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Article

Comparative Analysis of the Mitochondrial Genome Sequences of Diaporthe longicolla (syn. Phomopsis longicolla) Isolates Causing Phomopsis Seed Decay in Soybean

by
Shuxian Li
1,*,
Xiaojun Hu
2 and
Qijian Song
3
1
United States Department of Agriculture, Agricultural Research Service (USDA, ARS), Crop Genetics Research Unit, 141 Experiment Station Rd., Stoneville, MS 38776, USA
2
USDA, Animal and Plant Health Inspection Service (APHIS), Plant Protection and Quarantine (PPQ), Plant Germplasm Quarantine Program (PGQP), Beltsville, MD 20708, USA
3
USDA, ARS, Soybean Genomics and Improvement Laboratory, Beltsville Agriculture Research Center, Beltsville, MD 20705, USA
*
Author to whom correspondence should be addressed.
J. Fungi 2024, 10(8), 570; https://doi.org/10.3390/jof10080570
Submission received: 27 February 2024 / Revised: 1 August 2024 / Accepted: 4 August 2024 / Published: 13 August 2024
(This article belongs to the Section Fungal Genomics, Genetics and Molecular Biology)
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Abstract

:
Diaporthe longicolla (syn. Phomopsis longicolla) is an important seed-borne fungal pathogen and the primary cause of Phomopsis seed decay (PSD) in soybean. PSD is one of the most devastating seed diseases, reducing soybean seed quality and yield worldwide. As part of a genome sequencing project on the fungal Diaporthe–Phomopsis complex, draft genomes of eight D. longicolla isolates were sequenced and assembled. Sequences of mitochondrial genomes were extracted and analyzed. The circular mitochondrial genomes ranged from 52,534 bp to 58,280 bp long, with a mean GC content of 34%. A total of 14 core protein-coding genes, 23 tRNA, and 2 rRNA genes were identified. Introns were detected in the genes of atp6, cob, cox1, cox2, cox3, nad1, nad2, nad5, and rnl. Three isolates (PL7, PL10, and PL185E) had more introns than other isolates. Approximately 6.4% of the mitochondrial genomes consist of repetitive elements. Moreover, 48 single-nucleotide polymorphisms (SNPs) and were identified. The mitochondrial genome sequences of D. longicolla will be useful to further study the molecular basis of seed-borne pathogens causing seed diseases, investigate genetic variation among isolates, and develop improved control strategies for Phomopsis seed decay of soybean.

1. Introduction

The seed-borne fungal pathogen Diaporthe longicolla (Hobbs) J. M. Santos, Vrandecic & A. J. L. Phillips (syn. Phomopsis longicolla T. W. Hobbs) is the primary cause of Phomopsis seed decay (PSD) in soybean [Glycine max (L.) Merrill] [1,2,3,4,5,6]. The PSD-causing pathogen was first identified as Phomopsis longicolla in 1985 [1]. Later, in 2011, the fungus was renamed Diaporthe longicolla [6]. However, the disease is still retained as Phomopsis seed decay although the fungus name has been changed.
PSD is one of the most destructive seed diseases, reducing soybean seed quality and yield worldwide. The most charactered symptoms of PSD include seed discoloration with cracked seed coats. The entire seed surface can be moldy and have a chalk-white color if soybean seeds are severely infected with D. longicolla. In some cases, D. longicolla-infected soybean seeds did not have visible symptoms [7,8]. Moreover, seed compositions could be adversely altered or reduced by the infection of D. longicolla [2]. In the mid-southern region of the United States, especially under the humid and warm conditions that favor the colonization of the pathogen, PSD has significantly economic impact on soybean production [8,9,10].
Breeding for host genetic resistance is one of the most effective means to manage PSD in an environmentally friendly approach [11,12,13]. In the past decades, extensive research efforts have been made to screen soybean germplasm and cultivars to identify sources of resistance to PSD [9,10,14,15]. However, development of resistant cultivars may depend on the variability of the pathogen, including isolate aggressiveness. Knowledge about the variability of the pathogen is crucial for understanding the pathogen population, which will also be important for selection of isolates for developing soybean lines with broad-based resistance to Phomopsis seed decay and high seed quality. Differences between D. longicolla isolates in their ability for infecting soybean have been reported. For an example, in evaluating the aggressiveness of 48 isolates of D. longicolla from different geographic origins, which included 35 D. longicolla isolates from soybean in eight states in the United States, 2 D. longicolla isolates from velvetleaf in Illinois [16], and 11 other Phomopsis spp. isolates from other hosts in four states in the United States as well as Canada and Costa Rica, it was found that there were significant differences in stem lesion length of a susceptible cultivar, Williams 82, after inoculation with different isolates, and the most aggressiveness isolates were identified [4]. Similar results of difference in isolate aggressiveness on soybean were also reported in another study [17].
To facilitate investigation of the genomic basis of the pathogenicity and variability of isolates and to understand the mechanism of disease development, as part of a genome-sequencing project on the fungal Diaporthe–Phomopsis complex, the genome of a D. longicolla isolate (MSPL10-6) from Mississippi, USA, was sequenced and analyzed [18]. Preliminary study on its mitochondrial genome was conducted [19]. It is well known that mitochondria are semi-autonomous organelles that have their own genetic material (DNA) and are capable of synthesizing proteins required for their functioning, existing in most eukaryotic cells. Mitochondria play crucial roles in essential energy production, respiratory metabolism, cell growth, and many other essential cellular processes [20]. Because of the relatively small size, high copy number, and high mutation rate, sequences of fungal mitochondrial genome (mitogenome) have been used as valuable tools to develop molecular markers for species identification and characterization [21,22]. However, the mitochondrial genome of different D. longicolla isolates from different geographic origins has not been well studied. Although variation of different isolates in aggressiveness and virulence has been reported [4,17], the genomic variation is unclear. Repetitive and transposable elements in mitochondrial genomes could play an important role in fungal diversity [23].
As a continuing effort to explore the mitochondrial genome of D. longicolla, eight isolates from different states in the U.S. (Table 1) were sequenced and analyzed in this study. The objectives of this study were (1) to study the gene content and organization of the mitochondrial genome of D. longicolla; (2) to compare differences in the occurrence and distribution of mobile genetic elements, such as the intron’s types and numbers, as well as transposable elements in different D. longicolla isolates; and (3) to identify repetitive elements in the mitochondrial genome. The mitochondrial genome sequences of D. longicolla will be useful to further study the molecular basis of seed-borne pathogens causing seed disease in soybean, investigate genetic variation among isolates from different geographic origins, and develop improved control strategies for Phomopsis seed decay of soybean.

