Accumulation of the spontaneous and random mutations is causative of fungal culture degeneration

Abstract

Filamentous fungi frequently degenerate during subculturing, which manifests as the reduction or loss of conidiation, sexuality, secondary metabolite production, and/or virulence against hosts. The underlying mechanism of spontaneous fungal degeneration is still elusive. In this study, the fluffy mycelium-type sector variants formed by three ascomycete fungi were transferred and found to show the typical features of culture degeneration. The variant cells were evidenced with the accumulation of reactive oxygen species (ROS), and the ROS-associated formation of hyphal coils. Genome resequencing of these sector cultures identified substantial random mutation sites in each variant in a trend associated with fungal reproduction style. The high bias towards transversions over transitions was similarly detected in degenerate genomes. Otherwise, a higher number of mutations were accumulated in the intergenic regions of the Metarhizium robertsii and Cordyceps militaris sector genomes, whereas the exonic regions of the Aspergillus nidulans variant genes were detected with a higher mutation rate. Unexpectedly, none of those mutated genes had orthologous relationships among the three sectors, while only a few of them were shared between two fungi. A few transcription factor genes with frameshift mutations in sectors were selected for deletions in parental strains, and the null mutants demonstrated the varied degrees of degenerate phenotypes. In addition to reasoning the causal mechanism of fungal degeneration, our data provide insights to better maintain and monitor fungal culture stability.

Graphical abstract

1. Introduction

During the subculturing maintenance of filamentous fungi, culture degeneration frequently and spontaneously occurs by showing the reduction or loss of conidiation, sexuality, secondary metabolite and enzyme production, and/or virulence against hosts [1], [2], [3], [4], [5], [6]. In the context of agricultural and industrial utilizations, fungal culture deterioration can lead to huge economic losses, especially for the mushroom industry [7], [8], [9], [10]. The practical experience to avoid the related losses is to reduce the frequency of culture subcultivation by bulk storage of seed cultures [6]. For entomopathogenic fungi, such as the Metarhizium and Beauveria species, “rejuvenation” of degenerate cultures was recommended and tested by passing through insect hosts [9,11]; however, which was contentious especially for the degenerate cultures that have lost their ability to produce conidial spores [12].

Considering the high frequency of occurrence, fungal degeneration was once questioned in association with genetic mutations [9,12,13]. However, genomic and/or mitochondrial DNA methylation/glycosylation that may cause mutagenesis were detected in the degenerate cultures [12], [13], [14]. Differential expression of a long noncoding RNA was related to the degeneration in virulence decline of the aphid-obligate pathogen Conidiobolus obscurus [15]. Chromosome abnormality and instability have also been found in association with fungal degeneration [8,16]. Otherwise, mycovirus infection can lead to fungal culture deterioration and hypovirulence of plant pathogens [17], [18], [19]. Comparative transcriptomic and proteomic investigations revealed that hundreds of genes or proteins could be differentially expressed in the degenerate cultures when compared with the parental strains [5,11,20,21]. Previous investigations also indicated that fungal degeneration demonstrated the sign of cell aging as denoted by the cellular accumulation of reactive oxygen species (ROS) and mitochondrial dysfunction [9,13,22,23]. In support, the overexpression of an antioxidant gene in the medicinal fungus Cordyceps militaris could reduce the rate of culture degeneration and allow degenerate strains to re-form sexual fruiting bodies [24]. Overall, the cause and effect of fungal degeneration remains disputed.

In this study, the sector variants were obtained from three ascomycete fungal species, i.e., Metarhizium robertsii (being heterothallic and asexual), C. militaris (heterothallic and frequently sexual) and Aspergillus nidulans (homothallic and frequently sexual). These cultures showed the typical features of culture degeneration. We performed genome resequencing of these degenerate variants and identified different but considerable numbers of site mutations in each variant. A few genes showing the frameshift or stop-codon loss/gain mutations in sectors were selected for deletion in the parental wild-type (WT) strains, and the null mutants demonstrated the varied degrees of degenerate features.

