Whole-genome sequencing and analysis of the endophytic fungus Alternaria alternata Y-2 from Leymus chinensis
Authors: Feifei Qin, Li Li, Haoyue Chen, Lixia Yang
Published: 2026

ABSTRACT
To explore the genetic basis and functional potential of beneficial symbiosis between the endophytic fungus Alternaria alternata Y-2 and its host Leymus chinensis, we performed Illumina-based draft whole-genome sequencing and systematic bioinformatic analysis. Although this assembly does not reach telomere-to-telomere completeness, it provides high-quality gene-level information for gene prediction, functional annotation, carbohydrate-active enzyme (CAZyme) identification, and secondary metabolite biosynthetic gene cluster analysis. The final genome size of A. alternata Y-2 was 34,383,676 bp with a GC content of 51.0%, containing 12,724 predicted protein-coding genes, 90 tRNAs, and 12 rRNAs. BUSCO assessment showed 98.9% completeness, supporting the high quality of this draft genome. A total of 12,627 genes were successfully annotated in the NCBI NR database, and 17,183 genes were functionally categorized using GO terms. In total, 448 CAZyme genes and 21 secondary metabolite biosynthetic gene clusters were identified, which are potentially involved in lignocellulose degradation, cellular redox homeostasis and biosynthesis of bioactive metabolites. Based on ITS sequence alignment, NR annotation, and phylogenetic analysis of single-copy orthologous genes, the strain was confidently identified as A. alternata. This study firstly reports the draft genome of an endophytic A. alternata strain derived from L. chinensis and provides valuable genetic resources for exploring the endophytic lifestyle, stress tolerance, and bioactive metabolite potential of this fungus.

Introduction

Endophytic fungi in plants are a type of microorganism that parasitize within the tissues and organs of healthy plants. Broadly speaking, they include mycorrhizal fungi, saprophytic and parasitic fungi, as well as latent pathogenic fungi that are temporarily harmless to the host1. Narrowly defined, they are a group of fungi that infect and colonize the host plant during certain stages or the entire life cycle without causing visible disease symptoms; the latter is the type of endophytic fungi commonly referred to in most studies. Through long-term evolution, endophytic fungi form stable mutualistic symbiosis with host plants. They obtain nutrients for growth while improving host resistance to biotic and abiotic stresses via bioactive metabolites and enzymatic systems2. Extensive studies confirm that endophytes promote plant growth3, regulate immunity, control pests and diseases4, enhance nutrient acquisition5, and strengthen host stress6. Exploring the symbiotic mechanism of endophytes and their roles in host stress resistance has important research significance.

Alternaria sp. is a common type of fungus found in ecosystems, belonging to the Fungi kingdom, Ascomycota phylum, Dothideomycetes class, Pleosporales order, Pleosporaceae family, and genus Alternaria7. The genus Alternaria exhibits strong environmental adaptability and wide ecological distribution, with more than 300 reported species8. Members of this genus can survive as saprophytes, plant pathogens or endophytes9, and possess prominent secondary metabolic capacity to produce insecticidal, antibacterial and antiparasitic metabolites that enhance host stress tolerance. Guo10 discovered that the fermentation broth of the A. alternata experimental strain could significantly inhibit the body length of Caenorhabditis elegans and reduce its lifespan by 55.2%. Although the specific active substance was not clearly determined in the study, it indicated that the metabolites produced by Alternaria fungi have inhibitory effects on C. elegans. Shi11 isolated two compounds, (±)-Alternarlactone A and (±)-Alternarlactone B, from fungus A. alternata P1210. (±)-Alternarlactone A has inhibitory effects on Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum, with IC50 (half maximal inhibitory concentration) values of 21.8, 37.8, 4.7, and 5.9 µg/mL respectively. (±)-Alternarlactone B shows inhibitory effects on T. brucei rhodesiense, L. donovani, and P. falciparum, with IC50 values of 36.9, 8.9, and 9.7 µg/mL respectively. Wang12 isolated six compounds from the endophytic A. alternata in tea fresh branches, namely Altenuene-2-acetoxy ester, Altenuene-3-acetoxy ester, (+)-(10R)−7-hydroxy-3-(2-hydroxy-propyl)−5,6-dimethyl-isochromen-1-one, Alternariol, Phialophoriol, and Altenuene. Among them, Altenuene-2-acetoxy ester, (+)-Altenuene-2-acetoxy ester (MIC(minimum inhibitory concentration)80 = 17.1 ± 1.2 µg/mL), (-) - Altenuene-2-acetoxy ester MIC80 = 15.4 ± 1.1 µg/mL, Altenuene-3-acetoxy ester ((+)-Altenuene-3-acetoxy ester MIC80 = 46.8 ± 2.0 µg/mL, (-)-Altenuene-3-acetoxy ester MIC80 = 45.0 ± 1.7 µg/mL) and Altenuene (MIC80 = 38.9 ± 1.5 µg/mL) have antibacterial activity against Staphylococcus aureus, among these, the compounds that exhibit antibacterial activity against Bacillus subtilis are (+)-(10R)−7-hydroxy-3-(2-hydroxy-propyl)−5,6-dimethyl-isochromen-1-one (MIC80 = 19.7 ± 1.0 µg/mL), Alternariol (MIC80 = 8.6 ± 0.7 µg/mL) and Phialophoriol (MIC80 = 16.7 ± 1.2 µg/mL). To date, no draft genome of endophytic A. alternata derived from L. chinensis has been publicly reported, and genome-wide mining of its functional genes and secondary metabolite biosynthetic genes is still lacking.

At present, the genomic background and functional gene resources of A. alternata symbiotic with L. chinensis are still poorly understood, and the molecular genetic mechanism of its endophytic beneficial characteristics has not been systematically elucidated. To fill this research gap and clarify the molecular genetic mechanism of its endophytic beneficial characteristics, the present study isolated the endophytic fungus A. alternata Y-2 from L. chinensis, and performed whole-genome sequencing and comprehensive functional annotation. We focused on the identification of candidate genes involved in endophytic habitat adaptation, plant cell wall degradation, redox homeostasis and secondary metabolite biosynthesis. The relevant genomic data and analytical findings will lay a scientific foundation for revealing the symbiotic interaction mechanism between A. alternata Y-2 and its host, as well as its potential roles in promoting plant growth and enhancing stress tolerance.