2. Material and Method

2.1. Fungal Isolates and DNA Extraction

Eight isolates of D. longicolla originating from six states of USA were used in this study, including the type culture of TWH P74 (ATCC 60325) obtained from the American Type Culture Collection in 2010 (Table 1). Total genomic DNA of each isolate was extracted using a Qiagen DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA) after culturing in potato dextrose broth (Difico Laboratories, Detroit, MI, USA) in an incubator statically for 4 days under 12-hour light and dark cycles at 24 °C. The quality of DNA was assessed by 1% agarose gel electrophoresis, and DNA was quantified using a Nanodrop 1000 (NanoDrop Technologies, Inc., Wilmington, DE, USA) and a Qubit v.2.0 fluorometer (Fisher Scientific, Waltham, MA, USA).

2.2. Genome Sequencing and Mitochondrial DNA Assembly

Genome sequencing was carried out either at the Genomics Core Facility, Purdue University at West Lafayette, Indiana, using the Illumina HiSeq 2500 (Illumina, Inc., San Diego, CA, USA) sequencer (for isolates MSPL10-6, TWH P74, and PL185E), as reported previously [18], or Novogene (for isolates PL1, PL6, PL7, PL10, and PL11) using HiSeq PE 150 (Illumina, Inc., San Diego, CA, USA). The D. longicolla genomes were assembled using the Spades program (v3.15.4) [24]. Mitochondrial contigs were picked out from assembly results by comparing with the published mitochondrial DNA of D. longicolla isolates at GenBank (accessions KP 137411.1 and MT 527962) using BLASTX [25]. Then, the reads mapped to those contigs were assembled again, using Spades (v3.15.4), into a circular mitochondrial genome.

2.3. Gene Annotation and Analysis

Gene annotation (including intron identification) was conducted using the online program MFannot (https://megasun.bch.umontreal.ca/apps/mfannot/, accessed on 1 November 2023). The intron in the nad2 gene of isolates (PL7, PL10, PL11, and PL185E) was identified in the alignment of the mitochondrial genome sequences D. longicolla, which was performed by MAFFT (v7.490) [26] with an automatic algorithm (parameter: mafft—auto). Its conserved intron domain (double LAGLIDADG_1) was searched using the online program Pfam (http://pfam.xfam.org/, accessed on 1 November 2023). The intron type was determined by the online program RNAweasel (https://megasun.bch.umontreal.ca/apps/rnaweasel/, accessed on 1 November 2023). The intron–exon border was identified by viewing the alignment using BioEdit (v7.2.0) [27].
The physical maps of the mitochondrial genome of eight D. longicolla isolates were drawn using the program Circos (v0.69-9). The secondary structure of tRNA genes was predicted and drawn using the MITOS2 web server (https://usegalaxy.org/root?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fiuc%2Fmitos2%2Fmitos2%2F2.1.3%20galaxy0, accessed on 1 November 2023). The codon usage was calculated by Sequence Manipulation Suite (https://www.bioinformatics.org/sms2/codon_usage.html, accessed on 1 November 2023) with genetic code 4.

