Extracellular Self-DNA Accumulation as a Potential Driver of Continuous Cropping Obstacle in Morchella sextelata and Morchella eximia

Abstract

Continuous cropping obstacle (CCO) is becoming a major restrictive factor limiting the sustainable development of morel industry. The species-specific autotoxicity of extracellular self-DNA (esDNA) may be one of the primary drivers underlying the occurrence of CCO. In this study, the effects of short fragments (≤250 bp) of esDNA or extracellular DNA (exDNA) on mycelial growth of cultivable Morchella eximia and M. sextelata were assayed. These effects were quantified using a response index (RI). The results indicated the dose-dependent, strain-specific, and conspecific autotoxicity of esDNA in cultivable morels. At ecologically relevant DNA concentrations, the strain-specific and conspecific growth inhibitory effects of esDNA in tested Morchella strains were consistently negative (RI < 0). Additionally, our study found that the growth-inhibitory effects of exDNA from M. sextelata on M. eximia strains were weaker than those observed in the reverse scenario. Taken together, our study suggests, for the first time, the conspecific autotoxicity of esDNA in cultivable Morchella under laboratory conditions, providing novel insights into the potential mechanisms of CCO and highlighting its prospective applications in morel production.

1. Introduction

True morels (Morchella spp., Pezizales, Ascomycota) are highly prized edible and medicinal mushrooms, with a long history of consumption and culinary appreciation worldwide. Their unique appearance, distinct savory flavor, and remarkable biological activities have motivated global scientific efforts toward their domestication and large-scale cultivation [1,2,3,4,5]. Successful cultivation largely hinges on the development of easy-fruiting varieties and the implementation of exogenous nutrient supplementation strategies [1,3,6]. Although soil conditions are essential for morel cultivation, this dependence inevitably leads to continuous cropping obstacles (CCO), which is known as soil sickness or replant disease, refers to the decline in crop yield and vigor caused by the repeated cultivation of the same plant species on the same land [7,8]. The CCO phenomenon involves a complex interplay of factors, including the buildup of soil-borne pathogens and pests, nutrient imbalances, degradation of soil physico-chemical structure, and the accumulation of autotoxic allelochemicals released from plant residues [8]. The CCO phenomenon has been observed in many crops and other soil-dependent cultivated fungi, including mushroom-forming species Ganoderma lucidum, Dictyophora rubrovalvata, Oudemansiella raphanipes, as well as the medicinal fungus Wolfiporia cocos [7,8]. In morels, CCO is characterized by a sharp decline in fruiting body yield, poor mycelial colonization in subsequent cultivation cycles, alterations in soil microbial community structure, and an increased abundance of soil-borne pathogens [7,9,10,11,12,13]. In China, the morel industry has reached an annual production of approximately 30,000 tons (Fresh mushrooms), with a direct economic value of around 4 billion CNY, making the effective resolution of CCO a current priority.

Also known as soil sickness or replant disease, CCO has been extensively studied in plant systems [14,15]. As a typical manifestation of negative plant–soil feedback (NPSF) in both natural and agricultural ecosystems, CCO can be defined as the self-induced deterioration of soil conditions that inhibit organismal growth and reproduction [16,17]. Proposed mechanisms for species-specific NPSF or CCO include nutrient deficiency, the buildup of soil-borne pathogens, and litter autotoxicity [14,18]. However, long-term CCO in agriculture often presents as highly species-specific inhibition, which cannot be fully explained by nutrient depletion or pathogen accumulation alone [14,15,18,19,20]. Furthermore, the short persistence (weeks or months) of low molecular weight allelopathic compounds—such as short-chain organic acids, tannins, and phenols—in soil contrasts sharply with the long-lasting nature (often years) of CCO [14,21]. Within this context, the species-specific inhibitory effect of extracellular self-DNA (esDNA) in higher plants has provided a compelling framework to reconcile the autotoxicity hypothesis with the occurrence of CCO [22,23,24,25,26].