2. Materials and methods

2.1. Fungal cultures and maintenance

The parental WT strains of M. robertsii ARSEF 2575, C. militaris Cm01 [25], and A. nidulans FGSC A4 were used and maintained on potato dextrose agar (PDA; BD Difco) at 25 °C. The degenerate cultures were obtained from non-sporulation sectors (Fig. 1a) and subcultured on PDA every two weeks over five generations before use for experiments. Both the WT and sector cultures were also inoculated into Sabouraud dextrose broth (SDB; BD Difco) and incubated for a week at 200 rpm and 25 °C in a rotary shaker for metabolite extraction and chromatographic analysis [26]. The sexual fruiting bodies of the C. militaris WT and sector cultures were induced on a rice medium and the pupae of Chinese Tussah silkworm (Antheraea pernyi) by injection with 100 µl of 1 × 10⁷ conidia/ml suspension per insect for 30 days as described before [27]. Total RNA was extracted from the two-week-old WT and sector PDA cultures using the TRIzol reagent (Thermo Fisher Scientific), treated with DNase I (New England Biolabs) and checked with 1% agarose gel to determine the presence or absence of mycovirus in each fungus [28].

Fig. 1.

Phenotyping of fungal culture degeneration. (a) Phenotyping three fungal cultures with and without the formation of sectors (arrowed). Fungi were inoculated on PDA for two weeks. (b) Phenotyping the sector cultures of three fungal species. The cultures were photographed after inoculation on PDA for two weeks. Each culture was isolated from the corresponding sector and subsequently transferred to PDA for five generations before use for experiments. (c, d) Fruiting-body induction of the C. militaris WT and sector on a rice medium (c) and caterpillar pupae (d). Fungi were inoculated on rice or by injection of the Tussah silkworm pupae and incubated at 22 °C for 30 days.

2.2. Cellular oxidative stress and viability assays

Both the WT and sector cultures of three fungal species were inoculated in SDB for three days, and the mycelia were collected by filtration and washing twice with sterile water. The samples were then treated with nitroblue tetrazolium (NBT; Sigma-Aldrich) at a final concentration of 0.3 mM, which can be reduced by ROS to form blue-black/purple precipitate formazan [22]. Fungal protoplasts were prepared for dihydroethidium (DHE; Beyotime) staining by digesting the mycelial samples using the 0.5% lytic enzyme Lywallzyme (Guangdong Institute of Microbiology, Guangzhou, China) dissolved in the osmotic SK buffer (18.22 g sorbitol, and 13.61 g KH₂PO₄ in 100 ml sterile water, pH 5.5). After adding the enzyme, the samples were incubated at 26 °C and 60 rpm for 4 h. The samples were then filtrated through two layers of microCloth (Millipore), and protoplasts were collected by centrifugation at 4 °C and 220 rpm for 10 min. The supernatant was carefully removed and the protoplasts were re-suspended in the osmotic buffer for staining with DHE at a final concentration of 10 µM for 30 min prior to observation with a fluorescence microscope (Nikon DS-Ri2). To determine the viability of the WT and sector protoplasts, we suspended the samples in 2 × SK buffer and fixed them with 8% polyoxymethylene for 30 min. The cells were then centrifuged, washed twice with SK buffer and re-suspended in 2 × SK buffer. The samples were then added with an equal volume of 0.6% Triton X-100 for 5 min, washed twice with SK buffer and then treated with the dye TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; Beyotime) for 60 min. After washing twice with SK buffer, protoplasts were photographed using a fluorescence microscope.

2.3. Induction of hyphal coils

We challenged the WT fungi on PDA amended with the ROS-generating agents paraquat (Aladdin) at a final concentration of 5 mM or H₂O₂ at a final concentration of 40 mM [29]. A sterile cover slip was inserted approximately 0.3 cm away from the inoculation site, and removed 7 days post inoculation to check the formation of hyphal coils under a microscope. The sector culture of C. militaris was also checked for hyphal-coil formation with a scanning electron microscope (SEM; Merlin VP Compact; Zeiss) [30].

