With the global heavy metal pollution becoming more and more serious, more and more researchers begin to focus on the harm of heavy metal pollution to human and plants 23 and the use of high concentration of heavy metal plants to adsorb heavy metals in soil 24. Among them, the problem of Cd exceeding the standard is a major problem affecting the food safety of A. brasiliensis industry. Heavy metal stress may be caused by oxidative stress, DNA damage, inhibition of enzyme function and destruction of protein functions that regulate cell proliferation, differentiation or apoptosis 25. A series of changes in agronomic characters of A. brasiliensis were observed under Cd stress, which indicated that low concentration of Cd could promote the proliferation and differentiation of A. brasiliensis cells, and when the concentration was high, the cell apoptosis would be inhibited 26. Plants absorb and transport Cd from soil through their roots 27, however, edible fungi absorb and transport Cd from soil through hyphae, and then accumulate in stipe and pileus. In this study, there were significant differences in weight and fruiting body numbers among different treatments of J1 and J77, which indicated that Cd stress affected the physiological growth of A. brasiliensis, and there was no significant difference in the length of stipe and the thickness of pileus. Therefore, the two mushrooms maintained the characteristics of the original strain and did not produce much difference due to Cd stress. This indicated that although Cd stress had an impact on its growth, the strains had unique characteristics.
These heavy metals in soil may gain entry from plant roots via passive diffusion along with water molecules. Alternatively, they may gain co-entry along with active uptake of trace essential metals via either ion exchange between clay particles and root surface or along a concentration gradient along cation channels28. The results showed that the uptake of Cd by microorganisms increased with the increase of Cd concentration29,and application of 200 mg/kg soil Cd concentration, led to a significant accumulation of Cd in shoots and roots 30. This is consistent with the increase of Cd concentration in A. brasiliensis fruiting body in this study. Different agronomic measures have certain effects on the ability of plants to absorb Cd. The results of Cai et al. Showed that compared to CK, Cd accumulation in root when Si applied at transplanting and tillering stages were elevated by 75% and 64%, respectively. While Cd accumulation in aboveground tissues were all declined by Si addition 31. Plant growth regulators (PGRs) are widely used in agricultural activities and have the potential to improve plant growth and plant tolerance against metal stress, and the application of exogenous PGR had reduced the combined U and Cd stress by stimulating photosynthesis, decreasing the levels of active oxygen and lipid peroxidation, and enhancing the activity of the antioxidant defence systems32.
Plants use a series of different enzymes, such as SOD, POD, CAT, APX, to remove different types of ROS, thus protecting potential cell damage and tissue dysfunction. Trace metals can induce oxidative stress by inducing reactive oxygen species (ROS), and antioxidant enzymes promote reactive oxygen species resistance and clearance. SOD plays an important role in antioxidant defense by catalyzing the disproportionation of superoxide to oxygen and hydrogen peroxide33. SOD is an antioxidant metal enzyme existing in organism. It can catalyze the disproportionation of superoxide anion to oxygen and hydrogen peroxide. However, fungi generally contain Mn SOD and Cu/Zn SOD. Mn, Cu and Zn ions are more reductive than Cd ions, which may replace Cd to form Cd precipitation 34. Under high concentration of Cd stress, the yield of J1 decreased, which indicated that SOD did not clear the excess free radicals in fruiting bodies in time under high concentration of Cd stress. Therefore, the activity of SOD was higher, but the yield and agronomic traits were poor. However, the high concentration of Cd in J77 increased the yield, but the activity of SOD decreased. It can be seen that SOD can remove free radicals in fruiting bodies, thus increasing the yield and decreasing the content of SOD.
POD mainly regulates the aging degree of plants. The content of POD is lower in young tissues and higher in senescent tissues. When the concentration of Cd increased to a certain extent, the activity of POD showed a downward trend, which indicated that when the Cd concentration reached the limit that the fruiting bodies could bear, it would also affect the activity of POD. POD can oxidize all kinds of toxic substrates, and the result of oxidation makes these toxic substances become non-toxic substances. When J1 and J77 were exposed to Cd stress, POD could also play a role, so the activity of POD decreased under high concentration of Cd stress.
The decrease of CAT activity is one of the toxic effects of Cd. Oxygen free radicals produced under heavy metal stress inhibit these enzymes by attacking antioxidant enzymes and oxidative damage. When CAT activity decreased, the accumulation of H increased and catalase was inhibited. It is reported that the decrease of CAT activity and the increase of Cd concentration in some plants are responsible for the decrease of protein content after metal toxicity and oxidative stress 35. In this study, the CAT activity of J1 and J77 increased with the increase of Cd concentration. When the concentration was high to a certain extent, the CAT activity showed a downward trend, but the CAT activity under stress was higher than the control. CAT activity can provide antioxidant defense mechanism for the body. Its biological function is to promote the decomposition of hydrogen peroxide in fruiting body cells, so that it will not further produce toxic hydrogen oxygen free radicals, so as to protect the function of antioxidant enzyme system, which is of great significance for the fruiting body under stress. The enhancement of CAT activity can help fruiting bodies to produce antioxidant function and resist Cd stress.
APX, a peroxidase, plays a regulatory role in intracellular ROS levels. APX is a heme peroxidase, found in all kingdoms of life, and typically catalyzes the one-and two-electron oxidation of a number of organic and inorganic substrates. Only ascorbate and cytochrome c peroxidases are typical monofunctional peroxidases with either ascorbate or cytochrome c as one-electron donor36. In Arabidopsis, ascorbate peroxidase 1 (APX1) also plays key roles in the reactive oxygen gene network response to abiotic stress 37. In this study, the change of APX activity of J1 was small, and with the increase of Cd stress concentration, APX enzyme activity showed a downward trend, and the activity of each treatment was lower than the control. The APX activity of J77 increased under a certain concentration of Cd stress, and then decreased. The APX activity was inhibited by higher concentration of Cd stress.
