DNA Polymerase Theta Regulates the Growth and Development of Fusarium acuminatum and Its Virulence on Alfalfa

Abstract

Fusarium acuminatum is a major pathogenic fungus causing root rot in alfalfa (Medicago sativa). DNA polymerase theta is known to play a crucial role in repairing DNA double-strand breaks. However, its biological function in F. acuminatum remains unknown. In this study, the POLQ gene was deleted by homologous recombination using Agrobacterium tumefaciens-mediated transformation. Compared to the wild type (with the POLQ gene), the mutants (without the POLQ gene) showed significant phenotypic changes: they produced brown-yellow pigments instead of pink, slowed mycelial growth, and exhibited changes in macroconidia size and shape. The virulence of the mutants was greatly reduced, inducing only mild symptoms in alfalfa. In addition, FITC-WGA staining showed impaired spore germination and hyphal growth. These results suggest that POLQ is a key gene regulating growth and development of F. acuminatum, indicating that DNA repair may play an essential role in the pathogenicity of the pathogen in alfalfa. The POLQ gene could thus be a promising target for limiting F. acuminatum infections in alfalfa.

1. Introduction

Fusarium, as a soil-borne fungus, is highly destructive and severely damages crop production [1]. It poses a significant threat to alfalfa (Medicago sativa) cultivation, causing global yield losses of 20% to 40% annually. Severe outbreaks can lead to mortality rates over 60%, severely impacting the industry’s growth. Fusarium root rot is widespread across different geographical regions in China, with regional variation in Fusarium species. Among the pathogens responsible for alfalfa root rot in Gansu Province, a major alfalfa-producing region in China, F. acuminatum shows the highest isolation frequency. Fusarium acuminatum is one of the most toxic species within the Fusarium genus, known for secreting mycotoxins like fumonisins and trichothecenes, which can cause plant tissue death and dysfunction [2]. These trichothecene mycotoxins inhibit peptidyl transferase activity in eukaryotic cells, significantly suppressing protein synthesis and thereby reducing host crop yield [3]. F. acuminatum is also capable of producing other toxins, including DAS (Diacetoxyscirpenol), NEO (Neosolaniol), and T-2 toxin (T-2 Mycotoxin) [4,5], which lead to host tissue necrosis. Its macroconidia are typically fusiform and can be quite large, reaching lengths of up to 114.7 μm [6]. F. acuminatum is highly adaptable, capable of parasitizing various hosts, including fruits and crops, resulting in varying levels of virulence [7]. In our previous research, we isolated F. acuminatum from alfalfa with root rot in Gansu Province, a major alfalfa production region in China. We developed a low-cost, rapid virulence test that confirmed F. acuminatum’s virulence in alfalfa, manifesting as typical root rot symptoms, such as leaf yellowing and brown lesions on the root and stem. Additionally, we discovered that F. acuminatum can extensively colonize the root and invade the xylem vessels within 48 h post inoculation [8]. Current research on F. acuminatum has mainly focused on morphological characterization, phylogenetic analysis, and toxin profiling [4,6,9], while molecular studies on its pathogenic mechanisms remain extremely limited. The genetic and molecular basis underlying the pathogenicity of F. acuminatum thus remains largely unknown.

DNA polymerase theta (encoded by the POLQ gene) is a crucial enzyme in the microhomology-mediated end joining DNA repair pathway (also called Theta Mediated End Joining), which serves as an alternative to the more accurate homologous recombination (HR) repair mechanism [10]. Polymerase theta is widely conserved in animals and plants, but not in fungi; for example, it is absent in some unicellular fungi like yeast [11]. Given the importance of DNA repair pathways in maintaining genome stability and regulating pathogenicity in fungi, investigating the potential role of POLQ in F. acuminatum is of particular interest. Studies in model organisms such as Caenorhabditis elegans, Arabidopsis thaliana, and Drosophila melanogaster have shown that POLQ or its homologs are essential for resistance to DNA interstrand cross-linking agents and for normal development [12,13,14]. In humans, increased POLQ mRNA expression has been observed in some tumor samples from the stomach, lung, and particularly colon cancers, compared to matched non-tumor tissues [15,16]. Consequently, POLQ is emerging as a promising target for cancer therapies, especially for treating HR-deficient tumors [17]. However, the functions of POLQ in fungi remain entirely unknown. Based on the above research, we propose that POLQ in F. acuminatum may not only be involved in DNA damage repair but may also potentially affect fungal development and pathogenicity.