Considering the adverse effects of CCO on artificial cultivation of Morchella, understanding its underlying mechanisms has become a research focus. Current evidence points to several driving factors, including soil nutrient imbalances, disruption of the soil microbial community, increased disease incidence, and the accumulation of allelochemicals and autotoxins [7,9,10,11,12,27,28]. Our previous studies identified 4-coumaric acid (p-CA) and vanillic acid as allelochemicals in morels, both of which can induce oxidative stress and accelerate strain aging [7,28]. However, these phenolic acids may not be the primary cause of morel soil sickness, as they are readily degraded by soil microorganisms [29]. Alternatively, extracellular self-DNA (esDNA) released into the soil from the degradation of the extensive morel mycelial network may play a critical role. The proposed mechanism suggests that self-DNA may act as a damage-associated molecular pattern (DAMP), potentially triggering innate immune responses or interfering with the cell cycle, leading to autotoxic effects [30,31]. Unlike short-lived metabolites, esDNA can persist in soil for extended periods by adsorbing to soil particles and clay minerals, which effectively protects it from microbial degradation [32,33,34,35]. Given the documented species-specific inhibitory effects of esDNA across diverse biological kingdoms [30,36,37,38,39], we hypothesized that the accumulation of morel esDNA could be a previously overlooked causal factor underlying CCO in morel cultivation.

The conspecific inhibitory effect of esDNA represents a fascinating and evolutionarily conserved biological phenomenon, having been documented across diverse kingdoms of life, from plants and insects to bacteria and slime molds [30,36,37,38,39]. However, a critical knowledge gap remains in macro-fungi, particularly in cultivated, soil-inhabiting species that are plagued by CCO, such as Morchella. Establishing this phenomenon in morels is novel within mycology, as it introduces a previously unrecognized, innate signaling mechanism—esDNA-triggered autotoxicity—that can regulate fungal population dynamics in the soil environment. This expands the ecological and molecular principles governing fungal-fungal interactions beyond the well-studied realms of resource competition and antibiotic secretion. Collectively, this study not only reports a fundamental biological discovery but also lays the groundwork for developing innovative, knowledge-based solutions to ensure the sustainable cultivation of morels and potentially other soil-cultivated fungi.

2. Materials and Methods

2.1. Plant Sampling

Equal fresh weights (250 g) of the stems and leaves of Digitaria sanguinalis (monocot) and Calystegia hederacea (dicotyledon) were collected. These plant species are common weeds co-occurring in the morel cultivation plastic house, making their nucleic acids a relevant and ecologically meaningful non-self DNA control. The composite plant sample was then placed into a sealed zip-lock bag, immediately transported to the laboratory, and stored at −20 °C until further use.

2.2. Fungal Strains and Culture Conditions

Three productive strains (7A6-2, SK, and J7) of M. eximia and two strains (LB-2 and 13) of M. sextelata domesticated from wild morels were used in this study. These strains are commercially representative and widely cultivated in China, making them agronomically relevant for investigating CCO. The taxonomic status of the five tested cultivable morel strains was confirmed through analysis of concatenated sequences of ribosomal DNA internal transcribed spacer (ITS), translation elongation factor 1-alpha gene (TEF1-α), and RNA polymerase II subunit 1 (RPB1) (Supplementary File S1). All strains are available upon request from Peixin He in Zhengzhou University of Light Industry. Strain culture was carried out using complete yeast extract medium (CYM) (glucose 20 g/L, yeast extracts 2 g/L, peptone 2 g/L, K₂HPO₄ 1 g/L, MgSO₄ 0.5 g/L, KH₂PO₄ 0.46 g/L, and agar 20 g/L) covered with autoclaved cellophane membranes, and cultured in the dark at 22 °C for 7 d. The mycelia were collected, ground into powder with liquid nitrogen, and stored at −20 °C for further use [11].

