Morchella Effectively Removes Microcystins Produced by Microcystis aeruginosa

Microcystins (MCs) produced by Microcystis aeruginosa are harmful to animal and human health, and there is currently no effective method for their removal. Therefore, the development of biological approaches that inhibit cyanobacteria and remove MCs is needed. We identified strain MB1, confirmed as Morchella, using morphological and molecular evolution methods. To assess the impact of strain MB1 on M. aeruginosa, we conducted an experiment in which we inoculated M. aeruginosa with Morchella strain MB1. After their co-cultivation for 4 d, the inoculation with 0.9696 g MB1 completely inhibited and removed M. aeruginosa while concurrently removing up to 95% of the MC content. Moreover, within 3 d of their co-cultivation, MB1 removed more than 50% of nitrogen and phosphorus from the M. aeruginosa solution. Therefore, the development of effective biological techniques for MC removal is paramount in safeguarding both the environment and human well-being. We herein successfully isolated MB1 from its natural habitat. This strain effectively inhibited and removed M. aeruginosa and also reduced the content of nitrogen and phosphorus in the M. aeruginosa solution. Most importantly, it exhibited a robust capability to eliminate MCs. The present results offer a new method and technical reference for mitigating harmful algal blooms.

Cyanobacterial toxins are metabolites released by cyanobacteria upon cell death or lysis.Among these toxins, microcystins (MCs) are the most widely distributed and toxic (Campos and Vasconcelos, 2010;Neilan et al., 2012).More than 300 MC isomers have been identified to date (Bouaicha et al., 2019;Du et al., 2019;Massey and Yang, 2020).MC-LR and MC-RR are relatively common and highly toxic compounds; MC-LR contains two variable amino acids, leucine (L) and arginine (R), while MC-RR contains two variable amino acids of arginine (R) (Hu et al., 2017;Massey and Yang, 2020).MCs are monocyclic heptapeptide hepatotoxins that remain stable, even under harsh conditions, such as high temperatures, extreme pH, and sunlight (de la Cruz et al., 2011).The inherent structural stability of MCs leads to their slow degradation in aqueous environments (He et al., 2022).
MCs may reduce water quality, affect the regulation of cell protein phosphorylation, promote apoptosis (Huo et al., 2021), and lead to cellular destruction and cytoskeletal damage (Zhu et al., 2022).Their toxicity extends to humans and animals (Massey et al., 2018;Cao et al., 2019;Alosman et al., 2021).Prolonged and frequent exposure to low concentrations of MCs may eventually lead to uncontrolled cell proliferation in the human body, promote the occurrence and development of tumors, and lead to primary liver cancer (Drobac et al., 2013;Lee et al., 2017;Woolbright et al., 2017).Physical methods and chemical reactions do not offer economical or effective solutions for MC removal (Li et al., 2017;Duan et al., 2018).In contrast, microbial methods are widely regarded for their cost-effectiveness, environmental friendliness, and ecological restoration capabilities (Neilan et al., 2012;Dziga et al., 2013;Teng et al., 2023).However, most MC-degrading bacteria cannot completely eliminate MCs (Massey and Yang, 2020;Zhan and Hong, 2022).Therefore, it is imperative to develop efficient biological approaches for MC removal in order to address water resource pollution and safeguard human health.
In the present study, we identified a Morchella strain (MB1) in the field that effectively inhibited and eliminated both M. aeruginosa cells and MCs.Additionally, strain MB1 exhibited the capacity to remove nitrogen and phosphorus in the M. aeruginosa solution tested.The present results provide evidence to support the effective management of M. aeruginosa.

Materials
In the present study, Morchella strain MB1 was isolated from alga-associated Morchella in moist soil on the campus of Jilin Normal University.M. aeruginosa used in this study was provided by the Institute of Hydrobiology, Chinese Academy of Sciences.The standard algal toxin products, MC-LR and MC-RR, were obtained from Beijing Biorebo Technology.