Material for testing

Test plant

The Leymus chinensis plant materials used in this study were collected from Xiertala, Hailar District, Hulunbuir City, Inner Mongolia Autonomous Region. The field sample collection activities have been approved by Hulunbuir City, and have strictly followed the general provisions on grassland protection and sustainable utilization stipulated in local regulations such as the “Regulations on Grasslands of Inner Mongolia Autonomous Region"13. It should be noted that L. chinensis, as a widely distributed grass species, is not included in the “List of National Key Protected Wild Plants"14 of China or the “List of Key Protected Wild Plants of Inner Mongolia Autonomous Region"15. The collection of this plant and plant name does not involve special permits for endangered or specially protected species, and no corresponding licenses are required. The entire research, field investigation and material collection process comply with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora16. All collected plant samples and specimens were expertly identified by Professor Xuewen Huang from Hulunbuir University. The voucher specimens identified have been permanently preserved in the Plant Specimen Collection of Hulunbuir University, which is a public collection institution open to researchers and the public.

Test strains

Alternaria alternata Y‑2 was isolated from the endophytic fungi collected from the L. chinensis in Xiertala, Hailar District, Hulunbuir City, Inner Mongolia Autonomous Region, and was stored in the Molecular and Cell Biology Laboratory of Hulunbuir University.

Methods

Strain isolation

Healthy plants of L. chinensis from Xiertala, Hailar District, Hulunbuir City, Inner Mongolia Autonomous Region were collected and brought back to the laboratory in sterilized sampling bags. They were temporarily stored in a refrigerator at 4℃. The collected L. chinensis leaves were subjected to surface sterilization. The specific procedure was as follows: First, in a ultra-clean workbench, sterilized scissors were used to cut the leaves into approximately 1 cm segments. The leaf segments were then placed in 70% alcohol for 5 s; followed by immersion in 5% sodium hypochlorite solution for 7 min, with continuous shaking during the process; then the sodium hypochlorite solution was discarded and replaced with sterile water, and the leaves were washed with sterile water at least 3 times, each for 30 s. After surface sterilization, the surface-sterilized leaves were inserted at an angle into the prepared potato glucose agar medium and placed at 25℃ for cultivation. Once the fungi grew on the medium, the single colonies were purified, and the purified strains were numbered.

Strain culture

Transfer the fungi A. alternata Y-2 preserved in a 4℃ refrigerator to the surface of a new potato glucose agar medium and incubate for 7 days. Then, use a sterile inoculation needle to pick the mycelia growing on the surface of the potato glucose agar medium and inoculate them into 250 milliliters of potato glucose liquid medium. Cultivate them in a shaking incubator at 25℃ at a speed of 180 revolutions per minute for 5 to 7 days. After filtering the cultured fungi solution with three layers of sterile gauze paper, collect the mycelia, rinse them three times with sterile water, and freeze the treated mycelia in liquid nitrogen for future use.

Extraction of genomic DNA

Genomic DNA was extracted using the Rapid Fungi Genomic DNA Isolation Kit (purchased from Sangon Biotech(Shanghai)Co.,Ltd.). The integrity of the DNA was determined using 1% agarose gel electrophoresis, and the concentration and purity of the DNA were determined using Qubit® (purchased from Life Technologies).

Library construction, sequencing, data quality control and assembly

The whole genome DNA of A. alternata Y‑2 was constructed and sequenced by Sangong Bioengineering (Shanghai) Co. In this study, we used Illumina short‑read sequencing to generate a draft genome assembly, which is sufficient for gene prediction, functional annotation, and comparative genomic analysis. After library construction, the library size was determined by 1% agarose gel electrophoresis, and the library concentration was measured by a Thermo Qubit 4.0 fluorescence quantification instrument17.

The raw image data files obtained by Illumina Hiseq were converted into raw sequenced reads by Base Calling analysis. The raw data quality values and other information were determined, and the quality of the sequencing data of the samples was evaluated visually using FastQC v0.11.218. The raw data was filtered using Trimmomatic v0.3619, which included removing the following: sequences with N bases; splice sequences in reads; low-quality bases (Q-value < 20) starting from the 3′ to 5′ direction of reads; low-quality bases (Q-value < 20) starting from the 5′ to 3′ direction of reads; bases with quality values below 20 in the tails of the reads using the sliding window method (window size of 5 bp); and cases where the read itself along with its paired read had a length of less than 35 nt17.

The second-generation sequencing data was spliced using SPAdes v3.5.020, which first corrects the sequence errors of the original sequence, then assembles the sequences by multiple Kmer values, and finally synthesizes the assembly results of each Kmer value to obtain the best results. Then GapFiller v1.1121 was used to complement GAP on the contig obtained from splicing, and finally PrInSeS‑G v1.0.022 was used for sequence correction to correct the editing errors and the insertion-deletions of small fragments during splicing17. Genome completeness was evaluated using BUSCO v5.8.023 with the lineage dataset pleosporales_odb10.

Gene element prediction

GeneMarkES24 and GlimerHMM25 were used for gene prediction of the assembly results, while RepeatModeler26 was used for the Denovo prediction of repetitive sequences of the assembly results to find the position and frequency of occurrence of each type of repetitive sequences on the genomic segments17.

Gene function annotation

The protein sequences of the predicted genes were aligned with the NR27, SwissProt28, TrEMBL28, COG29, PFAM30, and CDD31 databases to obtain protein functional annotation using NCBI BLAST+ v2.2.2832 software17. GO (Gene Ontology)33 functional annotations were obtained using the SwissProt and TrEMBL databases19, and KEGG (Kyoto Encyclopedia of Genes and Genomes, Kanehisa Laboratory, copyright reserved) annotations34,3536 were obtained using KAAS v2.117,37.

Analysis of resistance assistance function and secondary metabolite‑related genes

The gene set protein sequences were aligned to the CAZy database38 using HMMER v3.1b139 to obtain their corresponding carbohydrate-active enzyme annotation information. The screening condition was E-value<1e − 5. Secondary metabolite synthesis gene clusters in strain Y-2 were predicted using the antiSMASH v6.0.040 online tool. (https://fungismash.secondarymetabolites.org)17.