2.4. Genome resequencing and analysis

The sector cultures of three fungi were grown in SDB for three days and the mycelia were collected by filtrations and washed thrice with sterile water. The samples were then homogenized in liquid nitrogen into fine powders for genomic DNA extraction using a cetyltrimethyl ammonium bromide (CTAB) method [31]. The Illumina sequencing libraries were prepared using the Illumina TruSeq™ Nano DNA Sample Prep Kit followed by sequencing using the Illumina Hiseq system at the Biozeron Company (Shanghai, China). To verify the single nucleotide variant (SNV) and indel site mutations at the exon regions, we amplified the fragments of different genes by PCR using different primers (Table S1) and the high-fidelity 2 × Phanta Max Master Mix (Vazamy) for Sanger sequencing. The obtained sequences were then aligned and compared with those of the parental strains.

Reads mapping and SNV/indel calling were conducted by reference to the respective genomic data of three fungal species as we described before with different programs [32]. Radar charts were generated to demonstrate SNV and indel site distributions among the intergenic, intron, exon, 5′-untranslated regions (UTR), 3′-UTR and 5′/3′-UTR regions of each genome. To determine whether mutations occurred at the orthologous genes of three fungi, we performed the OrthoMCL analysis of those genes with the exonic mutants [33]. Overlap analysis of mutated genes was performed using the program InteractiVenn [34].

2.5. Gene deletions

To determine the association of gene mutations with sector phenotype variations, we selected the genes with frameshift mutations in sector variants for deletion in the parental strains of three fungi. The putative C2H2-zinc finger transcription factor (TF) gene AN0273 of A. nidulans was detected with multiple indel sites, however, its orthologous genes in C. militaris (CCM_05808, 40% identity at the amino acid level) and M. robertsii [EXV03578 (X797_003378); 47% identity] sector cultures had no mutations (Dataset S1a-S1f). These homologous TF genes were then individually deleted in the WT strains of three fungi. The additional TF genes selected for deletion in the WT strains included EXU95116 (X797_011801, a putative kinase gene) and EXV05492 (X797_000207, a putative GAL4-like Zn2Cys6 TF) of M. robertsii, and CCM_04286 (a putative APSES-type TF) and CCM_05556 (a putative GAL4-like Zn2Cys6 TF) of C. militaris. Gene deletions in M. robertsii and C. militaris were performed by homologous replacement via Agrobacterium-mediated transformation as we described before [35]. The flanking regions of the targeted genes were individually amplified by PCR using different primers (Table S1). Deleting AN0273 was performed in the WT strain of A. nidulans via the CRISPR-Cas9 system by direct transformation of protoplast cells with the Cas9 ribonucleoprotein and guide RNA sequences (Table S1) [36]. In addition, the flanking regions of AN0273 were amplified and fused with a benomyl-resistance gene (Fig. S1a), and the cassette was co-transformed for the selection of the drug-resistance colonies [37]. The successful deletion was confirmed by PCR and sequencing of PCR products.

2.6. Insect bioassays

To determine the effect of gene deletions on M. robertsii virulence, we performed insect survival assays using the spore suspensions (5 × 10⁵ conidia/ml in 0.05% Tween 20) of the WT and null mutants obtained above against the male adults of Drosophila melanogaster [38]. After immersion in spore suspension for 30 s, flies were recorded for mortality every 12 hrs. There were 70–75 flies used for each treatment. The significance of difference in fly survivals was determined by log-rank test [39].

2.7. Data availability

Genomic resequencing data of three sector variants have been deposited at NCBI under the accession PRJNA758737 (SRX11962147 for M. robertsii sector; SRX11962148 for C. militaris sector; and SRX11962146 for A. nidulans sector).

3. Results

3.1. Colony sectorization shows the feature of culture degeneration

During the maintenance of three fungi on PDA, the formation of fluffy and asporogenic mycelium-type sectors could be frequently observed on cultures (Fig. 1a). After transferring and subculturing the sectors for up to five generations, sector cultures completely lost the ability of conidiation and pigmentation (Fig. 1b). We performed the inoculation of the sexually-feasible C. militaris on a rice medium or injection of caterpillar pupae. As a result, in contrast to the parental strain, sector culture failed to form sexual fruiting bodies (Fig. 1c, d). We also examined the difference in production of secondary metabolites between the WT and sector variants after fungal growth in SDB. Consistent with the loss of pigmentation on solid media, the production and accumulation of potential small molecules considerably varied in both culture filtrates and mycelial cells of three fungi (Fig. S2). Our extraction and examination of total RNAs showed that the sector variants and parental cultures of three fungi had the similar 18S and 28S rRNA bands without the indication of mycovirus infection (Fig. S3). Taken together, the sector cultures of three fungi demonstrated the characteristics of culture degeneration.