Isolation and identification of Morchella strain MB1
Purified strain MB1 was inoculated on Potato Dextrose Agar (PDA) culture medium for 3-20 d, and colony morphology was observed.A fungal genomic DNA extraction kit (Beijing Solaibao Technology) was used to extract total DNA from samples.The Internal Transcribed Spacer (ITS) region is a non-coding DNA sequence located between the 18S and 28S rRNA genes within the fungal genome.This sequence is used as a molecular marker for fungal identification and phylogenetic analyses (Tretter et al., 2013;Ao et al., 2019).The ITS of the nuclear rRNA genes fragment of samples was amplified and sequenced using the universal primers ITS1 (5′-TCCGTAGGTGAACCTGC-3′) and ITS4 (5′-TC CTCCGCTTATTGATATGCG-3′) (Ramadurai and Balasundaram, 2020), and a phylogenetic tree was constructed by the maximumlikelihood method using MEGA7 software (Liu et al., 2018a).Sequences generated in the present study were deposited in GenBank under the accession number PP256506.
Inhibitory activity of strain MB1 against M. aeruginosa M. aeruginosa was cultured in 250 mL of BG11 liquid medium (Wang et al., 2013a;Kucala et al., 2021) and incubated under a 12-h dark/light cycle at 25°C with a light intensity of 500 μmol m -2 s -1 for a duration of 15 d (Chen et al., 2014;Omidi et al., 2019).After collecting M. aeruginosa in the logarithmic growth phase, the concentration of the algal solution was adjusted to 2-2.5×10 7 cells mL -1 as the initial concentration.The control group contained an inactivated M. aeruginosa solution (CK1) and active M. aeruginosa solution (CK2).Stationary phase strain MB1 was added to both CK1 and CK2.We collected 250 mL of the M. aeruginosa solution and co-cultured it with dry weights of 0, 0.2424, 0.4848, 0.7272, 0.9696, 1.2120, and 1.4544 g MB1, designated as M0, M1, M2, M3, M4, M5, and M6, respectively.The experiment was repeated three times for each group on days 0, 1, 2, 3, and 4, respectively.The solution was aerated using an air pump to maintain the activity of strain MB1.

Assessment of the removal of nitrogen and phosphorus
The M. aeruginosa solution was co-cultured with strain MB1 as previously reported (Cao et al., 2021).Following treatment, the culture medium was collected on days 0, 1, 2, and 3. Nitrogen and phosphorus contents in the culture medium were detected using a high-precision COD multiparameter tester (HI83099; HANNA Instruments Model) according to the manufacturer's instructions.This experiment was repeated three times, and nitrogen and phosphorus removal rates were calculated using the following formula: removal rate=(initial concentration-treated concentration)×100%/ initial concentration Assessment of the ability of strain MB1 to remove MC-LR and MC-RR M. aeruginosa cultured for 15 d was frozen at -20°C for 24 h and then dissolved by heating in a water bath at 50°C, followed by ultrasonication for 30 min.The algal liquid was then centrifuged at 5,000 rpm at 4°C for 20 min, and the resulting supernatant was collected as MC MC-LR and MC-RR samples.Morchella strain MB1, represented as M0, M1, M2, M3, M4, M5, and M6, was individually introduced into an MC solution for a 3-d cocultivation period.HPLC was used to detect the contents of MC-LR and MC-RR.A total of 0.9696 g of Morchella was then inoculated into the MC solution, and the MC content was measured over a 5-d co-cultivation with strain MB1.This procedure was repeated in triplicate, and continuous aeration was ensured using an air pump to maintain the activity of strain MB1.

Statistical analysis
Statistical software SPSS 20.0 (IBM SPSS) was used to analyze the significance of differences in the present study.P<0.05 was considered to be significant.