Genomic Feature Comparison

The genome assembly and annotation information of the reference strain A. alternata SRC1lrK2f were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001642055.1/). The basic genomic parameters of A. alternata Y-2, including genome size, GC content, contig number, contig N50 length, and the number of predicted protein-coding genes, were collected and sorted according to the whole-genome sequencing results. Genome completeness of strain Y-2 and the reference strain was assessed using BUSCO v5.8.023 based on the lineage dataset pleosporales_odb10.

Phylogenetic Analysis Based on Single-Copy Orthologs

Genomic protein sequences of A. alternata Y-2 and twelve closely related Alternaria species were retrieved from the NCBI RefSeq database. OrthoFinder241 was employed to identify orthologous groups and screen single-copy orthologous genes shared across all selected strains. Multiple sequence alignment of single-copy orthologs was performed using MAFFT42, and poorly conserved regions were trimmed subsequently. The concatenated aligned sequences were used to construct a phylogenetic tree by FastTree43 with 1000 bootstrap replicates to assess topological reliability.

Gene Family Expansion and Contraction

Gene family classification of A. alternata Y-2 and related Alternaria species was performed based on the clustering results of OrthoFinder241. A time-calibrated species tree was constructed using single-copy orthologous genes. CAFE544 software with a random birth-and-death model was used to estimate gene family expansion and contraction events at each phylogenetic node. Functional enrichment of expanded and contracted gene families was annotated using KOG, GO, and KEGG databases.

Whole-Genome Collinearity Analysis

Whole-genome collinearity analysis was conducted between A. alternata Y-2 and the reference strain A. alternata SRC1lrK2f. Genome-wide pairwise alignment was performed using MUMmer 445 to detect homologous regions and structural variations.

Results and analysis

Genome assembly

The total base length of all contigs of A. alternata Y‑2 was 34,383,676 bp, with a GC content of 51%. The assembly comprised 421 contigs with an average length of 81,671.44 bp and an N50 of 439,307 bp. The genome showed a moderate contiguity typical for Illumina‑only short-read-based draft assemblies of fungal genomes(Table 1). The assembled genome size and GC content were consistent with the genomic characteristics of most reported A. alternata strains, reflecting the relatively conserved base composition and genome scale within this species. The contig number and N50 value indicated that the Illumina short-read assembly achieved moderate fragmentation and acceptable continuity, which could fully meet the subsequent requirements of gene prediction, repetitive sequence annotation and functional gene mining.

BUSCO assessment indicated 98.9% complete conserved genes, including 98.7% single-copy and 0.2% duplicated BUSCOs, with only 0.5% fragmented and 0.7% missing genes among 6641 searched ortholog groups (Table 2).Such high BUSCO completeness demonstrated that the assembled draft genome had high integrity and low gene loss rate, proving the reliability and high quality of the sequencing and assembly results, and providing a solid data basis for subsequent comparative genomics and secondary metabolite gene cluster analysis.

To visually assess the sequencing uniformity and assembly reliability, the GC-depth distribution of the A. alternata Y‑2 genome was analyzed and plotted (Fig. 1). The points in the plot showed a concentrated distribution pattern, indicating homogeneous sequencing depth and stable GC content across the genome. This visualization further supports the reliability of the current draft genome assembly. Morphological images of A. alternata Y‑2 cultured on PDA medium were supplemented to show the colony and mycelial characteristics (Fig. 2).The results of the genome sequencing of A. alternata Y‑2 were uploaded to NCBI, with BioProject accession number PRJNA1400307 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1400307), Genome accession number JBUZNR000000000 (https://www.ncbi.nlm.nih.gov/nuccore/JBUZNR000000000).

Table 1Assembly results of Alternaria alternata Y‑2.Essential featureY−2Sum of base lengths of all contigs34,383,676Sum of N-containing bases and fuzzy bases8Maximum contig length1,733,055Average contig length81,671.44Number of contigs421N50439,307GC ratio0.51

Table 1Assembly results of Alternaria alternata Y‑2.Essential featureY−2Sum of base lengths of all contigs34,383,676Sum of N-containing bases and fuzzy bases8Maximum contig length1,733,055Average contig length81,671.44Number of contigs421N50439,307GC ratio0.51

Assembly results of Alternaria alternata Y‑2.

Table 2BUSCO evaluation of genome completeness of Alternaria alternata Y-2 based on the pleosporales_odb10 database.BUSCO metricPercentageNumberComplete BUSCOs (C)98.90%6566Complete and single-copy BUSCOs (S)98.70%6555Complete and duplicated BUSCOs (D)0.20%11Fragmented BUSCOs (F)0.50%31Missing BUSCOs (M)0.70%44Total BUSCO groups searched—6641

Table 2BUSCO evaluation of genome completeness of Alternaria alternata Y-2 based on the pleosporales_odb10 database.BUSCO metricPercentageNumberComplete BUSCOs (C)98.90%6566Complete and single-copy BUSCOs (S)98.70%6555Complete and duplicated BUSCOs (D)0.20%11Fragmented BUSCOs (F)0.50%31Missing BUSCOs (M)0.70%44Total BUSCO groups searched—6641

BUSCO evaluation of genome completeness of Alternaria alternata Y-2 based on the pleosporales_odb10 database.

Fig. 1GC-depth distribution plot of the Alternaria alternata Y-2 genome. The horizontal axis represents the GC content of fixed bins across the genome, and the vertical axis indicates the corresponding sequencing depth. Each dot stands for a single bin. The dots show a concentrated distribution, and darker colors denote a higher density of bins. This plot indirectly reflects the quality of genome assembly.

GC-depth distribution plot of the Alternaria alternata Y-2 genome. The horizontal axis represents the GC content of fixed bins across the genome, and the vertical axis indicates the corresponding sequencing depth. Each dot stands for a single bin. The dots show a concentrated distribution, and darker colors denote a higher density of bins. This plot indirectly reflects the quality of genome assembly.

Fig. 2Colonial morphology of Alternaria alternata Y-2 strain cultured on potato dextrose agar (PDA) medium at 25 ℃.

Colonial morphology of Alternaria alternata Y-2 strain cultured on potato dextrose agar (PDA) medium at 25 ℃.

Gene element predictions

Coding gene predictions

GeneMarkerES and GlimerHMM were used to predict the genes of the assembly results (Table 3). A total of 12,724 genes were predicted in the genome of A. alternata Y‑2, and the total length of the genes was 20,373,155, which accounted for 59.25% of the length of the whole genome of A. alternata Y‑2. The minimum gene length was 123 bp, the maximum gene length was 29,591 bp, and the average gene length was 1,601.16 bp.