In this study, we aimed to investigate the function of the POLQ gene in F. acuminatum by generating gene knockout mutants through homologous recombination via Agrobacterium tumefaciens-mediated transformation. We focused on analyzing the phenotypic changes associated with POLQ gene inactivation, particularly its impact on conidia growth, development, and virulence in alfalfa. To assess the virulence of the POLQ mutant strains, we conducted infection tests on alfalfa seedlings using the water agar root immersion method. Root rot severity served as an indicator of pathogenicity, with more extensive decay reflecting higher virulence. Additionally, we performed FITC-WGA labeling on the roots of alfalfa seedlings infected with the POLQ mutant and wild-type strains, and examined the hyphal invasion state using laser scanning confocal microscopy 48 h post inoculation. This approach provided insights into the infection mechanisms and the role of POLQ in fungal virulence. Our findings contribute to a deeper understanding of DNA repair pathways involved in fungal pathogenesis and highlight POLQ as a potential target for controlling fungal diseases in plants.

2. Materials and Methods

2.1. Plant Material

The plant material used in this study was alfalfa (Medicago sativa L.) cultivar ‘VISION’, a commonly used forage variety known for its high yield and disease resistance. Seeds of the ‘VISION’ variety were obtained from CLOVER Ecological Technology Co., Ltd. (Beijing, China). To prepare for germination, alfalfa seeds were initially sterilized by a brief 15 s exposure to 70% ethanol, followed by 3 short rinses in sterile distilled water (10 s each). The ethanol-treated seeds were then disinfected using a 10% household bleach solution supplemented with 1% Triton X-100 for 15 min. Afterward, seeds were thoroughly rinsed three times with sterile water to remove any remaining bleach. The sterilized seeds were placed on water agar (1% agar) and incubated in a growth chamber set to 22 °C with a 16 h light and an 8 h dark cycle.

2.2. Fungal Material

The Fusarium acuminatum strain 1A used in this study for pathogenicity and mechanistic analyses was isolated from infected alfalfa roots by our research group at Beijing Forestry University. To verify the strain’s identity and ensure its purity, single-spore isolation was conducted, followed by morphological examination and sequencing of the internal transcribed spacer (ITS) region. The isolate was routinely cultured on potato dextrose agar (PDA) at 25 °C in the dark and transferred to fresh medium every two or three weeks for maintenance.

2.3. Phylogenetic Analysis

To investigate the evolutionary relationships of polymerase theta proteins, multiple sequence alignment was performed using the MUSCLE algorithm implemented in the MEGA X software package [18]. This alignment ensured accurate comparison of conserved and variable regions among the selected protein sequences. Following alignment, a phylogenetic tree was generated using the maximum likelihood (ML) approach, which estimates the tree that most likely reflects the observed sequence data under a specified model of evolution. The LG substitution model with a gamma distribution (LG + GAMMA) was selected to account for variation in substitution rates across sites. Statistical support for the tree topology was evaluated by conducting 1000 bootstrap replicates, providing a robust measure of confidence for each branch in the resulting phylogeny.