Identification of strain MB1
Strain MB1 was cultured on PDA to observe colony and mycelial morphologies.After an incubation at 25°C for 20 d, mycelia gradually transitioned from transparent to light yellow, with some turning dark brown (Fig. 1A).The central older mycelia secreted a brown pigment that caused the discoloration of the medium (Fig. 1B), while maintaining a smooth surface.Thick mycelia exhibited numerous interlaced branches ranging from 1.5-13.5 μm in diameter (Fig. 1C).The sclerotium consisted of closely packed mycelia of various shapes and sizes, lacking an evident epidermal structure, leading to the presumption that these sclerotia were microsclerotia (Fig. 1D, E, and F).To analyze the phylogenetic relationship of MB1 within Morchella, a phylogenetic tree was constructed using ITS gene sequences.All analyzed sequences were classified into three groups (Fig. 2), which was consistent with previous studies (Richard et al., 2015), with MB1 and M. elata placed in the same clade, indicating a close relationship, and MB1 isolated from M. semilibera and M. tridentina.

Inhibitory effects of MB1 against M. aeruginosa
To investigate the inhibitory effects of Morchella on the growth of M. aeruginosa, we co-cultured strain MB1 in the stationary phase and M. aeruginosa in the logarithmic phase.Notably, the M. aeruginosa solution co-cultured with M2, M3, M5, and M6 exhibited gradual clarity, while the solution co-cultivated with M4 became almost clear on day 1 (Fig. 3A, B, C, D, E, F, G, H, I, J, K, and L).The number of M. aeruginosa cells cultured with strain MB1 was calculated and the results obtained revealed significant reductions in M2, M3, M4, M5, and M6 on day 1 (Fig. 3M).M. aeruginosa cell counts significantly decreased in the M1, M2, M3, M4, M5, and M6 groups on days 2-4.Moreover, M4 resulted in zero M. aeruginosa cells on day 4 (Fig. 3M  and S1).These results indicated that Morchella effectively inhibited the growth of M. aeruginosa.

Removal of nitrogen and phosphorus by Morchella strain MB1
To investigate the capability of Morchella to remove nitrogen and phosphorus from the M. aeruginosa solution, two control groups were established: CK1 and CK2.M4 was inoculated into CK1 and CK2, and nitrogen and phosphorus concentrations in the solutions were measured.The nitrogen content in the inactivated M. aeruginosa solution significantly decreased 2 d after the inoculation (CK2+strain MB1 vs. CK2).Additionally, the nitrogen content in the M. aeruginosa solution was significantly lower 1-2 d after the inoculation with the CK1+strain MB1 than that with CK1 (Fig. 4A).
Following the inoculation, the phosphorus content significantly decreased in CK1+strain MB1 vs. CK1 and CK2+strain MB1 vs. CK2 (Fig. 4C).Furthermore, the removal ratios of nitrogen and phosphorus revealed that the nitrogen clearance rate significantly increased on days 1 and 2 after the inoculation with the inactive M. aeruginosa and active M. aeruginosa solutions (Fig. 4B), whereas the phosphorus clearance rate significantly increased on days 1 and 2 after the M. aeruginosa inoculation.(Fig. 4D).These results indicate that MB1 removed nitrogen and phosphorus from the M. aeruginosa solution.

Removal of MCs by strain MB1
To investigate the ability of strain MB1 to remove MCs from M. aeruginosa, it was introduced into the MC solution.In M0, peak areas for MC-LR were measured at 815,589, while MC-RR registered at 237,606 (Fig. 5A).Across various volumes of the MB1 inoculation (M1 to M6), a consistent decrease in peak areas for both MC-LR and MC-RR was observed against M0 (Fig. 5A, B, C, D, E, F, and G).The minimum peak area of MC-LR occurred with M4, measuring 11,577.Similarly, M4 exhibited the largest decrease in the MC-RR peak area, registering at 9,497 (Fig. 5E).Furthermore, we analyzed the contents of MC-LR and MC-RR in the MC solution.The initial concentrations of MC-LR and MC-RR, 27.39 and 6.05 μg mL -1 , respectively, significantly decreased with varying amounts of the MB1 inoculation (M1-M6) into the solution.Notably, the lowest concentrations of MC-LR and MC-RR in the MC solution were achieved with M4 (Fig. 6A and B).Furthermore, M4 exhibited the most effective removal of MC-LR and MC-RR on day 5 after the inoculation (Fig. S2).These results Fig. 2. Maximum-likelihood phylogenetic tree generated from sequences of the nuc rRNA genes ITS region.