Table 3Statistics of coding gene prediction results of Alternaria alternata Y‑2.FeatureFeature typeRegion CountBase CountMinimum LengthMaximum LengthAverage LengthPercentage InGenome(%)Coding Genesgene12,72420,373,15512329,5911,601.1659.25

Table 3Statistics of coding gene prediction results of Alternaria alternata Y‑2.FeatureFeature typeRegion CountBase CountMinimum LengthMaximum LengthAverage LengthPercentage InGenome(%)Coding Genesgene12,72420,373,15512329,5911,601.1659.25

Statistics of coding gene prediction results of Alternaria alternata Y‑2.

Repeat sequence prediction

The results of genome repeat sequence predictions of the sequenced strains are shown in Table 4. The results showed that A. alternata Y‑2 contained 4,699 repeat region count, with a total length of 437,822 bp, accounting for 1.27% of the total genome length.

Table 4Repeat sequence statistics.RepeatFamilyRegionCountBaseCountMinimumLengthMaximum LengthAverageLengthPercentage InGenome(%)Unknown946192,576135,038203.570.56Simple repeat3,222135,582626642.080.39Low complexity38317,9951415646.980.05DNA: TcMar-Fot114891,669382,112619.390.27All RepeatTypes4,699437,82265,03893.171.27

Table 4Repeat sequence statistics.RepeatFamilyRegionCountBaseCountMinimumLengthMaximum LengthAverageLengthPercentage InGenome(%)Unknown946192,576135,038203.570.56Simple repeat3,222135,582626642.080.39Low complexity38317,9951415646.980.05DNA: TcMar-Fot114891,669382,112619.390.27All RepeatTypes4,699437,82265,03893.171.27

Repeat sequence statistics.

Non‑coding RNA predictions

Non-coding RNAs are RNAs that do not code for proteins. Different strategies were used to predict the different non-coding RNAs with respect to their structural characteristics. An analysis of the results of the genomic data of A. alternata Y‑2 revealed 90 transporter RNAs (tRNAs) and 12 ribosomal RNAs (rRNAs).

The prediction of genetic elements clearly clarified the basic composition characteristics of the genome of A. alternata Y‑2. The coding genes occupied a relatively high proportion of the whole genome, presenting a wide span of gene length and reasonable average gene size, which reflected the integrity of gene structure and the abundance of genetic information. The repetitive sequences only occupied a small proportion of the genome, among which simple repeats and unknown repetitive elements constituted the main components, while transposable element sequences were relatively few in composition and proportion. The identified non-coding RNAs contained abundant tRNA and rRNA members, which could support the normal translation process and basic life activities of the strain. Collectively, these genetic element characteristics intuitively reflected the structural composition characteristics of the strain Y-2 genome, and laid a reliable data foundation for subsequent functional gene annotation and metabolic pathway analysis.

Gene function annotation

The predicted protein sequences of the genes were compared with the functional databases, and the annotation results of the gene functional analysis are shown in Table 5. The numbers of annotated genes and the corresponding databases were: NR 12,627, KOG 5,501, CDD 4,322, PFAM 6,066, GO 3,733, KEGG 2,285,CAzy 448, VFDB A 159, VFDB B 321 and CARD 100 (Fig. 3).

Table 5Gene function analysis annotation results.DatabaseNumber of genesPercentage(%)Annotated in NR12,62799.24Annotated in KOG5,50143.23Annotated in CDD4,32233.97Annotated in PFAM6,06647.67Annotated in GO3,73329.34Annotated in KEGG2,28517.96Annotated in CAzy4483.52Annotated in VFDB A1591.25Annotated in VFDB B3212.52Annotated in CARD1000.79Annotated in at least one database12,63099.26Annotated in all database00Total genes12,724100

Table 5Gene function analysis annotation results.DatabaseNumber of genesPercentage(%)Annotated in NR12,62799.24Annotated in KOG5,50143.23Annotated in CDD4,32233.97Annotated in PFAM6,06647.67Annotated in GO3,73329.34Annotated in KEGG2,28517.96Annotated in CAzy4483.52Annotated in VFDB A1591.25Annotated in VFDB B3212.52Annotated in CARD1000.79Annotated in at least one database12,63099.26Annotated in all database00Total genes12,724100

Gene function analysis annotation results.

Fig. 3Line chart of database annotation rate. The horizontal axis represents the database name, and the vertical axis represents the proportion of genes annotated to that database.

Line chart of database annotation rate. The horizontal axis represents the database name, and the vertical axis represents the proportion of genes annotated to that database.

NR annotation results

Comparing the genomic genes of A. alternata Y‑2 with the NR database (Fig. 4), a total of 12,627 genes were annotated to the genus Alternaria, indicating that strain Y-2 indeed belongs to this genus. Among the top BLAST hits, 5,091 genes were annotated to A. tenuissima, followed by 4,343 genes matched to A. alternata. The higher gene number annotated to A. tenuissima is due to the extremely close phylogenetic relationship and high sequence similarity among closely related species within Alternaria.

Fig. 4Pie plot of species distribution. NR database species annotation statistical graph. Each sector represents a species, and the larger the sector, the greater the number of sequences aligned to that species.

Pie plot of species distribution. NR database species annotation statistical graph. Each sector represents a species, and the larger the sector, the greater the number of sequences aligned to that species.

GO functional classification annotation results

The predicted genes were categorized into biological process, cellular component and molecular function according to their functions in the GO database. The statistical results of gene functions and numbers of genes of A. alternata Y‑2 annotated in the GO database are shown in Table 6; Fig. 5. There were 17,183 genes belonging to biological process, 26 categories; 6,886 genes belonging to cellular component, 9 categories; and 4,356 genes belonging to molecular function, 12 categories. Among them, the most annotated genes in biological processes are in the cellular process, with 3,479 genes; the most annotated genes in cellular components are in organelle, with 2,941 genes; and the most annotated genes in molecular functions are in catalytic activity, with 1,749 genes.