2.3. DNA Extraction and Fragmentation by Ultrasonication

Genomic DNA was isolated from mycelial and plant powders using a protocol based on the SDS method with minor modifications [40]. Briefly, powdered tissue (5 g) was homogenized with 15 mL of lysis buffer [Triton X-100 2% (v/v), SDS 1% (v/v), NaCl 100 mM, Tris 10 mM (pH 8.0), EDTA 1 mM]. Samples were then incubated for 1 h at 65 °C and mixed periodically. Then 10 mL of PC reagent (phenol:chloroform = 1:1) was added, followed by centrifugation at 4 °C for 5 min at 8000× g. The supernatant was collected and transferred to a new tube for precipitation with two volumes of absolute ethanol at −20 °C for 2 h. Samples were centrifuged at 4 °C for 5 min at 8000× g. The DNA precipitates were treated with RNase A (50 µg/mL) at 37 °C for 30 min to remove residual RNA, followed by re-precipitation with ethanol. The precipitated DNA was washed with 20 mL of cold 75% ethanol and then subjected to centrifugation at 4 °C for 5 min at 8000× g. After air-drying, the precipitates were dissolved in 1 mL of ddH₂O. This protocol, including organic solvent (phenol–chloroform) purification and ethanol precipitation, is specifically designed to remove proteins, lipids, and small-molecule metabolites, yielding high-purity DNA suitable for downstream bioassays.

The DNA samples extracted from morel mycelia and plants were initially assessed using NanoDrop standard quality parameters (260/280 and 260/230 ratio above 1.8) to evaluate DNA purity. The DNA quantity was determined using a Qubit fluorimeter (Thermo Fisher, Waltham, MA, USA) fluorimeter, and its integrity was evaluated by electrophoresis in 1% agarose gel. The extracted DNA samples were fragmented by ultrasonication using an ultrasonic crusher (JY92-IIN, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) at 90% power with a 0.9 s pulse for 10 min in order to reach an average size approximately 200 bp. Verification of sonicated fragment sizes was performed on a 2% agarose gel.

2.4. Effects of Extracellular DNA on Fungal Growth

For clarity, the following terms are used throughout this manuscript: extracellular self-DNA (esDNA) refers to fragmented DNA applied to the same species or strain from which it was extracted; extracellular non-self DNA refers to DNA from a different species (e.g., plant DNA), and extracellular DNA (exDNA) is used as a general term.

The extracted and fragmented DNA samples from morel mycelia and control plants were filtered through a 0.22 μm membrane prior to bioassays. Seven DNA concentrations (0, 0.01, 0.1, 0.5, 1.0, 3.0, and 5.0 μg/mL) were tested. This range was selected to include sub-ecological doses, the measured in situ DNA concentration in our field soil (~2.0 μg/mL, see Supplementary File S2), and higher concentrations to ensure that a clear dose–response relationship could be established. Five milliliters of sterile distilled water or different concentrations of DNA fragments were added to 15 mL of CYM medium prior to solidification, resulting in a total volume of 20 mL per Petri dish. A mycelial plug (4 mm in diameter) from respective activated culture was placed at the center of each tested CYM plate (9 cm in diameter). The inoculated plates were incubated at 22 °C in the dark and observed every 12 h. Colony diameters were recorded when the mycelia of any treatment approached the edge of a CYM plate. Each treatment had three replicates, and the mean of five measures per replicate was calculated [7].