Morchella Removes Microcystins
suggest that strain MB1 removed MCs.The potential of Morchella to enhance water quality by reducing nitrogen and phosphorus levels and removing MCs positions it as a valuable asset for wastewater treatment.

Discussion
The majority of biological methods use bacteria and fungi to inhibit the growth of algae and destroy MC structures   Morchella Removes Microcystins (Bouaicha et al., 2019).Algae-lyzing microorganisms, including fungi, including Trichoderma and Penicillium (Mohamed et al., 2014;Han et al., 2021), bacteria, such as Pseudomonas (Wang et al., 2013b;Liu et al., 2014), Bacillus (Shao et al., 2013), and Streptomyces sp.(Kong et al., 2014), as well as MC-degrading Acinetobacter (Yi et al., 2015), have demonstrated the ability to inhibit the growth of M. aeruginosa and even remove MCs.However, it may take up to 8 d for these algal-bacteria to dissolve algae, achieving a removal rate of approximately 90% (Kong et al., 2014).In contrast, Morchella strain MB1 used in the present study exhibited a removal rate of approximately 100% against M. aeruginosa cells within just 4 d.This result suggests that Morchella exhibits superior efficiency and efficacy for the removal of M. aeruginosa to those of algae-lytic bacteria.
Morchella is a common edible fungus that generally thrives in challenging environments characterized by low temperatures, darkness, and humidity.It is also known for its richness in polysaccharides (Zhang et al., 2023), and previous studies showed that these microorganisms produced polysaccharides in the stationary phase (Xu et al., 2008;Qian et al., 2023).However, the precise mechanisms underlying the effects of Morchella on MCs, whether it involves degradation or polysaccharide adsorption, remain unclear.Morchella may use nitrogen and phosphorus in water as nutrients for its growth, indicating its consumption of nitrogen and phosphorus in the M. aeruginosa solution used in this experiment.Morchella exhibits robust vitality and has the capability to form sclerotia in order to sustain growth in nutrient-deprived environments without undergoing natural apoptosis (Liu et al., 2018b;Sun et al., 2020).Moreover, it does not produce spores that may lead to secondary pollution, making it an optimal choice for managing M. aeruginosa.However, due to its classification as a soil fungus, the survival and reproduction of Morchella are limited in the natural aquatic environment where M. aeruginosa, a producer of MC toxins, thrives.
The potential of Morchella to enhance water quality by reducing nitrogen and phosphorus levels and removing MCs positions it as a valuable asset for wastewater treatment.Therefore, Morchella has potential as a promising biological technique to remove Microcystis in a short time and even MCs, thereby safeguarding both the environment and human well-being.Nevertheless, further investigations are needed to establish whether Morchella inhibits MC biosynthesis as well as the underlying mechanisms.Additionally, it is important to elucidate the specific mechanisms responsible for the inhibitory effects of Morchella on the growth and reproduction of M. aeruginosa.

Fig. 1 .
Fig. 1.Morphological characteristics of strain MB1.(A) Strain MB1 was cultured at 25°C on PDA medium for 20 d. (B) Older mycelia in the center of the medium secreted brown pigment.(C) The mycelia of strain MB1 cultured for 20 d. (D, E, and F) Sclerotia of strain MB1 cultured for 20 d.