Table 6GO functional classification statistics.OntologyTermGene NumberBiological processbehavior1biological adhesion36biological phase1biological regulation1492cell aggregation36cell killing2cellular component organization or biogenesis1640cellular process3479detoxification60developmental process298growth193immune system process13localization1097locomotion8metabolic process2806multi-organism process318multicellular organismal process42negative regulation of biological process594positive regulation of biological process655regulation of biological process1244reproduction406reproductive process377response to stimulus1041rhythmic process4signaling393establishment of localization947Cellular componentcell junction1extracellular matrix16extracellular region104protein-containing complex1661membrane1159membrane-enclosed lumen943nucleoid22organelle2941supramolecular fiber39Molecular functionantioxidant activity22binding1591catalytic activity1749electron transfer activity19metallochaperone activity4molecular function regulator213molecular transducer activity15protein tag6structural molecule activity205translation regulator activity57transporter activity262enzyme regulator activity213

Table 6GO functional classification statistics.OntologyTermGene NumberBiological processbehavior1biological adhesion36biological phase1biological regulation1492cell aggregation36cell killing2cellular component organization or biogenesis1640cellular process3479detoxification60developmental process298growth193immune system process13localization1097locomotion8metabolic process2806multi-organism process318multicellular organismal process42negative regulation of biological process594positive regulation of biological process655regulation of biological process1244reproduction406reproductive process377response to stimulus1041rhythmic process4signaling393establishment of localization947Cellular componentcell junction1extracellular matrix16extracellular region104protein-containing complex1661membrane1159membrane-enclosed lumen943nucleoid22organelle2941supramolecular fiber39Molecular functionantioxidant activity22binding1591catalytic activity1749electron transfer activity19metallochaperone activity4molecular function regulator213molecular transducer activity15protein tag6structural molecule activity205translation regulator activity57transporter activity262enzyme regulator activity213

GO functional classification statistics.

Fig. 5GO categorical statistics. The horizontal axis indicates the secondary classifications of GO, and the two vertical axes indicates the number of genes in each classification (right) and their percentage in the total number of annotated genes (left). Different colors represent different orthologs.

GO categorical statistics. The horizontal axis indicates the secondary classifications of GO, and the two vertical axes indicates the number of genes in each classification (right) and their percentage in the total number of annotated genes (left). Different colors represent different orthologs.

KOG Functional classification annotation results

The genomic genes of A. alternata Y‑2 were annotated to the KOG database (Table 7; Fig. 6). The metabolic pathway with the highest number of annotated genes was General function prediction with 1,013; followed by Posttranslational modification, protein turnover, chaperones with 510; Signal transduction mechanisms with 411; and Secondary metabolites biosynthesis, transport and catabolism with 360.

Table 7KOG functional classification statistics.KOG CategoryGene NumberRNA processing and modification223Chromatin structure and dynamics90Energy production and conversion351Cell cycle control, cell division, chromosome partitioning172Amino acid transport and metabolism311Nucleotide transport and metabolism89Carbohydrate transport and metabolism339Coenzyme transport and metabolism100Lipid transport and metabolism356Translation, ribosomal structure and biogenesis339Transcription276Replication, recombination and repair219Cell wall/membrane/envelope biogenesis110Cell motility5Posttranslational modification, protein turnover, chaperones510Inorganic ion transport and metabolism152Secondary metabolites biosynthesis, transport and catabolism360General function prediction only1013Function unknown308Signal transduction mechanisms411Intracellular trafficking, secretion, and vesicular transport285Defense mechanisms47Extracellular structures8Nuclear structure25Cytoskeleton111

Table 7KOG functional classification statistics.KOG CategoryGene NumberRNA processing and modification223Chromatin structure and dynamics90Energy production and conversion351Cell cycle control, cell division, chromosome partitioning172Amino acid transport and metabolism311Nucleotide transport and metabolism89Carbohydrate transport and metabolism339Coenzyme transport and metabolism100Lipid transport and metabolism356Translation, ribosomal structure and biogenesis339Transcription276Replication, recombination and repair219Cell wall/membrane/envelope biogenesis110Cell motility5Posttranslational modification, protein turnover, chaperones510Inorganic ion transport and metabolism152Secondary metabolites biosynthesis, transport and catabolism360General function prediction only1013Function unknown308Signal transduction mechanisms411Intracellular trafficking, secretion, and vesicular transport285Defense mechanisms47Extracellular structures8Nuclear structure25Cytoskeleton111

KOG functional classification statistics.

Fig. 6KOG categorical statistical chart. Each category on the horizontal axis represents a functional classification of a KOG, and the vertical axis represent the number of genes (left) annotated to each classification.

KOG categorical statistical chart. Each category on the horizontal axis represents a functional classification of a KOG, and the vertical axis represent the number of genes (left) annotated to each classification.

KEGG functional classification annotation results

The genes of the A. alternata Y‑2 genome were annotated to the KEGG database in five major categories (Table 8; Fig. 7), including Cellular Processes, Environmental Information Processing, Genetic Information Processing, Metabolism, and Organismal Systems, which is consistent with the standard KEGG pathway classification system. The number of annotated genes in each category was 422, 276, 806, 1951, and 501, respectively. A total of 2285 genes were successfully annotated in KEGG pathways.

Table 8KEGG functional classification statistics. Gene function annotation and pathway classification are based on the KEGG database (Kanehisa Laboratory).TypeSubgroupGene NumberCellular ProcessesCell growth and death130Cell motility22Cellular community53Transport and catabolism217Environmental Information ProcessingMembrane transport12Signal transduction260Signaling molecules and interaction4Genetic Information ProcessingFolding sorting and degradation248Replication and repair99Transcription145Translation314MetabolismAmino acid metabolism317Biosynthesis of other secondary metabolites85Carbohydrate metabolism363Energy metabolism161Glycan biosynthesis and metabolism86Lipid metabolism170Metabolism of cofactors and vitamins167Metabolism of other amino acids96Metabolism of terpenoids and polyketides34Nucleotide metabolism117Overview235Xenobiotics biodegradation and metabolism120Organismal SystemsAging58Circulatory system30Development18Digestive system40Endocrine system131Environmental adaptation22Excretory system36Immune system77Nervous system79Sensory system10

Table 8KEGG functional classification statistics. Gene function annotation and pathway classification are based on the KEGG database (Kanehisa Laboratory).TypeSubgroupGene NumberCellular ProcessesCell growth and death130Cell motility22Cellular community53Transport and catabolism217Environmental Information ProcessingMembrane transport12Signal transduction260Signaling molecules and interaction4Genetic Information ProcessingFolding sorting and degradation248Replication and repair99Transcription145Translation314MetabolismAmino acid metabolism317Biosynthesis of other secondary metabolites85Carbohydrate metabolism363Energy metabolism161Glycan biosynthesis and metabolism86Lipid metabolism170Metabolism of cofactors and vitamins167Metabolism of other amino acids96Metabolism of terpenoids and polyketides34Nucleotide metabolism117Overview235Xenobiotics biodegradation and metabolism120Organismal SystemsAging58Circulatory system30Development18Digestive system40Endocrine system131Environmental adaptation22Excretory system36Immune system77Nervous system79Sensory system10

KEGG functional classification statistics. Gene function annotation and pathway classification are based on the KEGG database (Kanehisa Laboratory).