2.5. Statistical Analysis

The effect of esDNA or exDNA on morel mycelial growth was evaluated using response indices (RI). RI was defined as 1 − (C/T) when T ≥ C, and as (T/C) − l when T < C, where T represents the treatments and C represents the control value. RI ranges from −1 to +1, with positive values indicating growth promotion and negative values indicating growth inhibition relative to the controls [7,41]. Data are represented as means ± standard deviation (SD) (n = 3). To account for the factorial nature of the experimental design, linear mixed-effects models (LMMs) were fitted using the lme4 package in R. The full model specification was: RI ~ DNA_source × Concentration + (1|Strain), where DNA source (self, plant, conspecific, congeneric) and concentration (0.01–5 μg/mL) were treated as fixed effects, and strain was included as a random intercept to account for repeated measures. Model significance was assessed using Type III ANOVA with Satterthwaite’s method. Post hoc comparisons were conducted using estimated marginal means with Holm adjustment for multiple testing. To further validate the robustness of the results given the modest replication (n = 3), permutation tests (1000 permutations) were performed, and bootstrap confidence intervals were calculated using 2000 resamples. Probability values p < 0.05 and p < 0.01 were considered statistically significant and highly significant, respectively. All analyses were carried out using IBM SPSS Statistics 22. Graphs were generated using Origin 2022 (OriginLab Corporation, Northampton, MA, USA) and composited with Adobe Photoshop CS6.

3. Results

3.1. DNA Sonication and DNA Fragment Analysis

To evaluate the responses of the cultivable Morchella to esDNA, we extracted total DNA from the tested Morchella strains and syntrophic plant species (D. sanguinalis and C. hederacea). Sonication of DNA yielded fragments of various sizes, which were analyzed by agarose gel electrophoresis. The results suggested that the size of DNA fragments was ≤ 250 bp (Figure 1). These fragment sizes are comparable to those detected in decomposing litter and have previously been shown to exert inhibitory effects in in vitro assays [23].

3.2. Factorial Analysis of DNA Source and Concentration Effects

To comprehensively evaluate the interactive effects of DNA source and concentration, we implemented a linear mixed-effects model analysis (Figure 2). This analysis revealed highly significant main effects of both DNA source (F[3, 513.46] = 46.17, p < 0.001) and concentration (F[5, 512.02] = 10.26, p < 0.001), while the interaction term was not statistically significant (F[15, 512.02] = 0.98, p = 0.477) (Supplementary File S3). The absence of a significant interaction indicates that the dose–response patterns were generally consistent across different DNA sources, although the magnitude of effects varied substantially (Figure 2A).

Post hoc analysis demonstrated that self-DNA consistently produced inhibitory effects (RI < 0) that were significantly different from plant and congeneric DNA across most concentration levels (Supplementary File S4). Specifically, self-DNA showed significant inhibition compared to plant DNA at concentrations ≥ 0.1 μg/mL (p < 0.05) and compared to congeneric DNA at all tested concentrations (p < 0.01). The robustness of these findings was confirmed through permutation testing (p = 0.477 for interaction) and bootstrap confidence intervals (Figure 2B).

3.3. Strain-Specific Autotoxicity of EsDNA

The mycelial growth of all five tested morel strains was promoted by plant-derived DNA fragments under all six assay concentrations (RI > 0) (Figure 3), suggesting that plant DNA fragments may be taken up by Morchella hyphae and utilized as a nutrient source. In contrast, among the tested Morchella strains, dose-dependent strain-specific growth-inhibitory effects of esDNA were observed in strain J7 (Figure 3B) and SK (Figure 3C) of M. eximia under all assay concentrations (RI < 0). Moreover, in M. eximia 7A6-2 (Figure 3A) and two tested strains of M. sextelata (LB-2 and 13) (Figure 3D,E), mycelial growth was promoted at the lowest assay concentration (0.01 μg/mL) of esDNA (RI > 0), whereas all other concentrations resulted in growth inhibition (RI < 0), with the overall response pattern resembling hormesis. Nonetheless, under the local soil DNA content (1.971 μg/g) measured when morel cultivation was nearly completed at our Shangqiu cultivation base in China (Supplementary File S2), the effects of esDNA on mycelial growth of all tested Morchella strains were consistently strain-specific and inhibitory (Figure 3). These results clearly confirm the strain-specific and dose-dependent autotoxicity of esDNA in cultivable Morchella. The inhibitory and promotive effects described herein are visually supported by representative photographs of mycelial colonies shown in Figure 4. Conspecific autotoxicity of esDNA is typically characterized by stimulation at low concentrations and inhibition at high concentrations. For instance, on day 7, enhanced sclerotia production for M. sextelata 13 and M. eximia 7A6-2 was observed at low concentrations (0.01 μg/mL and 0.1 μg/mL), whereas at high concentrations, in addition to reduced growth rate (Figure 4A), the colonies exhibited markedly irregular margins (Figure 4A) and failed to produce sclerotia (Figure 4B).

