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. 2024 Sep 2;14:20394. doi: 10.1038/s41598-024-70994-3

Ginseng rusty root symptoms result from nitric oxide stress in soil

Peng-cheng Yu 1, Wei Zhang 1, Li-yang Wang 1, Wen-fei Liu 1, Xiu-Bo Liu 2, Yao Yao 1, Xiao-wen Song 1, Zhao-Ping Meng 1, Xiang-cai Meng 1,✉
PMCID: PMC11368917  PMID: 39223197

Abstract

Ginseng, from the roots of Panax ginseng C. A. Meyer, is a widely used herbal medicine in Asian countries, known for its excellent therapeutic properties. The growth of P. ginseng is depend on specific and strict environments, with a preference for wetness but intolerance for flooding. Under excessive soil moisture, some irregular rust-like substances are deposited on the root epidermis, causing ginseng rusty symptoms (GRS). This condition leads to a significant reduce in yield and quality, resulting in substantial economic loses. However, there is less knowledge on the cause of GRS and there are no effective treatments available for its treatment once it occurs. Unsuitable environments lead to the generation of large amounts of reactive oxygen species (ROS). We investigated the key indicators associated with the stress response during different physiological stages of GRS development. We observed a significant change in ROS level, MDA contents, antioxidant enzymes activities, and non-enzymatic antioxidants contents prior to the GRS. Through the analysis of soil features with an abundance of moisture, we further determined the source of ROS. The levels of nitrate reductase (NR) and nitric oxide synthase (NOS) activities in the inter-root soil of ginseng with GRS were significantly elevated compared to those of healthy ginseng. These enzymes boost nitric oxide (NO) levels, which in turn showed a favorable correlation with the GRS. The activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase first rose and then decreased as GRS developed. Excess soil moisture causes a decrease in oxygen levels. This activated NR and NOS in the soil, resulting in a production of excess NO. The NO then diffused into the ginseng root and triggered a burst of ROS through NADPH located on the cell membrane. Additionally, Fe2+ in soil was oxidized to red Fe3+, and finally led to GRS. This conclusion was also verified by the Sodium Nitroprusside (SNP), a precursor compound producing NO. The presence of NO from NR and NOS in water-saturated soil is responsible for the generation of ROS. Among these, NO is the main component that contribute to the occurrence of GRS.

Keywords: Ginseng rusty root symptoms, NO, ROS, Environmental stress, Antioxidant oxidase

Subject terms: Plant physiology, Plant stress responses

Introduction

Ginseng, the dried root of Panax ginseng C. A. Meyer, contains saponins, polysaccharides, and volatile oils. It has pharmacological effects such as nerve regulation, cardiovascular regulation, anti-oxidant, anti-inflammatory activity, anti-tumor activity, and anti-obesity properties1,2. It has a long history of therapeutic use. P. ginseng has a long growth cycle and requires precise and specific ecological conditions. It thrives in wet, slightly acidic (pH 4–6), well-aerated soil, showing a preference for moisture while being intolerant to excessive water levels3. In the presence of excessive soil moisture and a large number of undecomposed branches, P. ginseng becomes highly susceptible to a common disease known as red rusty symptom (GRS). This disease is characterized by the roots’ epidermis being covered with irregular rust-like red rough patches4,5. The presence of high contents of compounds such as Fe2O3, Al2Si3O3, Al3Cl (CH3COO)5·1.5CH3COOH, C13H21Al3O13 and C14H10Fe2NS2, which are characteristic components of GRS, contribute to this symptom by containing significant amount of Al3+ and Fe3+6–10. GRS not only reduces the yield but also diminish the levels of bio-active compounds11. In the 1990s, GRS resulted in an economic loss exceeding seven million yuan12. While there is lack of precise contemporary information, the incidence of GRS remains constant13. Despite the long history of ginseng cultivation, the etiology of GRS remains unclear. Some believe that GRS is a physiological disorder14,15, while others consider it an infectious disease caused by rhizobacteria such as acrophialophora and doratomyces16.