Proline is an amino acid, which has a very beneficial effect on plants exposed to various stress conditions. The PRO concentration in cells, tissues and plant organs is regulated by the interaction of biosynthesis, degradation and intercellular transport. It is well known that PRO protects plants from stress, protects membrane integrity and stabilizes antioxidant enzymes by acting as a cell osmotic regulator between cytoplasm and vacuole, by detoxifying free radicals and buffering cell redox potential, and by stabilizing mitochondrial electron transport complex II, membranes, proteins and enzymes (such as Rubisco)38. The results showed that the level of ROS and other free radicals could be reduced by a certain concentration of PRO, so as to reduce the oxidative damage of plants. In addition, exogenous PRO can enhance the activity of antioxidant enzymes and affect the accumulation of endogenous plant hormones, thus improving the adaptability to various stresses. In this study, the PRO content of J1 and J77 increased with the increase of Cd concentration, which indicated that PRO content accumulated in fruiting bodies, which could better deal with the damage caused by Cd stress.
MDA is a final product of lipid peroxidation, and it has been extensively used to evaluate metal-induced OS. MDA content can be used as an indicator of the severity of stress, and the accumulation of MDA will bring certain damage to the membrane and cells, thus affecting the growth and development of fruiting bodies 39. Oxidative stress can lead to the increase of MDA content, which is caused by membrane lipid peroxidation, which ultimately affects the function and integrity of membrane 40. In this study, the MDA content of J1 fluctuated greatly. With the increase of Cd stress, the MDA content showed a trend of first increasing and then decreasing. After the fruiting body was stressed, the oxidative stress reaction occurred, and the MDA content accumulated. When the stress exceeded the defense ability of the fruiting bodies to a certain extent, the yield decreased significantly, and the accumulation of MDA content decreased. The MDA content of J77 fluctuated slightly, showing a trend of decreasing first and then increasing, which indicated that the effect of Cd stress on J77 strain was small, and J77 had strong resistance to stress, so the MDA content fluctuated less. The above results indicate that a decrease in invertase activity is accompanied by an increase in MDA content. The increase of reactive oxygen species will cause membrane lipid peroxidation, which will increase the content of MDA to inhibit the activity of invertase41.
Microorganisms can survive in high concentrations of heavy metals, which indicates that they can form an effective defense system and reduce the toxicity of heavy metals. These defense systems are based on extracellular and intracellular metabolites and can chelate with heavy metal ions. Microorganisms contain a variety of bioactive substances, such as polysaccharides, proteins and so on. These bioactive substances contain carboxyl group, phosphate group, hydroxyl group, mercaptan group, amino group and other functional groups. Through electrostatic adsorption, complexation, chelation, ion exchange and covalent adsorption, these bioactive substances combine with the surrounding heavy metal ions, or form extracellular sediments with heavy metal ions to prevent them from entering the cells. Polysaccharides and proteins play an important role in antiviral activity.
Under stress conditions, plants need adaptation to survive, which ranges from simple phenotypic to complex physiochemical alteration and differential protein expression42. Mycorrhizal plants are able to improve their resistance against protein degradation under Cd stress and maintain natural metabolism of proteins. Stress results in a myriad of alterations in plants, like changes in soluble protein content and electrolyte leakage of the plasma membrane, which in turn decrease the efficiency of the photosynthetic apparatus giving rise eventually to a reduction of crop yield43. The results showed that the protein content of strain J1 decreased with the increase of Cd concentration, and increased at high concentration, which was basically the same as that of control group. The protein content of J77 increased with the increase of Cd concentration, which indicated that J77 produced a series of oxidative stress response when exposed to Cd stress, so its protein content increased to resist external stress.
Extracellular polysaccharides play a role in the response, free survival and symbiosis of legume rhizobia to zinc stress. Zinc stress can stimulate the synthesis of extracellular polysaccharide and effectively protect cells from zinc stress 44. The polysaccharide played a vital role on enhancing tolerance of Trichoderma asperellum under Pb2+ stress. The high concentration of Pb2+ can promote the synthesis of polysaccharide in Trichoderma asperellummycelia. And the pure polysaccharide can interact with Pb2+ to adsorb or transform it. Under Pb2+ stress, the polysaccharide had response changes in chemical composition, primary structure and advance structure to reduce the amount of free Pb2+ and enhance the tolerance of Trichoderma asperellum45. High concentration of Cd promoted the synthesis of polysaccharide in J1. The interaction between polysaccharide and Cd resulted in the transformation of Cd, which indicated that the polysaccharide of J1 provided adsorption sites for Cd ions and reduced the toxicity of Cd to cells. The polysaccharide content of J77 increased under low concentration of Cd stress, but when the concentration increased, the polysaccharide content decreased and then increased, which indicated that the polysaccharide was more sensitive to 4 mg·kg− 1 Cd concentration.
The increase in isoleucine, phenylalanine and tyrosine under combined Cd and N stress may be because they are both glucogenic and ketogenic amino acids, while proline, histidine, glutamine, valine and asparagine on the other hand are glucogenic amino acids. The nature of the different amino acids and their possible roles in lipid and carbohydrate biosynthesis through the tricarboxylic acid cycle (TCA cycle) can explain the positive relationship they had with total lipid and carbohydrates production in C. vulgaris46. The content of total amino acids was greatly affected by Cd stress. Both J1 and J77 showed a trend of decreasing first and then increasing. This may be due to the stress response of fruiting bodies to Cd stress. The decrease of amino acid content may be due to the increase of protein synthesized in fruiting bodies and other biomolecules responding to Cd stress, so amino acids were consumed Substance.