Fig. 7KEGG categorical statistics.The vertical axis indicates the name of each metabolic pathway involved, and the horizontal axis indicates the number of genes annotated to each pathway. Gene function annotation and pathway classification are based on the KEGG database (Kanehisa Laboratory).

KEGG categorical statistics.The vertical axis indicates the name of each metabolic pathway involved, and the horizontal axis indicates the number of genes annotated to each pathway. Gene function annotation and pathway classification are based on the KEGG database (Kanehisa Laboratory).

Comprehensive analysis of NR, GO, KOG and KEGG annotation results showed that NR database annotation successfully matched most genes to the genus Alternaria, clearly reflecting the species attribution of strain Y-2 at the molecular level. GO annotation distributed annotated genes into three major functional categories and multiple subcategories, covering a complete range of biological processes, cellular component composition and molecular function types, and reflecting the abundance and diversity of gene functions involved in the growth and physiological activities of the strain. KOG annotation revealed the functional distribution of genes involved in basic physiological metabolism, post-translational modification, signal transduction and secondary metabolite synthesis, reflecting the functional configuration characteristics of the genome in maintaining life activities and metabolic regulation. KEGG pathway annotation further allocated genes to multiple primary and secondary metabolic pathways, covering material metabolism, genetic information processing, environmental response and systematic physiological processes, systematically revealing the overall pathway composition and functional metabolic characteristics of the A. alternata Y‑2 genome. All annotation results complemented and supported each other, fully reflecting the functional richness and systematicness of the genome and laying a solid foundation for subsequent exploration of endophytic adaptation and metabolite synthesis characteristics.

Analysis of secondary metabolite-related genes

Carbohydrate‑active enzymes (CAZymes)

Endophytic fungi secrete a variety of carbohydrate-active enzymes, which can be subdivided into different families according to their functions, such as Glycoside Hydrolases (GHs), Glycosyl Transferases (GTs), Polysaccharide Lyases (PLs), and Carbohydrate Esterases (CEs), Auxiliary Activities (AAs), and Carbohydrate-Binding Modules (CBMs). The protein encoding genes for A. alternata Y‑2 were annotated to the CAZy database with a total of 448 genes (Table 9). The largest number of genes were annotated to the Auxiliary Activities (AAs) family with 185, while the smallest number of genes were annotated to the Carbohydrate-Binding Modules (CBMs) family with 0. The greatest number of genes annotated to the AA family of A. alternata Y‑2 were genes encoding AA3 (60), followed by AA7 (48) and AA9 (34), respectively. The most frequently annotated gene in the GH family was GH43 (14 genes), followed by GH5, GH2, GH31 and GH47 (11, 8, 8 and 8 genes, respectively).

Table 9CAZy annotation of Alternaria alternata Y‑2 genes.CAZy_ClassGene_countGlycoside Hydrolases (GHs)155Glycosyl Transferases (GTs)26Polysaccharide Lyases (PLs)15Carbohydrate Esterases (CEs)67Auxiliary Activities (AAs)185Carbohydrate-Binding Modules (CBMs)0

Table 9CAZy annotation of Alternaria alternata Y‑2 genes.CAZy_ClassGene_countGlycoside Hydrolases (GHs)155Glycosyl Transferases (GTs)26Polysaccharide Lyases (PLs)15Carbohydrate Esterases (CEs)67Auxiliary Activities (AAs)185Carbohydrate-Binding Modules (CBMs)0

CAZy annotation of Alternaria alternata Y‑2 genes.

Secondary metabolic gene clusters

Secondary metabolites are crucial factors for endophytic fungi to assist host plants in resisting adverse external environments. Various bioactive secondary metabolites, including polyketides, non-ribosomal peptides, terpenoids, and alkaloids, are all utilized to help the host resist adverse external conditions. A total of 21 gene clusters were identified in the genome of A. alternata Y‑2. The highest percentages were in the polyketide synthase gene clusters of type I (T1PKS), non-ribosomal peptide synthase like gene clusters (NRPS-Like), non-ribosomal peptides (NRPS), terpenes (TERPENE) and T1PKS/NRPS ( Table 10; Fig. 8).

Table 10Secondary metabolite annotation of Alternaria alternata Y‑2.T1PKSNRPSNRPS-likeNRPS, T1PKSterpeneGenes1035085812Gene Clusters84612

Table 10Secondary metabolite annotation of Alternaria alternata Y‑2.T1PKSNRPSNRPS-likeNRPS, T1PKSterpeneGenes1035085812Gene Clusters84612

Secondary metabolite annotation of Alternaria alternata Y‑2.

Fig. 8Secondary metabolite annotation of Alternaria alternata Y‑2.

Secondary metabolite annotation of Alternaria alternata Y‑2.

A BLAST comparison of all A. alternata Y‑2 gene clusters with known secondary metabolite gene clusters revealed that the ctg00001 genes in the T1PKS, NRPS gene cluster type had 100% similarity to the cluster encoding melanin/alternariol. The ctg00009 gene in the T1PKS gene cluster type had 25% similarity to the cluster encoding abscisic acid. The ctg00014 gene in the NRPS, T1PKS gene cluster type showed 45% similarity to the cluster of genes encoding equisetin. The ctg00022 gene in the T1PKS gene cluster type showed 80% similarity to the cluster of genes encoding alternapyrone. The ctg00026 gene in the NRPS gene cluster type showed 100% similarity to the cluster of genes encoding dimethylcoprogen. The ctg00044 gene in the terpene gene cluster type showed 40% similarity to the gene cluster encoding squalestatin S1, and the ctg00107 gene in the terpene gene cluster type showed 62% similarity to the gene cluster encoding betaenone A/betaenone B/betaenone C.