3.2. Fungal degeneration is associated with cellular oxidative stress

After growing the parent and sector cultures in SDB, we performed the staining of mycelial cells with NBT. As a result, the formation of purple precipitates was more clearly evidenced in sector cells than in the WT hyphae of each fungus (Fig. 2a). We also harvested the protoplast cells for DHE (a superoxide indicator) staining. The following fluorescent microscopy observations indicated that a higher number of sector cells were detected with a strong fluorescent signal than those of the parental strain cells (Fig. 2b). In addition, the TUNEL staining assay revealed that the sector cells of each fungus demonstrated a sign of apoptosis when compared with those of the WT strains (Fig. 2c).

Fig. 2.

Evident oxidative stress in degenerate fungal cultures. (a, b) Staining of mycelial cells with NBT (a) and protoplasts with DHE (b) showing the difference in cellular oxidative stress between the WT parent and sector cultures. The fungi were grown in SDB for three days for NBT staining or protoplast isolation for DHE staining. (c) TUNEL staining showing the difference in protoplast apoptosis between the WT and sector samples. Bar, 5 µm.

3.3. Formation of hyphal coils by the degenerate cultures

During microscopic observations, we found that hyphal coils could be formed by three sectors but not by the parental strains when grown on PDA (Fig. 3a). Having shown above the cellular accumulation of ROS in sector cells, we grew the WT cultures on the PDA amended with the oxidative reagents paraquat and H₂O₂ to determine the association between the oxidative stress and hyphal-coil formation. After challenges, the circular or oval coils could also be formed by the WT cultures of three fungi after either treatment (Fig. 3a). A SEM analysis of the C. militaris sector mycelia showed that coil formation started from the branching of a hyphal tip towards a near-by hypha and then the formation of rope-like strands that could be fused as circular coils (Fig. 3b).

Fig. 3.

Formation of hyphal coils by sectors and by WT strains under oxidative stress. (a) Microscopy imaging showing the formation of hyphal coils by the sector cultures of three fungal species in association with oxidative stress. Both WT and sectors were inoculated on PDA or the WT cultures inoculated on PDA amended with paraquat (para, at a final concentration of 0.5 mM) and H₂O₂ (40 mM) for a week before imaging. (b) SEM observation of hyphal-coil formation by the sector culture of C. militaris. B1 panel, the massive coils (e.g., arrowed) formed on the mycelial mat; B2-B7 panels, the selected images showing the process of circular coil formation that started from the branching of a hyphal tip towards a nearby hypha (B2), the formation of hyphal strands (B3-B5) to the fusion of hyphal strands as coils (B6, B7). The culture was inoculated on PDA for a week. Bar, 5 µm.

3.4. Massive mutations detected in degenerated cultures

To unveil the potential genetics associated with fungal culture degeneration, we performed genome resequencing of these three sector variants. By referring to the achieved genome sequences of three fungi (treated as the parental WT genomes) [25,40,41], calling for SNVs and indels identified substantial numbers of mutations in the sector genomes of M. robertsii (6183 in total: 3512 SNVs and 2671 indels), C. militaris (3278: 935 SNVs and 2343 indels), and A. nidulans (1837: 1358 SNVs and 479 indels) (Dataset S1a-S1f). Within each species, a higher number of SNVs were detected in the M. robertsii and A. nidulans sectors while the C. militaris sector had more insertion-type mutations than other types (Fig. 4a). Overall, the mutation numbers demonstrated a trend in association with fungal reproduction style. The homothallic and frequently sexual A. nidulans sector had the lowest spontaneous mutation rate at a genome wide of approximately 62 sites per megabyte (Mb), which was followed by the mutations in the heterothallic and frequently sexual C. militaris sector (101 sites per Mb), and then the heterothallic but rarely sexual M. robertsii variant (148 sites per Mb). The mapping of reads to the available mitochondrial DNA (mtDNA; NC_017896) of A. nidulans identified 34 SNVs and 0 indels (Dataset S1g). This mutation rate (1023 sites per Mb) was significantly higher (> 17-fold) than that of nuclear DNA. The detected SNVs showed a similar pattern of a high rate of transversion over transition in each sector genome: M. robertsii (83.8%), C. militaris (92.9%) and A. nidulans (67.7%) (Fig. 4b).