2.4. Identification of Repetitive Elements

Four methods were used to analyze repetitive elements in the mitochondrial genomes of eight D. longicolla isolates: (1) a BLASTn search [28] of the whole mitochondrial genome against itself to determine if there was intra-genomic duplication of large fragments at an E value of <10−10; (2) the online program Tandem Repeat Finder, with the default parameter to detect tandem repeats (>10 bp in length) [29]; (3) the REPuter tool (https://bibiserv.cebitec.uni-bielefeld.de/reputer/, accessed on 1 November 2023) to identify forward, reverse, complemented, and palindromic repeats [30]; and (4) the EMBOSS (v6.6.0) Suite to identify inverted repeats [31].
Simple sequence repeats (SSRs) with di- and tri-nucleotide repeats (> 5 bp) were identified with the Perl script MISA tool [32]. The sequences were aligned by Muscle [33] in the Mega6 program [34], and the aligned sequence file was imported into the DNASP6 program [35,36] for identification of single-nucleotide polymorphisms (SNPs). SNPs were verified by Sanger sequencing the PCR fragments containing the SNPs.

3. Results

3.1. General Feature of D. longicolla Mitochondrial Genome

The mitochondrial genomes of eight D. longicolla isolates were assembled into a single circular, double-stranded DNA molecule (Figure 1). The sizes of the mitochondrial genomes ranged from 52,534 bp to 58,280 bp. Isolate PL6 had the smallest size of 52,306 bp, while isolate PL185E had the largest size of 58,234 bp (Table 2). The isolates (PL7, PL10, and PL185E) had more introns than the other isolates. The mean GC content was 34%. The GC skew was positive (0.10), while the AT skew was slightly negative (−0.03), indicating that there was no significant strand-specific bias of nucleotide composition in the mitochondrial genome of D. longicolla.
A total of 14 core mitochondrial protein-coding genes were identified. In addition to the core protein-coding genes, four and five free-standing open-reading frames of unknown function (uORFs) were annotated from two and six isolates, respectively (Table 2). The mitochondrial genomes of D. longicolla isolates also contained 23 tRNA genes, except isolate PL185E, which had 22 tRNA genes. The mitochondrial genomes of D. longicolla also contained two rRNA genes, a large subunit ribosomal RNA gene (rnl), and a small subunit of ribosomal RNA gene (rns) as well as a gene coding the 40S ribosomal protein S3 (rps3).

3.2. Protein-Coding Genes and Codon Usage

The 14 conserved protein-coding genes encoded three subunits of the ATP-synthase (atp6, atp8, and apt9), seven subunits of electron transport chain of complex I (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), three subunits of complex III (cox1, cox2, and cox3), and one subunit of complex IV (cob). Of the 14 protein-coding genes identified, 9 contained introns, which were located at the atp6, cob, cox1, nad2, cox2, cox3, nad1, nad5, and rnl genes (Table 3).
The cox1 gene had the highest number of intron sequences (four in five isolates and five in three isolates). The total number of introns ranged from 9 to 13 among isolates. Isolates PL7 and PL185E had the highest number of 13 introns, and isolate PL10 had 12, while isolates PL1 and PL6 had nine introns. Both MSPL10-6 and TWH P74 had 10 introns, respectively. Isolates MSPL10-6, TWH P74, PL1, and PL6 did not have an intron in nad2 and cox3 genes (Table 3). Additional information about introns identified in the mitochondrial genome of D. longicolla isolates is shown in Table 4.
The most common start codon for the 14 protein-coding genes was ATG, while AAA, ACA, ACG, ATA, ATG, TTA, and TTT were also utilized. The most frequent stop codon was TAA, but TAG was also used. Codon usages in the isolate of PL185E, as an example, are presented in Figure 2.
The gene order for the D. longicolla mitochondrial genome, starting with cox1, was cox1–nad2–nad3–cox2–nad4L–nad5–atp8–apt6–rns–apt9–cox3–nad6–rnl–rps3–nad1–nad4–cob-1–cob-2. In comparison of gene order across eight isolates of D. longicolla, no gene rearrangement was found in those isolates.