2.4. Construction of Knockout Plasmids and Fungal Transformation

To generate the pch-KOpoltheta knockout plasmid, genomic regions flanking the target gene were amplified by PCR using gene-specific primers (listed in Table S1). (The PCR cycling conditions were as follows: 94 °C for 5 min; 94 °C for 30 s; 55 °C for 35 s; 72 °C for 1 min; and 72 °C for 10 min; repeated for 35 cycles.) These included approximately 1.0–1.5 kb of both the upstream (5′) and downstream (3′) sequences surrounding the poltheta gene to ensure homologous recombination. The amplified fragments were cloned into the EcoRI and XbaI restriction sites on either side of the hygromycin B resistance gene within the pch vector. This strategy allowed for the replacement of the native poltheta coding sequence with the resistance cassette through double-crossover recombination.

For fungal transformation, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed [19]. The confirmed pch-KOpoltheta plasmid was first introduced into A. tumefaciens strain EHA105 by the freeze-thaw method. Agrobacterium cultures harboring the binary vector were grown to an appropriate optical density and then co-cultivated with fungal spores or mycelial fragments on induction medium containing acetosyringone to activate the virulence genes necessary for T-DNA transfer. After 2–3 days of co-cultivation at 22–25 °C in the dark, the fungal tissue was transferred to selective medium containing hygromycin B and cefotaxime to inhibit Agrobacterium growth. Putative transformants were isolated after 7–10 days and subcultured onto fresh selection plates. Successful gene disruption was confirmed by PCR screening using primers flanking the integration sites.

2.5. Fungal DNA Extraction

Genomic DNA was extracted from Fusarium mycelium grown on potato dextrose agar (PDA) (standard composition) for seven days. The fungal cultures were first harvested and flash-frozen in liquid nitrogen, then ground into a fine powder using a pre-chilled mortar and pestle. Approximately 100 mg of the frozen mycelial powder was used for DNA extraction following a modified cetyltrimethylammonium bromide (CTAB) protocol as described by [20]. Briefly, the ground mycelium was suspended in preheated CTAB extraction buffer incubated at 65 °C for 30–60 min to lyse the cells and release nucleic acids. After incubation, an equal volume of chloroform:isoamyl alcohol (24:1) was added to the mixture and centrifuged to separate the aqueous phase containing DNA. The supernatant was carefully transferred to a new tube, and DNA was precipitated using isopropanol. The DNA pellet was washed with 70% ethanol, air-dried, and resuspended in TE buffer. The quality and concentration of the extracted DNA were assessed using agarose gel electrophoresis and spectrophotometric measurement.

2.6. Colony Observation and Spore Morphology

The fungal transformants, along with the wild-type strains, were cultured on PDA agar plates in the dark at a constant temperature of 25 °C for a period of two weeks. Colony phenotypes were carefully observed and documented by photographing the colonies at regular intervals. Starting from the point of transfer, the diameters of the colonies were measured every three days using a ruler and recorded. Each strain was analyzed with three biological replicates to ensure statistical reliability. The colony growth was quantified using ImageJ software, which enabled precise measurement of colony diameters. From these measurements, the average growth rate of the transformant and wild-type mycelium was calculated over the experimental period.

For microscopic observation, a small sample of mycelium from the transformant cultures was collected and placed on a glass microscope slide. The mycelium was then rehydrated in 10 μL of double-distilled water (ddH₂O) and covered with a cover slip. Spore morphology was assessed by examining the sample under an optical microscope at 100× magnification. At least 20 spores from each strain were measured for their length and width using ImageJ software to ensure accurate and consistent measurements. The spore dimensions were then statistically analyzed, and violin plots comparing the spore size distributions between the transformant and wild-type strains were generated using Origin 2021 software.

2.7. Virulence Analysis

The virulence test was performed as described by Yang et al. (2024) [8]. For the inoculation, fungal conidia were harvested from five-day-old liquid potato dextrose cultures by filtering through two layers of Miracloth (Millipore, Burlington, MA, USA). The conidial concentration was determined using a hemacytometer under a light microscope at 10× magnification. The concentration was calculated using the following formula:

$$\mathrm{Conidia\; concentration}=\mathrm{N}/5\times 25\times {10}^4\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{L}$$

where N is the total number of conidia counted in five squares (four corner squares and the central square) of the hemacytometer grid [8]. The conidial solution was adjusted to a final concentration of approximately 1 × 10⁶ conidia per ml. A 10 μL aliquot of the conidial suspension was then applied to the root tips of the alfalfa seedlings.