Fig. 4.

Statistics of site mutations detected in the sector variants of three fungal species. (a) Summary of SNV and indel sites detected in the genomes of three fungal sector variants. (b) The transition/transversion rates of the detected SNVs in three fungal variants. (c, d) Venn diagram analysis of the orthologous genes bearing the exonic SNV (c) and indel (d) mutations among the sectors of three fungi. The numbers in parentheses indicate the total number of corresponding mutations. (e-g) Radar charts showing the distribution of SNVs and indels at the different regions of sector genomes of M. robertsii (e), C. militaris (f) and A. nidulans (g). UTR, untranslated region.

All these mutations in each sector were then mapped to the annotated genome of each parental strain. Our PCR amplification and sequencing of four selected genes from each fungus (Table S2) confirmed the mutations in each sector variant (Fig. S4). OrthoMCL analysis unveiled that the genes with either SNV and/or indel-type mutations were largely species-specific (Fig. 4c, d). In particular, no single orthologous gene was detected with mutations in the sectors of three fungi. Otherwise, four orthologous genes with SNVs were shared between the M. robertsii and A. nidulans sector variants, and two orthologous genes had indels occurring in the M. robertsii and A. nidulans sector genomes. Even being closely related to each other [42], the entomopathogenic fungi M. robertsii and C. militaris had no orthologous gene mutated and accumulated in their degenerated cultures.

Radar chart analysis unveiled the disparate patterns of site mutations in the genomes of three sectors (Fig. 4e-g). Within the genome of the M. robertsii variant, both the SNV and indel mutations largely occurred in the intergenic regions followed by SNV mutation in exons and the insertions/deletions in the 3′-UTRs of different genes (Fig. 4e). The C. militaris sector, however, had SNV and indel mutations largely in the intergenic regions (Fig. 4f). The A. nidulans sector variant genome was detected with SNVs being mainly in exons while the indel sites were rather evenly distributed in different regions (Fig. 4g). In total, C. militaris had the fewest numbers whereas M. robertsii had the highest numbers of mutations in the exons of structural genes (Fig. S5a).

Aside from the largely single-site mutation per gene, there were individual genes mutated with multiple SNV and/or indel sites in each variant (Fig. S5b; Dataset S1a-S1f). For example, the hypothetical gene EXU95690 (X797_011205) of M. robertsii was detected with 25 exonic SNVs and one single-nucleotide deletion in sector, and the putative kinase gene AN10019 of A. nidulans had 12 exonic SNVs in sector variant. Multiple SNVs were also detected in the mt genes of the A. nidulans sector: cytochrome c oxidase subunit I had 9 exonic SNVs while the NADH dehydrogenase subunits 4 (4 nonsynonymous SNVs) and 5 (2 nonsynonymous SNVs) also had multiple mutations in gene exonic regions (Dataset S1g). These site mutations mostly resulted in nonsynonymous SNVs and frameshift indels in each variant (Fig. S5c,d), which would cause the variation or loss of gene functions in sectors.