3.3. Transfer RNA and Ribosomal RNA Genes

A total of 23 transfer RNA (tRNA) genes were identified in the mitochondrial genomes of D. longicolla coding for 20 amino acids, except for isolate PL185E, which had 22 tRNA genes coding for 19 amino acids. The length of the tRNA genes ranged from 69 bp (trnF) to 85 bp (trnS). Two tRNA genes (trnM and trnF) were duplicated. The trnM (cat) had three copies in all eight D. longicolla isolates. Isolate PL185E contained two copies of trnF: One was trnC (gca) as other isolates had, and another was trnF (gaa). Most of the tRNA genes were located between rnl and nad1. No introns were found in tRNA genes. The tRNA molecules can fold into a common cloverleaf secondary structure comprised of the acceptor stem, D-loop, anticodon, and TψC loop (Figure 3). There was a large loop structure on the extra arm of trnL2, trnS1, trnS2, and trnY. In trnC, there were two loops on the anticodon arm, while other tRNA genes had only one loop on the anticodon arm. Their graphic depictions are shown in Figure 3.
There were two ribosomal RNA (rRNA) genes in the mitochondrial genome of D. longicolla (rnl and rns). The rnl was 6050 bp in length, which was interrupted by an intron with a length of 2123 bp. The rns was 1637 bp, without an intron. The rnl was located between trnP and trnT, while the rns was placed between apt6 and atp9.

3.4. Repetitive Elements

A sequence BLASTn search against itself of each mitochondrial genome revealed 28 to 32 repetitive sequences among eight isolates, accounting for approximately 6.4% of the D. longicolla mitochondrial genomes. The size of repeats ranged from 33 bp to 336 bp (Table S1). Analysis with the Tandem Repeat Finder (TRF) program identified seven or eight tandem repeats with consensus sizes from 8 to 37 bp in each isolate. There were seven repeat motifs in isolates PL1, PL6, MSPL10-6, and TWH P74 (ATCC 60325) and eight repeat motifs in isolates PL7, PL10, PL11, and PL185E (Table S2). In addition, there was a tandem repeat element in the nad2 intron in the genomes of PL7, PL10, PL11, and PL185E. Using the REPuter program, 30 or 31 forward and 19 or 20 palindromic repeats were detected among isolates. (Table S3). A total of 20–22 short, inverted repeats with a size range from 17 to 54 bp were identified by the EMBOSS program (Table S4). Dispersed and inverted repeat sequences of eight isolates are shown in Figure 4. No reverse repeat sequences were identified.
Using Perl script MISA [32] to search the simple sequence repeats (SSRs) with di-, tri-, and tetranucleotide repeat units larger than 4 bp in the mitochondrial genome sequences of eight D. longicolla isolates, 24 SSRs with di- and tri-nucleotide repeats (>5 bp) were identified, 16 of which had AT motifs, and 8 SSRs had ATT motifs. Each mitochondrial sequence contains two SSRs with AT motifs and one SSR with ATT motifs. There was no polymorphism in these SSRs found among the isolates. However, single-nucleotide polymorphisms (SNPs) were detected. There were 48 polymorphic sites, including 20 singleton variable sites and 28 parsimony informative sites (Table 5), when the mitochondrial genome sequences were aligned by Muscle [33] in the Mega6 program [34]. Of eight isolates studied, seven had the same SNP at 14 sites. Isolate MSPL 10-6, originating from Mississippi, had more SNPs than all other isolates including two more isolates from Mississippi.