In addition to the reduction in radial growth, esDNA exposure led to distinct alterations in colonial morphology. At inhibitory concentrations (≥0.5 μg/mL), colonies exhibited irregular margins and a less compact, more diffuse mycelial texture compared to the dense, circular colonies of the control (Figure 4A). Furthermore, the formation of sclerotia—a key developmental structure—was markedly suppressed or delayed at high esDNA concentrations (Figure 4B). These morphological changes indicate that esDNA impacts not only mycelial expansion but also its vitality and differentiation capacity.

3.4. Conspecific Autotoxicity of EsDNA

Under all six assay concentrations, the mycelial growth of M. eximia 7A6-2 was suppressed by esDNA from M. eximia SK in a concentration-dependent manner (RI < 0). Meanwhile, the mycelial growth of M. eximia J7 was inhibited by esDNA from the other two M. eximia strains, and that of M. eximia SK was also inhibited by esDNA of M. eximia J7 (RI < 0) (Figure 5A). In contrast, the effects of esDNA from M. eximia J7 on M. eximia 7A6-2, those of M. eximia 7A6-2 on M. eximia SK (Figure 5A), and the reciprocal effects between the two M. sextelata strains (LB-2 and 13) (Figure 5B) all exhibited hormesis-like dose–response patterns, albeit with different autotoxic thresholds. Furthermore, under local soil DNA concentrations (Supplementary File S2), the autotoxic effects of esDNA on different conspecific strains were consistent (Figure 5). These results further confirm the dose-dependent conspecific autotoxicity of esDNA in cultivable Morchella.

3.5. Effect of Congeneric ExDNA on Mycelial Growth Morchella spp.

Overall, the effects of the exDNA from the two tested M. sextelata strains on the three M. eximia strains were predominantly promotive (RI > 0). ExDNA from M. sextelata LB-2 promoted mycelial growth of all three M. eximia strains (Figure 6A–C). Moreover, exDNA from M. sextelata 13 also promoted the mycelial growth of M. eximia 7A6-2 under all six assay concentrations (Figure 6A). Meanwhile, the effect of exDNA from M. sextelata 13 on the mycelial growth of M. eximia J7 and SK exhibited hormesis-like responses, with promotion at 0.01, 0.1 and 0.5 μg/mL (RI > 0) and inhibition at concentrations of 1.0, 3.0 and 5.0 μg/mL (RI < 0) (Figure 6B,C). Conversely, the overall effects of exDNA from M. eximia on the mycelial growth of M. sextelata were predominantly inhibitory (RI < 0). ExDNA from M. eximia 7A6-2 suppressed the mycelial growth of both M. sextelata strains (Figure 6D,E). Furthermore, the effect of exDNA from M. eximia J7 and SK on the mycelial growth of M. sextelata LB-2 (Figure 6D), as well as those of M. eximia SK on M. sextelata 13 (Figure 6E), were also consistent with hormesis-like responses. The only exception was the promotive effect of exDNA from M. eximia J7 on the mycelial growth of M. sextelata 13 (RI > 0) (Figure 6E). Taken together, our results indicate that the growth-inhibitory effects of exDNA from M. sextelata on M. eximia strains are weaker than those observed in the opposite direction, implying that the risk associated with replanting M. eximia after cultivation of M. sextelata may be lower than that of the reverse scenario. This asymmetric congeneric inhibitory pattern has direct implications for practical morel cultivation and crop rotation strategies.