Virulence was assessed by monitoring the root decay symptoms that developed after inoculation. Over time, alfalfa roots exhibited various lesions, which appeared in different colors, including brown, yellow, and red. Some roots softened and rotted at the crown, while others showed signs of chlorosis. The severity of root decay typically stabilized 4 weeks after inoculation. At that time, virulence was quantified based on a Disease Grade scale, as follows: Grade 1 (slightly rotten): 0–25% of the root area affected by lesions or rotting, with minimal disease symptoms; Grade 2 (rotten): 25–50% of the root area affected by lesions or rotting; Grade 3 (moderately rotten): 50–75% of the root area affected by lesions or rotting; Grade 4 (severely rotten): 75–100% of the root area affected by lesions or rotting, and the seedling was either wilted or dead [8]. For each fungal isolate, the experiment was conducted with 30 alfalfa seedlings per trial, and the experiment was repeated three times for each isolate. The virulence of each Fusarium isolate was visually assessed by examining the extent and severity of the root lesions.

2.8. Microscopic Observation of Infected Tissues

Histopathological analysis was carried out following the protocol described by Yang et al. (2024) [8]. After inoculation with Fusarium conidia, the alfalfa seedlings were harvested for fungal tissue observation. The seedlings were first immersed in a 10% potassium hydroxide (KOH) solution to macerate the plant tissue and clear the cell walls, allowing better visualization of the fungal structures. After soaking in KOH, the tissues were neutralized by immersing them in a 2% hydrochloric acid (HCl) solution to halt the KOH action. Following neutralization, the seedlings were thoroughly washed in 1× phosphate-buffered saline (PBS) buffer to remove any excess KOH or HCl.

Subsequently, the seedlings were stained using a fluorescein isothiocyanate-labeled wheat germ agglutinin (FITC-WGA) solution to visualize fungal structures. The FITC-WGA staining solution was prepared by combining 1 mL of 1× PBS, 2 μL of FITC-WGA, and 20 μL of 0.02% Tween 20 to facilitate the binding of WGA to fungal cell walls. The seedlings were then immersed in this staining solution and stored overnight at 4 °C to ensure adequate dye uptake by the fungal hyphae and spores.

Following staining, the seedlings were carefully rinsed with PBS to remove any unbound dye. FITC-WGA selectively labels fungal hyphae and spores by binding specifically to chitin in fungal cell walls, which is absent in plant tissues. This selective binding significantly enhances imaging contrast at the fungal–plant interaction interface. Fungal structures were then visualized using a Confocal Laser Scanning Microscope (CLSM) to obtain high-resolution images of fungal colonization and interaction with plant tissues. Images were acquired using 488 nm excitation, 500–550 nm emission, and a 40×/1.30 NA oil-immersion objective.

2.9. Reverse Transcription and Polymerase Chain Reaction

Mycelium from the isolated strains was scraped and placed into centrifuge tubes, rapidly frozen in liquid nitrogen, and quickly ground into powder. Total RNA was extracted using RNA Isolator Total RNA Extraction Reagent (Vazyme, R401-01, Nanjing, China). RNA was reverse transcribed into cDNA using the BeyoRT™ II First Strand cDNA Synthesis Kit with gDNA Eraser, which was then used as the template for RT-PCR. β-tubulin was used as the internal reference gene, with primers for Tubulin gene (Table S1). RT-qPCR was performed using Taq PCR Mix (Beyotime Biotechnology, Shanghai, China) and primers for the POLQ gene (Table S1). The reaction program was 95 °C for 2 min; 95 °C for 15 s; 60 °C for 30 s; and 72 °C for 30 s, for a total of 40 cycles.