4. Discussion

With the advancement of high-throughput sequencing technologies and facilities, the feasibility of mitochondrial genome sequences has been increasing. At present, at least 4201 fungal mitochondrial genome sequences are available at GenBank (https://www.ncbi.nlm.nih.gov/nuccore/?term=fungi%5BOrganism%5D+AND+mitochondrion%5Bfilter%5D+AND+complete%5Btitle%5D+AND+ddbj_embl_genbank%5Bfilter%5D+NOT+unverified, accessed on 28 January 2024). However, information about the mitochondrial genome of the fungal pathogens that cause soybean diseases is still limited. Most of the reported studies focused on either one specific species or compared two or more closely related species using only one fungal isolate or strain of each, such as the isolate Pg-21 of Coniothyrium glycines that causes red leaf blotch of soybean [37] and two Phakopsora species, namely P. pachyrhizi (isolate Taiwan 72-1) and P. meibomiae (an isolate from Puerto Rico), the causal agents of soybean rust [38].
Our present study was the first report of analysis of the mitochondrial genome sequences of eight D. longicolla isolates collected from different states in the U.S. Eight assembled mitochondrial genomes were compared to reveal a general structure, gene content, repetitive and transposable elements, and a possible pattern of variation. Like most other fungi, the mitochondrial genomes of eight D. longicolla isolates were assembled into a single circular, double-stranded DNA molecule. It contains a small set of conserved genes, including 14 protein-coding genes, 22 or 23 tRNA genes, as well as the small and large ribosomal RNA genes.
In the mitochondrial genomes of fungi, duplicated copies of conserved protein-coding genes are quite common. For example, there was an extra copy of atp9 in the mitochondrial genome of the phytopathogenic fungus Sclerotinia borealis [39]. The gene duplication was also reported in Botryotinia fuckeliana [39], Phlebia radiata [40], as well as many other fungal species. In our present study, the mitochondrial genomes of all eight D. longicolla isolates contained a truncated extra copy of cob, which was 160 bp of exon 1 in cob. Besides the conserved protein-coding genes, the tRNA gene trnM (cat) had three copies in all eight D. longicolla isolates, and isolate PL185E also contained two copies of trnF. The cause of those gene duplication and their impact on the function of mitochondria are not well understood and remain for further study.
There were remarkable size variations of mitochondrial genomes in numerous fungi [41]. The small mitochondrial genomes included those of about 18.84 kp in Hanseniaspora uvarum [42], whereas the larger mitochondrial genomes in fungi were more than 200 kb, such as those of 203 kp in Sclerotinia borealis [39] and 272.2 kb in Morchella importuna [43]. Results from the analysis of the mitochondrial genomes of nine Aspergillus and Penicillium species indicated that the number of introns and their length difference represent one of the major factors contributing to variations in the mitochondrial genome size of fungal species [44]. Analysis of 11 complete mitogenomes of Polyporales species revealed the great variations in intron distribution and content [45]. In our study, the sizes of the mitochondrial genomes of eight D. longicolla isolates ranged from 52,534 bp to 58,280 bp. Three isolates (PL7, PL10, and PL185E) had more introns and larger size than the other isolates. More research should be conducted to address if the differences in the mitochondrial genome size relate to the genetic diversity and evolutionary dynamics of this fungal species.
The numbers of intron in mitochondrial genomes vary in different fungal species [20]. In general, mitochondrial introns in fungi can be mainly classified into two groups (groups I and II) according to their conserved RNA secondary structures [46,47]. Group I introns are large self-splicing ribozymes. They catalyze their own excision from mRNA, tRNA, and rRNA precursors in a wide range of organisms and are rich in fungi [48]. Group I introns are further divided into subgroups such as IA, IA3, IB, IC1, IC2, and ID based on phylogenetic analyses [46]. In general, group I introns are prevalent in fungal mitochondrial genes with greater association for genes, e.g., cox1, cob, and rnl, while group II introns are predominant in plant mitochondrial genomes [46]. The mitochondrial cox1 gene has been reported to be the richest in group I introns [49]. In contrast, the Fusarium oxysporum species complex (FOSC) did not possess any introns in its cox1 gene [50]. In the case of D. longicolla, most introns located in the cox1 gene were all IB type, while IA introns were found in cob, cox3, nad2, and rnl; IB introns were found in cox2, nad1, nad3, and nad5; and IC introns were found in in Atp6 genes. These D. longicolla introns carried either LAGLIDADG or GIY-YIG homing endonuclease genes, which have been reported to facilitate the movement of introns into previously intron-less genes or certain regions, resulting in enlargement of the mitochondrial genome size [46]. Although introns are often found in mitochondrial genomes, we are still uncertain about their origins and the modes of transmission in fungi.