The analysis of CAZyme and secondary metabolite biosynthetic gene clusters reflected the inherent functional genetic characteristics of the A. alternata Y‑2 genome. A large number of CAZyme genes were distributed in multiple enzyme families, among which auxiliary activity and glycoside hydrolase genes occupied the main proportion, carrying the functional basis for carbohydrate decomposition and cell wall component utilization. The genome contained abundant secondary metabolite synthesis gene clusters dominated by T1PKS and NRPS-like types, and these clusters shared different degrees of sequence similarity with known synthetic gene clusters, covering the synthesis of melanin, alternariol, alternapyrone and other bioactive compounds. The composition and quantity of carbohydrate-active enzyme genes and secondary metabolic gene clusters jointly reflected the genetic potential of the strain in material degradation, metabolic synthesis and physiological adaptation, and provided key genetic resources for exploring the metabolic function and endophytic interaction characteristics of A. alternata Y-2.

Genomic Feature Comparison

Genomic characteristics of endophytic A. alternata Y-2 were compared with the NCBI reference strain A. alternata SRC1lrK2f (Table 11). Strain Y-2 had a genome size of 34.38 Mb, marginally larger than the 32.99 Mb of the reference strain. In total, 12,724 protein-coding genes were predicted in Y-2, slightly less than the 13,577 genes annotated in the reference strain. The two strains exhibited highly similar GC contents of 51.0% and 51.5%, reflecting conserved genomic base composition. In terms of assembly performance, Y-2 yielded 421 contigs with a contig N50 of 439.31 kb, whereas the reference strain contained only 91 contigs and achieved a higher contig N50 of 1,097.88 kb, demonstrating superior assembly continuity in the reference genome. BUSCO evaluation confirmed a genome completeness of 98.9% for Y-2, comparable to 99.1% of the reference strain. The highly consistent GC content and BUSCO completeness between the two strains indicated stable and conserved genomic composition and genetic integrity within A. alternata. The slight difference in genome size and gene number reflected intraspecific genomic variation, which may be related to host adaptation and endophytic lifestyle differentiation. The lower contig N50 and more fragmented contigs of strain Y-2 were mainly attributed to the limitation of Illumina short-read sequencing, whereas the comparable completeness guaranteed the reliability of subsequent functional annotation and comparative genomic analysis.

Table 11Comparison of genomic characteristics including genome size, GC content, number of genes, N50, and BUSCO completeness among Alternaria alternata Y‑2 and the reference strain Alternaria alternata SRC1lrK2f.StrainGenome size (Mb)Gene numbersContigsContig N50(kb)GC percentBUSCO completenessA. alternata Y-234.3812,724421439.315198.9%A. alternata SRC1lrK2f32.9913,577911,097.8851.599.1%

Table 11Comparison of genomic characteristics including genome size, GC content, number of genes, N50, and BUSCO completeness among Alternaria alternata Y‑2 and the reference strain Alternaria alternata SRC1lrK2f.StrainGenome size (Mb)Gene numbersContigsContig N50(kb)GC percentBUSCO completenessA. alternata Y-234.3812,724421439.315198.9%A. alternata SRC1lrK2f32.9913,577911,097.8851.599.1%

Comparison of genomic characteristics including genome size, GC content, number of genes, N50, and BUSCO completeness among Alternaria alternata Y‑2 and the reference strain Alternaria alternata SRC1lrK2f.

Phylogenetic Analysis Based on Single-Copy Orthologs

A phylogenetic tree was constructed using A. alternata Y-2 and ten related Alternaria species, with two Trichoderma species as outgroups (Fig. 9). Strain Y-2 formed a sister clade with A. alternata SRC1lrK2f, with the node connecting these two strains showing low bootstrap support (BP < 70). The larger clade containing A. alternata Y-2, A. alternata SRC1lrK2f, and A. arborescens FERA_675 received moderate bootstrap support (BP = 75). These results indicate that strain Y-2 is evolutionarily closer to A. alternata SRC1lrK2f than to A. arborescens, supporting its placement within the A. alternata species complex. The phylogenetic tree constructed based on single-copy orthologous genes provided robust molecular evidence for the taxonomic identification of strain Y-2. The low bootstrap value at individual nodes was attributed to the very short evolutionary differentiation time among closely related species in the Alternaria species complex, rather than defects in phylogenetic reconstruction. The clustering result further confirmed that Y-2 belongs to A. alternata, and clarified its evolutionary status and genetic relationship with other related species.

Fig. 9Phylogenetic tree constructed with single-copy orthologous genes showing the evolutionary relationship of Alternaria alternata Y‑2 with related Alternaria species. In the figure, Yang_2 represents Alternaria alternata Y-2.

Phylogenetic tree constructed with single-copy orthologous genes showing the evolutionary relationship of Alternaria alternata Y‑2 with related Alternaria species. In the figure, Yang_2 represents Alternaria alternata Y-2.

Gene Family Expansion and Contraction

Gene family gain/loss analysis was conducted along the phylogenetic tree to explore the evolutionary dynamics of Alternaria species. Compared with its most recent common ancestor, A. alternata Y-2 presented evident gene family expansion and contraction. The magnitude of gene family changes in strain Y-2 was notably larger than that in its closely related strain A. alternata SRC1lrK2f, suggesting a distinct pattern of gene family evolution. Divergence time estimation indicated that the diversification of the A. alternata complex occurred in a relatively recent period, which is consistent with the low bootstrap support of some internal phylogenetic nodes. The remarkable turnover of gene families in strain Y-2 may be an important genomic adaptive strategy to adapt to the endophytic living environment of L. chinensis. The extensive gene family contraction tended to eliminate redundant and non-essential functional genes, while gene family expansion enriched genes related to host colonization, stress tolerance and secondary metabolite biosynthesis. Such differential evolutionary changes further reflect the long-term adaptive coevolution between the endophytic fungus and its host plant, and also lay a genetic foundation for its unique symbiotic lifestyle and environmental adaptability (see Fig. 10).

Fig. 10Gene family expansion and contraction analysis among Alternaria species. The phylogenetic tree illustrates the contraction and expansion of all gene families. The gray text at each node indicates the divergence time, while the red text denotes the values of gene family contraction and expansion between species. In the figure, Yang_2 represents Alternaria alternata Y-2.

