13 ORFs, six encode LAGLIDADG endonucleases, while three encode GIY endonucleases (Table 1). Phylogenetic analysis reveals that C. farinosa belongs to the family Bionectriaceae and forms a sister clade with C. rosea (Figure 3). Collinearity analysis confirms the conservation of genetic order within the Clonostachys genus in the mitochondrial genome (Figure 4).

Figure 2. Circular maps and genetic compositions of Clonostachys farinosa strain CSC22A0184. Genes are located outside and color-coded by the functional classification. GC contents are shown on the inner circle. The names of genes containing introns are marked with an asterisk, and the names of genes or ORFs assigned inside the intron are located inside the outer circle.

Figure 3. Maximum likelihood (ML) tree for 10 species within the order hypocreales based on 14 concatenated, conserved protein-coding genes (atp6, atp8, atp9, cob, cox1–3, nad1–6, and nad4L). The ML tree was constructed using the PROTGAMMA + MTZOA model and 1000 bootstrap replicates. The scale bar indicates the number of substitutions per site. The following mitogenomes were used for the analysis: Clonostachys rosea (Wang et al. 2017), C. compactiuscula, C. rogersoniana, C. solani, Clonostachys sp. YFCC 8591 (Zhao et al. 2021), Hapsidospora chrysogena (Eldarov et al. 2015), Emericellopsis fuci (Konovalova and Logacheva 2016), Trichoderma harzianum (Kwak 2021) and T. lixii (Venice et al. 2020). Accession numbers of GenBank are indicated after the species name.

Figure 4. The collinearity analysis of mitochondrial genomes of Clonostachys species. The diagram is generated using genoPlotR package (Guy et al. 2010) based on the mitochondrial genome used in the phylogenomic analysis. The color represents the type of gene features.

Table 1. Classification of intron and ORF annotated in the mitochondrial genome of Clonostachys farinosa.

No.	Name	Start position	End position	Type
1	cox1 intron 1	387	1642	Group IB
2	cox1 intron 2	1988	3045	Group IB
3	cox1 intron 3	3373	3604	Group IB
4	cox1 intron 4	3672	4779	Group IB
5	cox1 intron 5	4917	5929	Group IB
6	nad4 intron 1	9243	10606	Group IC2
7	cox3 intron 1	17095	18249	Group IB
8	rnl intron 1	23888	25665	Group IA
9	nad2 intron 1	35117	37609	Group II
10	nad5 intron 1	40908	42277	Group IB
11	cob intron 1	44928	47491	Group IB
12	ORF368	388	1494	LAGLIDADG
13	ORF329	1989	2978	LAGLIDADG
14	ORF399	3672	4727	LAGLIDADG
15	ORF265	4918	5715	GIY
16	ORF148	9245	10459	LAGLIDADG
17	ORF317	17095	18048	LAGLIDADG
18	ORF821	28676	29587	–
19	ORF177	43838	43906	–
20	ORF144	44043	44339	–
21	ORF400	45214	46416	LAGLIDADG
22	ORF179	46577	47116	GIY
23	ORF340	47685	48704	GIY
24	ORF240	50137	50841	–

Discussion and conclusion

During the project on the diversity of endolichenic fungi in South Korea, I isolated the strain of C. farinosa and assembled its complete mitochondrial genome to elucidate its genomic characteristics. The length of the C. farinosa mitogenome (51,551 bp) is considerably longer than it of the five other Clonostachys species, which range from 27,410 to 42,075 bp (Wang et al. 2017; Zhao et al. 2021). Furthermore, the genetic component order remains consistent across all Clonostachys species, indicating an absence of gene rearrangement events within the genus Clonostachys. Phylogenetic analysis, based on the amino acid sequence of 14 conserved protein-coding genes, supports the close relationship between C. farinosa and C. rosea. This phylogenetic tree topology aligns with previous findings based on the DNA markers (Wang et al. 2017; Zhao et al. 2021). The mitochondrial genome of C. farinosa stands as a valuable resource to deepen our understanding of the evolution and phylogeny within the family Bionectriaceae, along with providing insights into the genus Clonostachys.

Ethics statement

Ethical approval is not required for this study. Because the material used in this study is a fungal strain rather than a vertebrate, there are no applicable experimental ethics for its use.

Author contributions

Conception and design: S-YO. Data analysis and interpretation: S-YO. Manuscript drafting and revision: S-YO. I agree to the publication of the final version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The sequence generated from this study has been deposited to GenBank under accession number OQ205181. The associated BioProject, BioSample, and SRA numbers are PRJNA1092717, SAMN40632650, and SRR28478378, respectively.

Additional information

Funding

This research is funded by the Rising Professor Financial Program at Changwon National University in 2023; the National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT) [NRF 2021R1C1C1008045]; and Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education [Grant No. 2023R1A6C101B022].