Although high variability of mitochondrial gene order among fungi has been reported [51,52,53], some closely related fungal groups appeared to be conserved. The order of protein-coding genes and tRNA was identical in two related soybean rust pathogens, Phakopsora pachyrhizi and P. meibomiae [38]. Results from the analysis of the distribution of mitochondrial protein-coding genes indicated no significant differences in gene order among Hypocreales species [54]. Comparison of Verticillium nonalfalfae and the closely related V. dahliae revealed a conserved gene order in their mitochondrial genomes [55]. In our present study, the gene order of all eight D. longicolla isolates was the same. No gene rearrangement was found among isolates. It is interesting to see such consistency of the gene order in their mitochondrial genomes. The absence of gene rearrangement among the D. longicolla isolates in their mitochondrial genomes contributes to our understanding of their genetic stability and lays the foundation for further investigation of their evolutionary patterns.
One of the most noticeable features of the fungal genome is the presence of repetitive elements. Generally, over 30% repetitive DNA was found in fungal Zygomycota, whereas more than 5% was reported in ascomycete and basidiomycetes fungi [56]. Results from analysis of a potato fungal pathogen Rhizoctonia solani AG-3 revealed that one-third of the mitochondrial genome was occupied by interspersed repeats, which included at least three types: short interspersed palindromic sequences < 40 bp, mid-length (50–95 bp) sequences, and longer elements (>100–963 bp) [57]. In our present study, approximately 6.4% of the D. longicolla mitochondrial genomes contained different types of repetitive sequences.
Simple sequence repeats (SSRs) have been considered as a valuable source of genetic markers and are widely used in population genetics, genetic diversity, fingerprinting, and forensic analysis in many organisms due to their abundance and inherent potential for variation [58]. Although there was no polymorphism in simple sequence repeats (SSRs) among the D. longicolla isolates, the SSRs identified in this study could be used to develop molecular markers to distinguish D. longicolla from other closely related fungal species, especially those in the Diaporthe–Phomopsis complex causing soybean diseases as well as D. nobilis, an alpine pathogen, which is close to D. longicolla based on the analysis of the mitochondrial genome sequences [59]. It is well known that single-nucleotide polymorphisms (SNPs) can be used to characterized similarities and differences within a species of different isolates or between different species [60,61,62]. Characterization of the SNP and structural variations in the mitochondrial genomes of a fungal pathogen Tilletia indica causing the disease Karnal bunt in wheat and its closely related species led to developing a simple and high-throughput diagnostic assay [63]. Results from analysis of the mitochondrial genomes of closely related Fusarium culmorum and F. graminearum demonstrated that the diversity of mobile genetic elements was the main distinctive trait for diagnostic purposes of Fusaria [64,65]. In our present study, 48 SNPs were detected in the mitochondrial genome sequences of eight D. longicolla isolates. This information can be utilized to develop molecular markers for the detection and identification of D. longicolla in soybean, especially those soybean seeds that have been infected by D. longicolla but do not show visible PSD symptoms.
It is well known that the occurrence and severity of plant diseases result from three factors: plant host, pathogen, and environment. This is called the “Disease Triangle” [66], a fundamental concept for disease causation and management. These three factors interact with each other. If any one of the three factors is missing, the “triangle” is not complete, and disease will not occur. The pathogen, D. longicolla, is one of the three factors in the PSD disease “triangle”. As the first step toward to disentangling the pathogenicity mechanism of D. longicolla causing PSD in soybean, we analyzed the mitochondrial genome sequences of eight D. longicolla isolates. This research was one of our efforts to investigate how genetic variations of the pathogen influence PSD development. Identification of genetic factors/genes associated with the pathogenicity of D. longicolla through a whole-genome analysis is underway.
Together, the results of the present study on the comparative analysis of the mitochondrial genome sequences of D. longicolla isolates will be useful to further study the molecular basis of seed-borne pathogens causing seed diseases, investigate genetic variation among isolates, and develop improved control strategies for Phomopsis seed decay of soybean.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof10080570/s1, Table S1. Local BLASTn analysis of the Diaporthe longicolla mitochondrial genome sequences against itself; Table S2. Tandem repeats detected in the mitochondrial genome of Diaporthe longicolla using Tandem Repeats Finder; Table S3. Number of repeats in the mitochondrial genome of Diaporthe longicolla searched by REPuter; Table S4. Summary of inverted repeats identified by EMBOSS (v6.6.0).