The observed asymmetric cross-species inhibition (Figure 6) provides insight into the phylogenetic specificity of the esDNA response. The fact that M. sextelata DNA can inhibit M. eximia indicates a conserved mechanism across the genus. However, the differential sensitivity—whereby M. eximia DNA exerts stronger inhibition on M. sextelata than vice versa—highlights species-level variation in response thresholds. This supports a model of a core, genus-wide recognition pathway whose downstream inhibitory output is fine-tuned by species-specific factors.

4. Discussion

4.1. Mechanism Underlying Autotoxicity of EsDNA Warrants Further Investigation

Our study provides preliminary evidence for conspecific autotoxicity induced by esDNA in macrofungi, specifically in cultivated morels. This discovery points to a previously unrecognized mechanism of soil sickness, distinct from the allelopathic effects of phenolic acids we reported previously [7]. The conserved autotoxic response we observed, including the hormetic effect at low concentrations, strongly suggests that esDNA functions as a DAMP in morels [30,31]. This is consistent with mechanisms proposed in plants, where esDNA perception can trigger a cascade of defense responses, such as ROS bursts [26,31,42,43,44], MAPK activation [31], and the generation and signaling of salicylic acid (SA) and jasmonic acid [26,45]. The dose-dependent shift from promotion to inhibition may reflect a transition from a priming signal to a full-scale, growth-suppressing immune response. On the other hand, the DNA damage response (DDR) pathway has been identified as playing a role in triggering SA-dependent plant defense responses to exDNA [45]. Cell-cycle arrest via the DDR pathway induced by esDNA through DNA damage mediated by ROS was observed in rice roots [46]. The mechanisms underlying the autotoxicity of esDNA in cultivable Morchella need further study. Additionally, a study investigating the effects of self-DNA, congeneric DNA, and heterospecific DNA from Brassica napus and Salmon salar on root elongation of seedlings confirmed a significantly higher inhibition by self-DNA and a magnitude of the effect consistent with the phylogenetic distance between the DNA source and the target species [24]. However, our study revealed an asymmetric inhibitory effect of congeneric DNA on mycelial growth of M. eximia and M. sextelata. The underlying mechanism remains to be disclosed.

The observed biphasic response, where certain low concentrations of esDNA led to significant growth promotion while higher concentrations caused inhibition, resembles the phenomenon of hormesis. This may be explained by the dual potential of extracellular nucleic acids. At very low concentrations, these fragments might be utilized as a nutrient source for nucleotides or phosphorus, providing a slight growth benefit [37]. Alternatively, sub-inhibitory levels of esDNA could act as a mild warning signal, priming the fungal defense system and transiently stimulating growth vigor as a compensatory adaptive response. However, once a critical threshold concentration is surpassed, this signal likely triggers a strong, inhibitory immune or autotoxic response, consistent with the established model of esDNA as a DAMP [26,31]. Such concentration-dependent hormetic effects are a common feature in biological systems and have been noted in previous studies on esDNA [23], reinforcing the biological relevance of our findings.