Whatever the reason is, both excessive soil moisture and a large amount of undecomposed branches and leaves are indispensable17. Excessive soil moisture reduces soil aeration, resulting in the depletion of localized O2 due to the decomposition of plant waste, hence stimulating the growth of anaerobic bacteria in the soil18. Under these conditions, the activities of nitrate reductase (NR) and nitric oxide synthase (NOS) were enhanced, resulting in the production of substantial quantities of NO19,20. NO is a reactive small molecule that can rapidly diffuse through the lipid membrane and cause stress responses in plant cells. Excessive soil moisture is also an environmental stress, that inevitably leading to over-production of reactive oxygen species (ROS)21,22. ROS, including O2·−, H2O2, ·OH, and others, are oxygen-containing substances with high oxidizing capacity. The mitochondrial respiratory chain is one of the main pathways to produce ROS. During electron transport, excessive production of ATP or obstruction of electron transport in the respiratory chain can lead to the leakage of single electrons. These electrons can then be transferred to O2, forming O2·−23. NADPH oxidase, an enzyme located in the cell membrane, plays a crucial role in generating ROS in response to external stresses24. High levels of ROS can cause the peroxidation of lipid in cell membranes, and produce large amounts of malondialdehyde (MDA), leading to permanent cellular damage. The antioxidant enzymes are essential for removing ROS. Superoxide dismutase (SOD) quickly converts O2·− into H2O2, which is then further converted into harmless H2O and O2 by catalase (CAT) or peroxidase (POD)25. In addition, the ASA-GSH cycle system, which includes ascorbic acid (AsA), and glutathione (GSH), glutathione oxidized (GSSG), ascorbate peroxidase (APX) and glutathione reductase (GR), is crucial for preserving protein stability, maintaining the integrity of biofilm structure integrity, and defending against membrane lipid peroxidation. Furthermore, this system also plays a significant role in scavenging ROS26.

To date, little is known about the etiology of GRS, which hinders the ability to successfully treat the condition once it occurs. It has been demonstrated that GRS is related to water stress, and ecological stress ultimately results in the excessive generation of ROS. Based on these, the etiology of GRS was investigated from the perspective of adversity physiological ecology in this paper.

Materials and methods

Plant material and tissue collection

All the plant experiments complied with relevant institutional, national, and international guidelines and legislation. Cultivated P. ginseng (RS20230710) collection was done with permission. All ginseng samples (5 years old), identified by Prof. Xiang-Cai Meng of the Heilongjiang University of Chinese Medicine, were collected in Baishan City, Jilin Province, China (42.17 N and 127.48 E) in July 2023. The farm is situated in a low-lying area, susceptible to flooding during the rainy season, and has a high GRS incidence. P. ginseng roots were excavated, cleaned, and the phloem tissue was isolated with a sharp blade. The phloem tissue from healthy ginseng (AG), phloem tissue around red-patches of GRS (BG), and red-patches tissue of GRS (CG) were collected, rapidly frozen in liquid nitrogen and then stored at -80 °C. Three independent biological replicates were prepared for each group, with each replicate consisting of individuals from five or more roots. The cultivated P. ginseng (RS20230710) has been deposited in a publicly available Heilongjiang University of Chinese Medicine herbarium. We complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Induction of GRS by SNP

Healthy ginseng and corresponding soil are collected from ginseng farm, transplanted in a pot, soaked in SNP (Sodium Nitroprusside) solution (0.5 mmol/L), prepared with ultrasonic treated water (to remove O2 of H2O), until the soil is saturated, covered with plastic wrap after each treatment to create an anoxic environment. After 30 days, the roots were dug out, cleaned, and photographed.