Author Contributions

Conceptualization, S.L.; investigation, S.L., X.H., and Q.S.; writing—original draft, S.L.; writing—review and editing, S.L., X.H., and Q.S.; All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the USDA-ARS Projects 6066-21220-014-000D and 6066-21220-016-00D, Crop Genetics Research Unit at Stoneville, USA.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Relevant data generated or analyzed during this study are included in this article. The mitochondrial genome sequences of D. longicolla isolates have been deposited in the NCBI GenBank database under the BioProject PRJNA1080574.

Acknowledgments

We are grateful to Phillip SanMiguel at Purdue Genomics Core Facility for genome sequencing and the sequencing staff at Novogene for their assistance with this research. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The findings and conclusions in this publication are those of the authors and should not be construed to represent any official USDA or U.S. Government determination or policy. The USDA is an equal opportunity provider and employer.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Circular maps of the mitochondrial genomes of eight Diaporthe longicolla isolates.
Figure 1. Circular maps of the mitochondrial genomes of eight Diaporthe longicolla isolates.
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Figure 2. Codon usage in the mitochondrial genome of Diaporthe longicolla isolate PL185E. Codon families are plotted on the X axis and represented by different color patches. Frequency of codon usage is plotted on the Y axis. The codon usage was calculated by the Sequence Manipulation Suite (https://www.bioinformatics.org/sms2/codon_usage.html, accessed on 1 November 2023) with genetic code 4.
Figure 2. Codon usage in the mitochondrial genome of Diaporthe longicolla isolate PL185E. Codon families are plotted on the X axis and represented by different color patches. Frequency of codon usage is plotted on the Y axis. The codon usage was calculated by the Sequence Manipulation Suite (https://www.bioinformatics.org/sms2/codon_usage.html, accessed on 1 November 2023) with genetic code 4.
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Figure 3. Putative secondary structures of the 23 tRNA genes from PL mitochondrial genome. The tRNAs are labeled with the abbreviations od their corresponding ammino acids. The tRNA arms are illustrated as from trnT. The map of tRNA structures was drawn using the MITOS web server (http://mitos.bioinf.uni-leipzig.de/index.py, accessed on 1 November 2023).
Figure 3. Putative secondary structures of the 23 tRNA genes from PL mitochondrial genome. The tRNAs are labeled with the abbreviations od their corresponding ammino acids. The tRNA arms are illustrated as from trnT. The map of tRNA structures was drawn using the MITOS web server (http://mitos.bioinf.uni-leipzig.de/index.py, accessed on 1 November 2023).
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Figure 4. Dispersed and inverted repeat sequences in Diaporthe longicolla mitochondrial genome. Colors: black, conserved protein-coding, rRNA and tRNA genes; grey, introns; white, intergenic regions. Red lines connect regions of significant (E-value < 1 × 10−10) nucleotide sequence similarity.
Figure 4. Dispersed and inverted repeat sequences in Diaporthe longicolla mitochondrial genome. Colors: black, conserved protein-coding, rRNA and tRNA genes; grey, introns; white, intergenic regions. Red lines connect regions of significant (E-value < 1 × 10−10) nucleotide sequence similarity.
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Table 1. A list of Diaporthe longicolla (syn. Phomopsis longicolla) isolates used in this study and their geographic origins in the U.S. and the year isolated from soybean.
Table 1. A list of Diaporthe longicolla (syn. Phomopsis longicolla) isolates used in this study and their geographic origins in the U.S. and the year isolated from soybean.
IsolateGeographic OriginYear Isolated
PL1Illinois2016
PL6Arkansas2009
PL7Missouri2009
PL10Mississippi2016
PL11Maryland2016
PL185EMississippi2015
MSPL10-6Mississippi2010
TWH P74 aOhio1983
a Type strain TWH P74 (ATCC 60325) was obtained from the American Type Culture Collection (ATCC) in 2010.
Table 2. General features of the mitochondrial genome of Diaporthe longicolla (syn. Phomopsis longicolla).
Table 2. General features of the mitochondrial genome of Diaporthe longicolla (syn. Phomopsis longicolla).
IsolateGenome Size (bp)GC (%)Number of Conserved Protein-Coding GenesNumber of rRNANumber of tRNANumber of IntronsIntronic ORFsFree-Standing ORFsTotal ORFs
PL152,53434.61422398513
PL652,30634.51422399514
PL758,28034.1142231312416
PL1057,06234.2142231210515