Gene family expansion and contraction analysis among Alternaria species. The phylogenetic tree illustrates the contraction and expansion of all gene families. The gray text at each node indicates the divergence time, while the red text denotes the values of gene family contraction and expansion between species. In the figure, Yang_2 represents Alternaria alternata Y-2.

Whole-Genome Collinearity Analysis

Whole-genome collinearity analysis was performed between A. alternata Y-2 and the reference strain A. alternata SRC1lrK2f (Fig. 11). Large collinear blocks were detected between the major contigs of strain Y-2 and the core chromosomes of the reference genome, indicating high macrosynteny in core genomic regions. Structural variations, including translocations and potential inversions, were observed in multiple contigs, particularly in the fragmented regions of the assembly. The high degree of genomic collinearity demonstrated that the core genetic framework was highly conserved between the endophytic strain Y-2 and the reference pathogenic strain of A. alternata. The existing local translocation and inversion events implied moderate genomic structural differentiation, which may be an important genetic basis for the formation of endophytic lifestyle and adaptive differentiation of strain Y-2. Meanwhile, the partial fragmentation of contigs also corresponded to the limitation of Illumina short-read sequencing in assembling complete chromosomal-level genomes.

Fig. 11Whole-genome collinearity analysis between Alternaria alternata Y‑2 and the reference strain A. alternata SRC1lrK2f. In the figure, Yang_2 represents Alternaria alternata Y-2.

Whole-genome collinearity analysis between Alternaria alternata Y‑2 and the reference strain A. alternata SRC1lrK2f. In the figure, Yang_2 represents Alternaria alternata Y-2.

Discussion

Alternaria fungi are widely distributed in natural ecosystems with rich species diversity and strong environmental adaptability. Some members act as plant pathogens, while many also exhibit prominent secondary metabolic capacity to produce antibacterial, insecticidal and antioxidant compounds, showing great potential for biological resource development46. Traditional isolation and identification methods are insufficient to reveal the molecular characteristics and functional mechanisms of endophytic A. alternata. Therefore, whole-genome sequencing and bioinformatic analysis were performed in this study to clarify the genomic basis of strain Y-2 isolated from L. chinensis. The obtained draft genome shows high BUSCO completeness and consistent genomic features with most reported A. alternata strains, providing reliable genetic data for gene prediction, functional annotation, CAZyme identification and secondary metabolite gene cluster mining.

As endophytic microorganisms colonizing internal plant tissues without causing visible disease symptoms, endophytic fungi can enhance host adaptability to biotic and abiotic stresses mainly by producing bioactive metabolites, regulating redox homeostasis and activating defense signaling pathways4748. Consistently, numerous genes encoding oxidoreductases and stress-related proteins were annotated in the strain Y-2 genome, which may participate in reactive oxygen species (ROS) scavenging and oxidative stress tolerance under drought, salinity and pathogen infection conditions. These genomic features imply a potential role of A. alternata Y-2 in assisting host stress adaptation, although further molecular and physiological validation is still required4950.

Comparative genomic analysis between A. alternata Y-2 and the reference strain A. alternata SRC1lrK2f revealed highly conserved GC content, BUSCO completeness and core genomic framework, whereas differences in contig number and N50 reflected the limitation of Illumina short-read assembly. Genome collinearity analysis showed high macrosynteny in core regions, while local translocation and inversion events indicated moderate structural differentiation. Compared with pathogenic Alternaria strains, the contraction of pathogenicity-related gene families in strain Y-2 further supports its beneficial endophytic lifestyle rather than pathogenicity.

Phylogenetic analysis based on single-copy orthologous genes clustered A. alternata Y-2 closely with A. alternata SRC1lrK2f within the A. alternata species complex. Low bootstrap values at several internal nodes were mainly attributed to the short evolutionary divergence time among closely related species, rather than defects in phylogenetic reconstruction. Combined with ITS sequence similarity, NR annotation and genomic characteristic comparison, multiple lines of molecular evidence consistently confirmed that strain Y-2 belongs to A. alternata.

Carbohydrate-active enzymes play essential roles in plant cell wall degradation, nutrient utilization and host colonization. A total of 448 CAZyme genes were identified in A. alternata Y-2, dominated by Auxiliary Activities (AAs) and Glycoside Hydrolases (GHs) families. The enrichment of AA3, AA7 and AA9 members suggests strong potential capability for lignocellulose decomposition and redox regulation, which may facilitate successful colonization in host tissues. The composition and quantity of CAZyme genes in strainY-2 are comparable to those of typical endophytic Alternaria strains, reflecting adaptive genomic characteristics for symbiotic lifestyle.

A total of 21 secondary metabolite biosynthetic gene clusters were predicted in A. alternata Y-2, including T1PKS, NRPS, NRPS-like and terpene clusters. Homology comparison showed that these clusters share high similarity with gene clusters responsible for the biosynthesis of alternariol, alternapyrone, dimethylcoprogen and other bioactive compounds. These metabolites have been reported to possess antibacterial, antioxidant and allelopathic activities, implying that strain Y-2 has considerable potential to synthesize bioactive secondary metabolites. The distinct gene arrangement of several clusters in strain Y-2 may contribute to its unique metabolic profile and ecological adaptation.

Gene family expansion and contraction analysis indicated obvious genomic evolutionary dynamics in A. alternata Y-2. Specific expansion of gene families related to stress tolerance and secondary metabolism, together with the contraction of redundant pathogenic genes, may represent important adaptive strategies for long-term coevolution with L. chinensis. These genomic variations lay a molecular foundation for the endophytic habit and environmental adaptability of strain Y-2.

This study still has certain limitations. All gene functions and metabolic pathways were predicted based on bioinformatics homology analysis; in vitro and in vivo functional verification, including gene knockout, transcriptome and metabolome detection, are needed to confirm actual biological roles. In addition, the current genome assembly based only on Illumina short reads remains fragmented, and long-read sequencing is recommended in future research to achieve chromosome-level assembly and more complete gene cluster analysis.

In conclusion, this study firstly reports the draft genome of endophytic A. alternata from L. chinensis. The genomic data, functional annotation and comparative analysis enrich the genetic resources of endophytic Alternaria, and provide a fundamental basis for further exploring the molecular mechanism of fungus–host symbiosis, stress tolerance and utilization of bioactive metabolites.