4.2. Degradation of EsDNA or ExDNA in Soil

EsDNA or exDNA has been found in large amounts both in soil and sediments [47]. Lysis of dead cells is the main source of esDNA or exDNA in soil [32]. The presence of active endonucleases can affect the postmortem integrity of DNA molecules before their release by cell lysis [48]. The activity and abundance of microbes (mainly bacteria) and extracellular nucleases (primarily bacterial DNases) significantly affect the esDNA/exDNA degradation [32,49,50]. Some bacteria belonging to genera Bacillus, Brevibacillus, Chryseobacterium, Fictibacillus, Flavobacterium, Microbacterium, Nubsella, Pseudomonas, Psychrobacillus, Rheinheimera, Serratia, Stenotrophomonas and so on have been reported as DNase producers [51,52]. On the other hand, the main extrinsic conditions influencing esDNA or exDNA degradation include soil mineralogy, organic components, pH, temperature and moisture [33]. The adsorption of DNA to clay minerals and silica protects it from degradation by extracellular microbial DNases and nucleases, which degrade unbound DNA in the soil solution [32,34,49]. The presence of organic matter and efficient adsorption of nucleases onto soil colloids and minerals appear to be responsible for the lower degradation of DNA in soil ecosystems [33,35]. Additionally, high bacterial abundance and increased microbial and enzymatic activity under moderate temperatures can accelerate the degradation of esDNA or exDNA [53,54]. The effects of pH on esDNA or exDNA persistence might be complex and may be related to other factors such as adsorption of nucleic acids to clay minerals and DNase activity. The absorption of esDNA or exDNA generally increases when the pH is below 5, and decreases as pH rises above 5 [55]. Moreover, DNase activity may decrease due to the declined availability of its Mg²⁺ cofactor in soil at low pH [33]. In addition, soil moisture determines microbial motility and activity, nutrient diffusion, waste removal, and the activity of extracellular enzymes [56].

4.3. Ecological and Evolutionary Implications

The autotoxicity of esDNA presents an intriguing ecological and evolutionary perspective. In natural settings, this phenomenon could act as a density-dependent negative feedback mechanism, preventing monocultural dominance of a single genotype and fostering spatial heterogeneity and genetic diversity. Indeed, accumulating evidence supports the view that conspecific esDNA released into the soil can inhibit the growth of the same species, thereby limiting intraspecific proliferation and promoting interspecific or genotypic coexistence [15,23,57]. For example, studies have shown species-specific growth inhibition by fragmented self-DNA in plants, which may underpin the negative plant–soil feedback observed in many natural communities [12,25]. In such contexts, esDNA accumulation can provide a competitive advantage to less related or different species, thereby promoting ecosystem biodiversity [15,57]. However, in the high-density, mono-strain conditions typical of agricultural cultivation, this naturally selected self-inhibition mechanism becomes maladaptive, translating directly into the significant economic losses associated with CCO or “soil sickness” [23,24,25,26].

4.4. Targeted Measures to Mitigate the Autotoxicity of EsDNA

In this study, the native DNA concentration (1.971 μg/g fresh soil) of a soil sample collected when morel planting was nearly complete at our Shangqiu morel cultivation base, China, was determined using real-time quantitative PCR (Supplementary File S2). The local DNA content may be largely attributable to the amount of mycelial biomass in the soil after the harvest of morel ascocarps. Along with the application of improved varieties or strains and advances in cultivation techniques, the yield of morel production in China is continuously increasing (˃1000 kg fresh products per 667 m² is common). Consequently, the mycelial biomass present in the upper 20–25 cm of soil, together with the residual stipes after morel harvest (about 3 cm long), may be substantial, and thus the native DNA content may also be considerable. Due to the dose-dependent autotoxicity and slow degradation of fragmented esDNA in soil, the species-specific inhibitory effects of esDNA might play a crucial role in NPSFs or CCO of morel production. Therefore, targeted measures should be taken to mitigate the autotoxicity of esDNA in the artificial cultivation of Morchella. First, the structure of DNA is very stable under dry, anoxic conditions, with an estimated half-life of ~500 years under ideal conditions [58]. However, exDNA decays rapidly in oxygenated environments due to processes such as hydrolysis and oxidation [59]. Meanwhile, tilled soils exhibit higher degradation rates and less stabilization of exDNA than no-till soils [33]. Accordingly, it is not beneficial to leave the soil idle and dry for extended periods after morel production. Alternatively, measures aimed at increase soil microbial community diversity and improve soil physicochemical properties, such as crop rotation, intercropping, application of organic fertilizers, and deep ploughing, may play an important role in mitigating morel CCOs [8]. Secondly, land flooding is beneficial. DNA is slightly soluble in water; thus, dissolved esDNA or exDNA can be transported out of the system within minutes by flowing water [60,61]. Moreover, DNA molecules can desorb from soil particles during flooding and subsequently enter the soil solution, where they are vulnerable to degradation by extracellular microbial DNases and nucleases [32]. Finally, soil disinfection is effective. The degradation of recombinant DNA accelerates with increasing temperature and requires approximately 4 d at 50 °C [62]. Hence, in addition to eliminating harmful microorganisms and pests, “greenhouse thermal pasteurization” can also accelerate the degradation of morel esDNA or exDNA accumulated in soil [8]. Meanwhile, bioremediation-based strategies, such as the enrichment or introduction of microorganisms with high DNase activity, as more sustainable and ecologically compatible approaches for mitigating extracellular DNA accumulation in morel cultivation systems. Furthermore, the use of disinfectants, such as quicklime [63] and chlorine-releasing agents, including sodium hypochlorite and sodium dichloroisocyanurate [64,65], which can directly oxidize DNA, will effectively mitigate the autotoxicity of esDNA.