PL1155,83734.3142231110515
PL185E58,23434.1142221312517
MSPL10-653,64634.314223109413
TWH P74 a53,43634.414223109514
a Type culture of Diaporthe longicolla (syn. Phomopsis longicolla) from American Type Culture Collection (ATCC 60325).
Table 3. Number of introns identified from the mitochondrial genome of Diaporthe longicolla (syn. Phomopsis longicolla) isolates.
Table 3. Number of introns identified from the mitochondrial genome of Diaporthe longicolla (syn. Phomopsis longicolla) isolates.
Isolateatp6cobcox1cox2cox3nad1nad2nad5rnlTotal
PL11041010119
PL61041010119
PL711511111113
PL1011411111112
PL1111410111111
PL185E11511111113
MSPL10-611410101110
TWH P74 a10510101110
a Type culture from American Type Culture Collection (ATCC 60325).
Table 4. Information of introns identified in the mitochondrial genome of Diaporthe longicolla isolates.
Table 4. Information of introns identified in the mitochondrial genome of Diaporthe longicolla isolates.
GeneIntronPositionIntron Size (bp)Intron TypeORFConserved DomainE-ValueIdentity (%)SimilarityAccession
cox1Intron 133 aa a1406IBorf343GIY-YIG1 × 10−15276Tuber melanosporum Mel28XP_002838532.1
Intron 2198 aa1373-orf305LAGLIDADG_11 × 10−17286Chrysoporthe cubensisYP_009262145.1
Intron 3206 aa1080IBorf328double LAGLIDADG_1088Candida oxycetoniaeYP_008475039.1
Intron 4251 aa1134IBorf349LAGLIDADG_1088Fusarium graminearumYP_001249331.1
Intron 5411 aa1340IBorf441GIY-YIG095Sordaria macrospora k-hellXP_003342392.1
cox2Intron 1385 aa1304IB(3′)orf317GIY-YIG4 × 10−14466Cryphonectria parasiticaAMX22275.1
nad5Intron 1239 aa1039IBorf312LAGLIDADG1 × 10−14471Chrysoporthe austroafricanaYP_009262028.1
atp6Intron 1193 aa1644IC2orf295GIY-YIG2 × 10−14888Podospora anserinaNP_074919.1
rnlIntron 13209 bp2116IAorf516rps3 gene062Chrysoporthe deuterocubensisYP_009262125.1
nad1Intron 1212 aa1201IBorf110GIY-YIG2 × 10−10281Neurospora crassa OR74AYP_009126720.1
cobIntron 1163 aa1316IAorf315LAGLIDADG3 × 10−17985Neurospora crassa OR74AYP_009126715.1
nad2Intron 1551 aa1814IA- bdouble LAGLIDADG_11 × 10−17877Chrysoporthe austroafricanaYP_009262006.1
cox3Intron 1141 aa1622IAorf390double LAGLIDADG_13 × 10−13368Annulohypoxylon stygiumYP_008964946.1
a Amino acid; b Values that were not present or not observed.
Table 5. Single-nucleotide polymorphism (SNP) position and alleles identified among the mitochondrial genomes of Diaporthe longicolla isolates.
Table 5. Single-nucleotide polymorphism (SNP) position and alleles identified among the mitochondrial genomes of Diaporthe longicolla isolates.
Isolates of Diaporthe longicolla
SNP Position in TWH P74 aSNP TypeTWH P74MSPL10-6PL1PL185EPL6PL10PL7PL11
1433G/T G.TTTTT
8256T/GGTGGGGGG
8732A/CCACCCC.C
10,944A/T.A.T..T.
11,734G/T.G.T..T.
11,735T/G.T.G..G.
11,736A/GGAG.....
11,747A/T.A...T..
11,747A/TTAT..T..
20,327C/G.G.CC.CC
22,989T/AATAAAAAA
27,771G/C.G.C.CC.
29,383G/CCG......
32,866G/C.G.CC.CC
33,430G/T.G....T.
33,447G/T.G.T..T.
33,448T/C.T.C..C.
34,426A/G.A....G.
34,427A/G.A....G.
35,718C/TTCT..T..
35,907A/C.A.CC.CC
37,809A/TTA..TT.T
37,809C/TTC..TT.T
38,229T/CCTCCCCCC
39,838T/GGTGGGG.G
40,164C/TTCTTTTTT
40,166G/TTGTTTTTT
40,170A/CCACCCCCC
40,198T/GGTGGGGGG
40,960A/CCACCCCCC
41,001T/GGTGGGG.G
44,675A/T.A.T..T.
44,676T/G.T.G..G.
47,659T/AATAAAAAA
48,512G/T.G....T.
48,513A/G.A....G.
49,744T/GGTGG.GG.
49,752A/GGAGG.GG.
49,753T/CCTCC.CC.
51,544T/C.T.CC.CC
51,948T/AATA.A...
51,949G/AAGA.A...
51,958A/CCAC.C...
51,960A/GGAG.G...
52,745A/TTATTTTTT
52,760A/T.A.T....
52,774T/C.T.C....
52,776T/C.T.C....
a The SNPs among the mitochondrial genome sequences of Diaporthe longicolla isolates were identified using the DNASP6 program [35], and the SNP positions on the type strain TWH P74 (ATCC 60325) were identified by blasting the flanking sequence to the TWH P74 mitochondrial genome sequence.
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Li, S.; Hu, X.; Song, Q. Comparative Analysis of the Mitochondrial Genome Sequences of Diaporthe longicolla (syn. Phomopsis longicolla) Isolates Causing Phomopsis Seed Decay in Soybean. J. Fungi 2024, 10, 570. https://doi.org/10.3390/jof10080570

AMA Style

Li S, Hu X, Song Q. Comparative Analysis of the Mitochondrial Genome Sequences of Diaporthe longicolla (syn. Phomopsis longicolla) Isolates Causing Phomopsis Seed Decay in Soybean. Journal of Fungi. 2024; 10(8):570. https://doi.org/10.3390/jof10080570

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Li, Shuxian, Xiaojun Hu, and Qijian Song. 2024. "Comparative Analysis of the Mitochondrial Genome Sequences of Diaporthe longicolla (syn. Phomopsis longicolla) Isolates Causing Phomopsis Seed Decay in Soybean" Journal of Fungi 10, no. 8: 570. https://doi.org/10.3390/jof10080570

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