3.5. Deletion of the mutated genes in the WT strains leading to the degenerated phenotypes

To determine the effect of spontaneous mutations, we performed the deletions of selected genes in the parental strains of three fungi. In particular, the putative TF and kinase genes with frameshift mutations were selected for deletions (Table S3). First, the C2H2 Zn finger-like gene AN0273 was deleted and verified in A. nidulans (Fig. S1b). The independent mutants had the similar phenotypes of non-sporulation and heavy pigmentation changes on PDA when compared with those of the WT strain (Fig. 5a). The phenotypes of these mutants were similar to the Aspergillus sector variant, a good indication of the connection between gene mutation and fungal degeneration. We found that the AN0273 orthologous gene of M. robertsii (EXV03578) and C. militaris (CCM_05808) had no mutation in their sectors. To examine whether a similar phenotype could be obtained after gene deletions, we also deleted these two genes in the parental strains of M. robertsii and C. militaris (Fig. S1b), respectively. It was found that the spore production of ΔEXV03578 (P < 0.001) and ΔCCM_05808 (P < 0.05) was significantly reduced but not completely lost when compared with those of WT strains (Fig. 5b-e). In addition, ΔCCM_05808 lost its ability to form sexual fruiting bodies (Fig. 5c).

Fig. 5.

Deletion and characterization of the selected genes in the WT strains of three fungi. (a) Characterizing the growth phenotypes of the parental WT and null mutants of A. nidulans on PDA for two weeks. Three independent mutants of ΔAN0273 are included and the lower panels show the corresponding back feature of the WT and each null mutant. (b) Characterizing the growth phenotypes of the WT and three null mutants of M. robertsii on PDA for two weeks. (c) Characterizing the growth phenotypes of the WT and three null mutants of C. militaris on PDA for two weeks (upper panels) or a rice medium for 30 days (lower panels). (d, e) Quantification and comparison of spore production between the WT and mutants of M. robertsii (d) and C. militaris (e) after growth on PDA for two weeks. There were five independent plates sampled for each strain. Two-tailed Student's t-test was conducted to determine the significance of difference between the WT and individual mutant: *, P < 0.05; **, P < 0.01; ***, P < 0.001. NS, not significant. (f) Survival assays of the WT and three null mutants of M. robertsii against Drosophila adults. Control (CK) insects were treated with 0.05% Tween 20.

Additional deletions of EXU95516 (a putative kinase gene) and EXV05492 (a Zn2Cys6-type TF) were conducted in the parental strain of M. robertsii. In comparison with that of the WT, the conidiation ability of ΔEXU95516 was significantly (P < 0.001) reduced whereas the sporulation of ΔEXV05492 was not obviously (P > 0.05) impaired (Fig. 5b,d). We also deleted two additional TF genes in C. militaris, i.e., CCM_04286 (an APSES-type TF) and CCM_05556 (a Zn2Cys6-type TF), and found that ΔCCM_04286 could similarly sporulate but could not produce fruiting bodies. However, relative to the WT, the null mutant of CCM_05556 was impaired (P < 0.05) in spore production but could still form the WT-like fruiting bodies (Fig. 5c,e). By using the WT and null mutants of M. robertsii, we performed the topical infection of Drosophila male adults. As a result, deletion of EXV03578 (Log-rank test: χ² = 10.36, P = 0.0013) or EXV05492 (χ² = 4.38, P = 0.0364) significantly reduced fungal virulence when compared with the WT strain. In contrast, fly survivals had no significant (χ² = 0.047, P = 0.8292) difference between the treatments with the WT and ΔEXU95516 spores (Fig. 5f). Overall, deletions of those genes mutated in sector variants could alter the parental strains with phenotype changes more or less similar to individual sector cultures.

4. Discussion

In this study, we performed phenotyping and genome resequencing of three sector variants of fungal species M. robertsii, C. militaris and A. nidulans. It was found that fungal variants demonstrated the similar and typical features of culture degeneration including the loss of sporulation, variation in secondary metabolisms, and cellular accumulation of ROS. Rather unexpectedly, varied but massive mutations were detected in the genomes of these degenerate cultures. For those detected SNVs, the highly biased transversions over transitions were similarly found in sector variants. Deletion of the TF genes with frameshift mutations in sectors could alter the parental strains with the phenotypes being largely similar to the degenerate cultures. Thus, the spontaneous occurrence and accumulation of random mutations would be causative of sector formation and fungal culture degeneration. We also found the causal link between hyphal coil formation and cellular oxidative stress. It was found previously that heavy metals could induce oxidative stress in the dark septate endophytic fungi and the formation of hyphal coils on solid media [43]. Taken together, it suggested that microscopic check of hyphal coil formation, or its absence, might be a feasible method for assessing the quality of seed cultures in economic fungi (e.g., C. militaris) prior to inoculation for mass production. However, the underlying mechanism requires further investigation to solidify the practical application.