2.10. Statistical Analyses

The size of the mycelium, as well as the length and width of the spores, were measured using ImageJ. Each experiment was performed in triplicate. Standard deviations were calculated, and violin plots, bar charts, and line graphs were generated using Origin 2021.

3. Results

3.1. Phylogenetic Analysis of DNA Polymerase Theta in Eukaryotes

We hypothesized that DNA polymerase theta may be present in fungi, contrary to the previous conclusion [11]. To test this, we selected 68 protein sequences of DNA polymerase theta—comprising 2 from plants, 30 from animals, and 36 from fungi (Table S2)—and constructed a phylogenetic tree (Figure 1). The tree reveals three main clades: one consisting of plant species, one of animal species, and one of fungal species. The fungal clade is more closely related to the animal clade than to the plant clade, consistent with the widely accepted evolutionary relationship among fungi, animals and plants. Within the fungal clade, DNA polymerase theta from Fusarium forms a subclade that groups with sequences from Hapsidospora chrysogenum and is closely related to another subclade containing Metarhizium anisopliae and Cordyceps militaris. Additionally, the DNA polymerase theta from F. acuminatum 1A is most closely related to that of F. sporotrichioides (Figure 1). The bootstrap support values for most branches are above 60%, providing strong statistical confidence in the tree’s structure.

3.2. Generation of POLQ Knockout Mutant

To investigate the function of POLQ, we generated a knockout mutant using homologous recombination via Agrobacterium-mediated transformation. Although the transformation efficiency was very low, we successfully obtained two colonies through selection on hygromycin plates. This low efficiency is likely due to the absence of established protocols for genetic manipulation in F. acuminatum, in contrast to the well-established and systematic transformation systems available for F. oxysporum. PCR analysis confirmed that the POLQ coding sequence was replaced by the hygromycin resistance cassette (Figure 2d), using primers targeting the upstream and downstream homology arms, as well as primers specific to the hygromycin cassette (Figure 2a). Negative selection with G418 further confirmed that the T-DNA was not randomly integrated into the genome (Figure 2b,c). RT-PCR showed that POLQ is expressed in the wild-type strain but not in the mutant, thereby verifying that the mutants are true knockouts (Figure S1). Together, these results demonstrate the successful generation of the POLQ knockout mutant in F. acuminatum 1A.

3.3. Effects of POLQ Gene Knockout on Mycelium and Conidia

During the cultivation of the POLQ knockout mutant, changes in the mycelium’s color and morphology were observed. As shown in Figure 3a,b, the upper surface of the mutant colony is light yellow with white edges, while the lower surface is tan with distinct brown concentric rings. In contrast, the wild-type 1A colony secretes a pink pigment, resulting in a pink upper surface with a cottony texture and abundant aerial mycelium (Figure 3c,d).

Quantitative analysis revealed that the average mycelial growth rate of the polq mutant was 7.648 ± 0.328 mm/day, compared to 8.325 ± 0.287 mm/day for the wild type (Figure 3e). In addition, on the third day of growth, the colony diameter of the mutant strains was significantly smaller than that of the wild type (p < 0.001) (Figure 3e).