Extraction of plant tissue

The samples of each group were added with the corresponding PBS buffer (pH = 7–7.4) at the ratio of weight (g): volume (mL) = 1:10 and grounded into homogenate under an ice water bath. The supernatant crude enzyme was obtained by centrifugation at 5000 rpm/min for 10 min, subpackaged, and stored at − 80 ℃ to detect various indexes.

Quantification of ROS and MDA

The levels of O2·− were determined using the O2·− assay kit (spectrophotometry; Beijing Solarbio Technology Co., Ltd., 20230609). O2·− can reacts with hydroxylamine hydrochloride to produce NO2−, then form a purplish red azo compound with a characteristic absorption peak at 530 nm under the action of p-aminobenzenesulfonamide and N-(1-Naphthyl) ethylenediamine dihydrochloride. The content was calculated according to A530.

H2O2 contents were determined using the plant H2O2 assay kit (colorimetric method; Nanjing Jiancheng Bioengineering Research Institute, 20230806), the absorbance of the reaction between crude enzyme solution and molybdic acid was measured at 405 nm, and the amount of H2O2 was calculated.

The concentration of MDA was detected with malondialdehyde kit MDA kit (Nanjing Jiancheng Bioengineering Research Institute, 20230422) by TBA method.

Determination of antioxidant enzyme activity

The activities of SOD were determined with SOD assay kits (Nanjing Jiancheng Bioengineering Research Institute, 20230624). The crude enzyme solution was added to the working liquid and incubated at 37 ℃ for 20 min, and its activity was calculated by absorbance at 450 nm.

The activities of CAT were determined with CAT assay kit (Nanjing Jiancheng Bioengineering Research Institute, 20,230,903). The activities were calculated by the reaction of H2O2 and ammonium molybdate at 405 nm.

The activities of POD were determined with POD assay kit (Nanjing Jiancheng Bioengineering Research Institute, 20230616). The activities were calculated by measuring the absorbance change at 420 nm, based on the principle of POD catalyzing hydrogen peroxide reaction.

Determination of the AsA-GSH cycle

The activities of APX were determined with APX assay kit (Nanjing Jiancheng Bioengineering Research Institute, 20230903). The crude enzyme solution mixed with the substrate, the activities were calculated by measuring the absorbance values at 290 nm for 10 s and 130 s.

The activities of GR were determined with GR assay kit (Beijing box labor Technology Co., Ltd., 1012304023). The crude enzyme solution was mixed with the substrate and, the activities were calculated by measuring the absorbance values for 10 s and 190 s at 340 nm.

AsA content assay kit (Beijing box labor Technology Co., 1012304011) was used to calculate the AsA content by calculating the difference in reaction between ascorbic acid and substrate at 265 nm.

The contents of GSH and GSSG were detected with corresponding assay kits (Beijing box labor Technology Co., Ltd., 1012304032; 1012304041) based on the principle that the product produced by the reaction of glutathione and 5,5′-dithiobis-2-nitrobenoic acid had a characteristic absorption peak at 412 nm.

Determination of NR and NOS in soil

While plant samples was collected, the rhizosphere soil of healthy and rusty ginseng was also collected. The soil was dried at 37 ℃, and ground to a powder with a particle size of less than 0.25 mm. The activities of NR and NOS were determined using the s-NR ELISA detection kit (Nanjing Jiancheng Bioengineering Research Institute, 20230220) and s-NOS (Jiangsu Jingmei Biotech Co., Ltd., 202303) ELISA detection kit.

Determination of NO content and NO synthesis-related enzyme activities

NO level was measured indirectly at 550 nm with NO assay kit (Nanjing Jiancheng Bioengineering Research Institute, 20230320) by colorimetry.

The activities of NR were calculated by the absorbance of the compounds formed by NR-reduced nitrite, p-amino benzene sulfonic acid, and 1-naphthylamine at 540 nm (NR assay kit, nitrate reductase assay kit Nanjing Jiancheng Bioengineering Research Institute, 20,230,320).