Fungal culture sectorization can occur at a high frequency. For example, more than 40% of the same batch cultures of an M. anisopliae strain could form the fluffy mycelium-type sectors [9,13]. This kind of high frequency once questioned the causal link of gene mutations to fungal culture degeneration [12,44]. For example, the spontaneous mutation rate was estimated to range from 7.9 × 10⁻⁹ to 6.9 × 10⁻¹⁰ per base pair per cell division in the budding yeast Saccharomyces cerevisiae [45,46]. The genome sizes of three fungi used in this study range within 30–50 Mb [41,42]. It is still technically challenging to determine the number of cell division cycles after inoculation of fungi on solid media for two weeks. Since mutations can occur per cycle of DNA replication, the lab-maintained parental WT strains might bear nucleotide differences to those achieved genomes that have been sequenced different years ago. This study is somehow similar to the mutation analysis in cancer biology to identify the driver mutations/genes that can promote cancer development whereas those passenger mutations/genes do not [47]. These concepts can be well applied here that the mutation occurrences, if any, in the parental strains without causing fungal degeneration would be the passenger mutations. Parallel sequencing of the start culture and concurrent degenerate variant is still required for comparative analysis to determine the driver gene(s)/mutation(s) that can essentially trigger fungal culture degeneration.

Aside from the detection of synonymous SNVs, the incidence of nonsynonymous SNVs and indels in the intergenic, exonic, intronic and/or 5′/3′-UTRs of drive genes might be the cause of the substantial physiological changes in sectors. In particular, the detection of SNVs and indels in TF genes, kinase genes and the genes with global regulation functions could feasibly lead to sector formation. In support, our gene deletions in parental strains resulted in the sector-like phenotypes. These genes can thus be drivers, at least to some extent, of fungal degeneration. Transversions have greater impacts on shaping DNA structures and larger regulatory effects than transitions [48]. In this respect, our observation of similar transversion biases could also explain the extensive phenotypic and physiological changes of sector variants.

Cellular accumulation of ROS can damage DNA including mtDNA [13]. In turn, the mitochondrial dysfunctions may accelerate the cellular increase of ROS, thus the start of a “vicious cycle” [9,22,49]. Indeed, more frequent mutations were detected in mtDNA than in the nuclear DNA (> 17-fold) of A. nidulans. Besides the upregulation of heat shock proteins, ROS accumulation could quickly trigger the production of pyruvate to scavenge ROS [50,51]. SNVs/Indels were detected in both the intergenic regions and/or introns of pyruvate metabolic genes in the sectors of three fungi. For example, two indels were detected in two introns of the putative pyruvate dehydrogenase kinase (CCM_01061) gene of C. militaris (Dataset S1d). These mutations might lead to the increase of pyruvate dehydrogenase enzyme activity to accelerate the catabolism of pyruvate [52], which would facilitate ROS accumulation as evident in sector cells. We once cloned the antioxidant glutathione peroxidase gene from A. nidulans and overexpressed it in degenerate C. militaris, which could restore the Cordyceps variant to reproduce fruiting bodies [24]. Eliminating deleterious mutation(s) by reducing cellular ROS remains to be determined. Taken together, the data imply that DNA mutation and oxidative stress can be the switchable cause/effect of fungal degeneration.

Mutation rates vary among taxa [53]. The genotype-dependent degeneration/instability has also been found in fungi [23]. Except for the similarly-biased transversions over transitions, the number, region and pattern of SNVs and indels differed substantially among the variants of three fungal species. In particular, only a few orthologous genes were shared between the sectors of two fungi whereas none was shared among the variants of three fungi. The observations thus provided clear evidence of random mutations in individual fungus. Our deletions of the orthologous C2H2 Zn finger-type TF genes in the WT strains of M. robertsii and C. militaris resulted in a substantial reduction but not the complete loss of sporulation as in A. nidulans. Even being mechanistically elusive, this kind of phenotypic divergence has been frequently observed after the deletions of orthologous genes in divergent fungal species [35,54,55]. It has been evidenced in yeast that the higher the transcription level the higher the mutation rate of a gene [56]. The possibility and magnitude of differences in orthologous gene transcription between fungal species remains to be determined.