Interestingly, we found that the macroconidia of the polq mutant were slender, transparent, flat-oval, blunt at both ends, and lacked septa (Figure 4a), with dimensions of 13.175–28.247 × 2.636–4.510 µm (mean ± SD = 18.407 ± 3.581 × 3.387 ± 0.495 µm) (Figure 4e,f). The microconidia were kidney-shaped or oval, transparent, tapering gradually at the apex, blunt at the base, and also lacked septa (Figure 4c), with dimensions of 4.637–8.343 × 1.296–2.882 µm (mean ± SD = 6.070 ± 1.017 × 2.002 ± 0.433 µm) (Figure 4g,h). In contrast, the wild-type macroconidia were typically fusiform with 5–7 septa, hook-shaped ends (Figure 4b), and dimensions of 20.709–54.330 × 3.297–6.370 µm (mean ± SD = 36.610 ± 9.418 × 4.416 ± 0.839 µm) (Figure 4e,f). The wild-type microconidia were predominantly fusiform or spindle-shaped, without septa, featuring hook-shaped tops and blunt bases (Figure 4d), with dimensions of 4.019–15.024 × 0.849–3.286 µm (mean ± SD = 8.204 ± 2.889 × 1.727 ± 0.735 µm) (Figure 4g,h).

In summary, the length and width of the polq mutant’s macroconidia were reduced compared to the wild type, while the width of the microconidia was slightly increased.

3.4. Essential Role of POLQ Gene in F. acuminatum 1A Virulence in Alfalfa

To assess whether the virulence of the mutant differed from that of the wild type, we conducted an inoculation test on 1% water agar medium, which has been demonstrated as a robust and reliable condition for virulence testing across different Fusarium species and isolates [8]. Spore suspensions of polq mutant and wild type were inoculated onto the roots and stems of alfalfa. As shown in Figure 5a, the virulence of the mutant was significantly reduced. After four weeks of infection, the alfalfa plants inoculated with the polq mutant spore suspension exhibited minimal disease symptoms. Only slight yellowing of the leaves and a few brown or yellow lesions on the roots were observed; the root systems remained mostly white and appeared much healthier compared to the seedlings inoculated with the wild-type spore suspension.

In contrast, most of the root systems of alfalfa seedlings inoculated with the wild-type spore suspension exhibited extensive browning, with some roots turning black and beginning to rot (Figure 5b). Statistical analysis of disease grades revealed that most seedlings inoculated with the polq mutant spore suspension were categorized as Grade 1, with a Grade 4 incidence rate of only 6.7%. Among the affected roots, the infection severity ranged from 75% to 100%. This was significantly lower than the Grade 4 incidence rate of 21.1% observed for the wild-type 1A (Figure 5c).

To further investigate why the polq mutant exhibited reduced virulence in alfalfa, we performed FITC-WGA staining and CLSM to observe fungal development during root infection. At 48 h post inoculation (hpi), wild-type hyphae densely colonized the xylem with almost none invading the xylem (Figure 5d). Moreover, even after 48 hpi, ungerminated spores or spores in the early stages of germination were still present on the alfalfa root. These results suggest that the polq mutation significantly impairs spore germination and hyphal growth on and within alfalfa roots, likely due to the increased sensitivity of the polq mutant to both abiotic and biotic factors, a characteristic commonly observed in mutants of DNA repair pathways.

4. Discussion

Previously, POLQ was thought to be present in plants, protists, and other multicellular eukaryotes but absent in yeast and other fungi [11]. However, we found that POLQ is present in certain fungal species, such as Fusarium oxysporum, Tolypocladium paradoxum, and Madurella mycetomatis. This absence in earlier studies may have been due to the lack of high-quality fungal genomes more than a decade ago [21,22]. POLQ may still be restricted to multicellular eukaryotes, as we could not identify POLQ homologs in yeast, consistent with previous findings [11]. Our phylogenetic analysis, together with prior studies, suggests that POLQ may have played a role in the evolution of multicellularity from single-celled ancestors. However, we identified POLQ homologs in only a limited number of fungal species, and even within the same genus, some species also lack POLQ homologs. How these fungi acquired POLQ remains unclear. Could it have been obtained through horizontal gene transfer? The availability of more high-quality fungal genomes may help resolve this question.