The activities of NOS were detected with Total Nitric Oxide Synthase assay kit (Nanjing Jiancheng Bioengineering Research Institute, 20230806) based on the principle that NOS catalyzes the reaction of L-Arg and O2.

Determination of NADPH oxidase activities

The activities of NADPH oxidase were determined with a plant NADPH oxidase ELISA detection kit (Jiangsu Jingmei Biotech Co., Ltd., 202303).

Statistical analysis

Statistical analysis was performed with SPSS 20.0 software. The data were expressed as the mean ± standard deviation (SD). p < 0.05 was considered to be statistically significant.

Results

Quantification of ROS and MDA

The levels of O2·− in the BG and CG groups were 166.2% and 101.0% higher than in the AG group, while the H2O2 levels were 99.8% and 67.4% higher, and MDA levels were 120.4% and 216.7% higher, respectively. Compared with the BG group, the CG group exhibited a decrease of 24.5% and 16.2% in O2·− and H2O2 levels, respectively. Additionally, the CG group showed a reduction of 43.7% in MDA. These findings suggest that the occurrence of GRS is related to environmental stress, and physiological characteristics of stress had emerged before the GRS. As shown in Fig. 1.

Fig. 1.

Fig. 1

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Changes of antioxidant indexes in tissues with different severity of GRS.

Activities of Antioxidant enzymes

Compared with the AG group, the activities of SOD, CAT, and POD were significantly increased in the BG and CG groups. Among them, the SOD activities in the CG group were the highest, with a 50.4% increase compared to the AG group. The BG group also showed a 30.0% increase in SOD activities. The CAT and POD levels in the BG group showed the most significant rise, with an increase of 168.0% and 104.7%, respectively. The CG group also exhibited a significant increase of 113.2% and 39.8%, respectively, indicating a response of P. ginseng roots to stress. As shown in the Fig. 2.

Fig. 2.

Fig. 2

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Key enzyme activities and antioxidant content in AsA-GSH cycle.

Changes in main indicators of the AsA-GSH cycle

The AsA-GSH cycle showed an increasing and then decreasing trend. The BG group presented significantly higher levels of APX and GR activity, as well as AsA content and the GSH to GSSG ratio, than the AG group. These increases were 199.8%, 110.9%, 111.1%, and 174.8%, respectively. As shown in Fig. 2.

Activities of S-NR and S-NOS in soil

The activities of S-NR and S-NOS in the rhizosphere soil of GRS were considerably greater compared to those in healthy ginseng. There was a 174.9% rise in SNR and a 27.8% increase in S-NOS, respectively, indicating a large amount of production of NO in the soil. As shown in Fig. 3.

Fig. 3.

Fig. 3

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Determination of NR and NOS activities in the soil, the NO content, NR and NOS activities and NADPH oxidase activity in plants.

NO contents and activities of NR and NOS in ginseng tissues

The NO contents in ginseng increased as the GRS increased. The highest rise of 240.0% was observed in the CG group, followed by a 133.3% increase in the BG group compared to the AG group. The activities of NR and NOS in the BG group showed the highest levels, with an increase of 180.0% and 38.7%, respectively, compared to the AG group. The activities of NR and NOS were slightly reduced in the CG group compared to the BG group, with no significant difference between them. This suggests that the NO in the soil was absorbed by plant tissues, resulting in further NO production in the plants. As shown in Fig. 3.

Activities of the NADPH oxidase

The BG group exhibited a significantly higher NADPH oxidase activity compared to the AG group, with a notable rise of 64.1%. Conversely, the CG group displayed a considerable decrease in the NADPH oxidase activity, with a notable decrease of 31.0%. As shown in Fig. 3.

Validation of GRS induction by SNP

The P. ginseng roots treated with SNP solution appeared to have irregular red rust-colored scars on the epidermis, which aligns with the GRS and suggests that NO is the essential cause of GRS formation. As shown in Fig. 4.

Fig. 4.

Fig. 4

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Verification of SNP-induced GRS.