It was found from a T-DNA insertion library of M. robertsii that the inactivating mutation of the vacuolar arginine exporter gene Vae (X797_006325) resulted in the fluffy sector-like phenotype [57]. With respect to the driver/passenger mutation speculation [47], Vae mutation can be a driver to trigger Metarhizium culture degeneration. Intriguingly, we checked and found that this gene was not detected with any mutation in the sequenced sector genome of M. robertsii. Taken together with the divergent mutations detected in three fungi, there would be multiple genes in controlling fungal culture stability, and different fungal species might have divergent driver genes associated with fungal degeneration. It is worth noting here that multiple sectors could be formed by each fungus after simultaneous batch inoculations. Further investigation is still required to determine whether the conserved (being drivers) or highly random (being passengers) mutations would occur among these independent sectors.

Taking into account of fungal reproduction style, the asexual species M. robertsii sector accumulated the highest number of mutations that roughly doubled the digit of the heterothallically-sexual C. militaris sector while tripling the mutation number detected in the homothallically-sexual A. nidulans variant. The data would imply that fungal sexuality might benefit the suppression or elimination of deleterious mutations. This assumption could be supported by the Fisher-Muller theory that sexual recombination promotes the suppression of the deleterious mutations as well as the combination of the adaptive/beneficial mutations in separate lineages into one genome [58,59]. In further support, we found that the locust-specific M. acridum culture was much more stable than the generalist species M. anisopliae after subculturing or challenge with oxidative stress [13]. As opposed to the asexual M. robertsii and M. anisopliae, host specialization of the basal Metarhizium species like M. acridum has been proposed in association with the retention of sexuality [40]. The higher mutation rates observed in C. militaris than in A. nidulans would suggest that homothallism might be better at limiting mutations than heterothallism. Nevertheless, the latter has been considered to be more advantageous over homothallism in accelerating genetic recombination [60]. It was intriguing to find that the loss of the opposite mating-type (e.g., MAT1–2) partner frequently occurred during subculturing C. militaris, which was associated with fungal degeneration in producing sexual fruiting bodies [61]. In this respect, our observation that C. militaris having higher mutation rates than A. nidulans might be due to the loss of sexuality during the successive transfer of C. militaris. In contrast to sexual species, the asexual isolates/lineages will undergo clonal interference by competing with each other [62,63]. Thus, the variant of M. robertsii might be good at outcompeting the parental cells, which would lead to the accumulation of more mutations than the sectors of sexual fungi. Having known the presence of a high level of genetic divergence among fungal strains [32], further investigations are also required to determine the difference and pattern in mutation and degeneration among fungal strains, which can facilitate the identification of essential driver gene(s) as well as the screening or engineering of genetically-stable isolates for uses in agriculture and/or industry.

In conclusion, we report that fungal culture degeneration is associated with the occurrence of spontaneous and random mutations during subculturing. By providing valuable insights, the results of this study pave the way for improved maintenance and monitoring of fungal lineages with economic significance.

Declaration of competing interest

The authors declare that they have no conflicts of interest in this work.

Biographies

Xuewen Wang is a PhD candidate at the Center for Excellence of Molecular Plant Sciences, Chinese Academy of Sciences. She is mainly focused on the investigations of fungal culture degeneration and genomics.

Chengshu Wang (BRID: 09597.00.07519) is a principle investigator at the Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence of Molecular Plant Sciences, Chinese Academy of Sciences. He received his PhD from China Agricultural University and had his postdoc trainings at the Swansea University and University of Maryland, respectively. His research interests include but are not limited to fungal genetics and fungus-insect interactions. He is the recipients of the National Science Fund for Distinguished Young Scholars of China and the Science Fund for Creative Research Groups of the National Natural Science Foundation of China.