These statistical findings are consistent with the proposed mechanistic framework. Our factorial analysis using linear mixed models provides statistical rigor to the observed patterns, confirming the significant independent effects of both DNA source and concentration while indicating consistent dose–response relationships across DNA types. The lack of a significant interaction suggests that the autotoxic mechanism operates similarly across concentrations, with self-DNA maintaining its inhibitory character regardless of dose level. This statistical framework strengthens our conclusion that esDNA functions as a conserved DAMP signal in morels, triggering dose-dependent immune responses that follow predictable patterns across the concentration gradient.

4.5. Methodological Considerations and Limitations

A pertinent consideration in this study is the potential co-extraction of metabolites alongside esDNA, which could theoretically influence the observed growth effects. While LC-MS analysis to conclusively rule out this possibility was beyond the scope of the present work, several lines of evidence strongly suggest that the species-specific inhibitory effects are attributable to esDNA itself. First, the DNA extraction protocol employed is a standard molecular biology method designed for high-purity DNA isolation, as confirmed by spectrophotometric ratios and clear electrophoretic bands (Figure 1). Crucially, if non-specific inhibitory metabolites were present in the fungal DNA extracts, one would expect the plant DNA extracts, prepared using the identical protocol, to also exhibit inhibitory effects. The consistent neutral or promotive effects of plant DNA on all Morchella strains (Figure 2) argue strongly against this possibility and highlight the species-specific nature of the inhibition, a hallmark of the self-DNA response that is unlikely to be mediated by general metabolites. This experimental approach, utilizing heterologous DNA as a control, aligns with established paradigms in the field [23,37]. Nonetheless, future studies incorporating DNase digestion controls and metabolomic profiling will be valuable for fully disentangle the role of pure DNA from other potential co-extracted compounds.

5. Conclusions

Our study reveals, for the first time, the strain-specific and conspecific autotoxicity of esDNA, as well as an asymmetric inhibitory effect of congeneric exDNA on the mycelial growth of cultivable M. eximia and M. sextelata. The underlying mechanisms, potentially involving conserved DAMP and DDR pathways, require further molecular investigation. These findings contribute to a deeper understanding of NPSF, or soil sickness, in the cultivation of morels and highlight prospective applications for the alleviation of CCO. From a practical standpoint, our results suggest that the risk of replanting M. eximia strains in fields previously cultivated with M. sextelata strains is lower than in the opposite scenario, which is conducive to practical morel production. Furthermore, agronomic practices such as deep ploughing, land flooding, and soil disinfection, which facilitate the degradation or removal of persistent esDNA, should be prioritized to alleviate morel CCO. Developing methods to accelerate extracellular DNA degradation in soil could be a novel strategy to overcome CCO in Morel cultivation.