Loss of POLQ in fruit flies and roundworms results in hypersensitivity to DNA mutagens [12,23]. Additionally, the frequency of micronuclei increases following ionizing radiation exposure and is significantly elevated in POLQ null mutant mice [24]. Interestingly, although these knockout mutants are sensitive to DNA mutagens, no major alterations in growth or development have been observed in POLQ null mutants under normal conditions [23]. These findings in animals align with our observations in Fusarium fungus, where the POLQ null mutant exhibited slightly slower growth than the wild type. For instance, while the wild-type strain typically requires 7 to 10 days to fully colonize a petri dish, the mutant strain takes approximately 2 weeks. Previous studies have shown that POLQ suppresses acute genomic instability—such as chromosome breakage—in response to mutagens through its error-prone DNA repair mechanism. However, this low-fidelity repair process may lead to the accumulation of mutations over time, potentially contributing to long-term genomic instability. However, whether fungal POLQ plays a role in fungal responses to DNA mutagens and contributes to genomic instability requires further investigation [25]. In fruit flies, loss of POLQ leads to reduced fertility and increased embryo mortality [11]. Interestingly, in Fusarium fungi, loss of POLQ results in a decrease in the size of both macro- and microconidia, which are essential for reproduction, with macroconidia also exhibiting a loss of septa. By comparing mutant phenotypes across species, we could identify phenotypic similarities associated with PolQ loss, suggesting a partially evolutionarily conserved role of POLQ genes.

F. acuminatum can cause root rot disease in alfalfa, Dianthus chinensis, Mongolian milkvetch, and Maidong [8,26,27,28], as well as postharvest rot in stored kiwi fruit [29], making it a devastating fungal pathogen. As a xylem-targeting pathogen, F. acuminatum is difficult to control, particularly with fungicides, as these chemicals are not easily able to penetrate the xylem [30]. Alternatively, RNAi-based approaches have emerged as powerful tools for targeting the genes of plant pathogens [31]. One such approach is host-induced gene silencing (HIGS), where transgene constructs producing dsRNA (typically hairpin RNA of pathogen genes) generate small RNA molecules that target the pathogens [32]. POLQ could be a promising candidate for HIGS in alfalfa, as its knockout in F. acuminatum significantly reduces virulence. Notably, this mutation alters the growth behavior of F. acuminatum on and within the root, making it difficult for the fungus to invade xylem tissues. It is quite possible that transgene expression of hairpin RNA targeting POLQ in F. acuminatum could produce plants with a lower susceptibility to F. acuminatum infection. While this method is promising, its application in agriculture is limited by the requirement for transgenic processes, which means the final products must be classified as GMOs [33,34]. Another RNAi-based method is spray-induced gene silencing (SIGS), which has been reported to be even more efficient than HIGS. SIGS involves the direct uptake of long, unprocessed precursor dsRNAs, which are then processed by the fungal RNAi machinery. Thus, SIGS targeting POLQ could be a better approach to controlling F. acuminatum, as it does not require transgenes. However, the high cost of dsRNA synthesis still limits its wide application. However, various alternative approaches and emerging technologies may help reduce costs in the future. For example, microbial synthesis technology—which involves engineering microorganisms such as yeast to produce dsRNA through fermentation—shows promising potential [35]. In addition, the loss of POLQ resulted in a marked alteration in pigmentation, suggesting that POLQ may play a role in regulating secondary metabolism. Microbial pigments have been reported to contribute to pathogenicity by interfering with host immune responses or exerting pro-inflammatory or cytotoxic effects [36]. However, the precise mechanisms by which pigments influence virulence in Fusarium remain to be elucidated and warrant further investigation.

5. Conclusions

In this study, we investigated the functional role of POLQ in F. acuminatum (1A) by generating a POLQ knockout mutant. Our findings demonstrate that POLQ is essential for fungal growth, conidial development, secondary metabolism, and virulence. Phylogenetic analysis further revealed that fungal POLQ sequences are more closely related to those of animals than to plants, within the evolutionary context of eukaryotic POLQ. These results suggest that POLQ may serve as a promising molecular target for the control of plant-pathogenic fungi.