Discussion

The essence of GRS occurred

It is inevitable for plants to produce large amounts of ROS under unfavorable conditions27. GRS has been acknowledged to have a strong correlation with both excessive soil moisture and the presence of un-decayed plant residues. Under severe stress, the balance between ROS generation and elimination is disrupted, resulting in a large amount of accumulation of ROS and subsequent oxidative damage. ROS consists of O2·−, H2O2, ·OH, and others. Among them, the ·OH is extremely active and has a short half-life, making it difficult to monitor28. O2·− is also extremely unstable and readily converted to H2O2, which has higher stability and a relatively long survival cycle. Therefore, H2O2 serves as a more precise indication for measuring the quantity of ROS29,30. MDA, which is produced as a result of damage to the cell membrane, is considered as a critical indicator for assessing cellular damage31. The levels of O2·−, H2O2, and MDA in the BG and CG groups were significantly increased compared to the AG group. This suggests that the cells experienced ecological stress and were severely damaged both before and during GRS development. Interestingly, the ROS in the CG group was slightly lower than that in the BG group, probably due to the ROS production by enzymatic reactions32. The excessive ROS impaired the activity of ROS-producing enzymes, resulting in a decrease in ROS. This observation is further supported by the fact that the severity of the GRS is most strongly correlated with the levels of MDA. Plants protect their cells from ROS through a complex defense system consisting of antioxidant enzymes and non-enzymatic antioxidants. Under stress, antioxidant enzyme activities in plants usually show a dynamic change, initially increasing and subsequently dropping. This pattern may be attributed to the damage of elevated levels of ROS on enzymes33,34. Figure 1 shows that activities of CAT and POD exhibited a similar change, reaching their highest level at the BG group prior to the GRS. The MDA content in the CG group was higher than the BG group (Fig. 1), indicating a lower activity of antioxidant enzymes in the CG group. Interestingly, SOD activity has been increased (Fig. 1). Under copper or drought stress conditions, the antioxidant enzyme activities of ginseng also showed similar changes, with SOD displaying higher stability compared to CAT and POD in ginseng35,36.

The ASA-GSH cycle is also a critical system for scavenging ROS. As the primary enzyme, APX uses AsA as an electron donor to eliminate H2O2 and generate DHA (dehydroascorbate). GSH reacts with DHA to produce AsA and oxidized glutathione (GSSG). GR catalyzes the decomposition of GSSG into GSH and returns to the cycle as the last essential enzyme in the cycle37,38. AsA and GSH have the ability to directly eliminate ROS as non-enzymatic components39,40. They are less susceptible to the effects of ROS. However, APX and GR in the CG group are still reduced, and excessive ROS are also difficult to eliminate through this pathway. As shown in Fig. 2.

In summary, large amounts of ROS have been accumulated before GRS occurred.

Sources of ROS

The occurrence of GRS is closely related to environmental stress. The acidic anoxic environment can directly activate NR, inhibit the function of nitrite reductase (NiR), and increase the level of nitrite substrate and the activity of NOS19,20,41,42. These enzymes are critical in catalyzing NO biosynthesis. A potential association between NO and GRS in the soil environment is possible. Figure 3 showed that the NR and NOS activities in the rhizosphere soil of GRS were significantly higher than the healthy P. ginseng, This suggests that there is a large amount of NO production around the rusty root. Usually, NO can be quickly oxidated to NO2 and diffused into the atmosphere. However, perishable plant residues in water-saturated soil can deplete the limited oxygen and prevent oxidation, resulting in nitric oxide accumulation43. In addition, at hypoxic conditions, NOS and NR have high activities44. Mitochondria can also produce NO45, while cytochrome C oxidase activity is reduced46, all leading to NO accumulation. As an active signaling molecule, NO can freely diffuse through membrane lipids within or between plant cells. Figure 3 showed that the activities of NR and NOS in the plant of the BG group peaked, whereas the CG group remained stable. However, despite this physiological condition, the NO contents in plants still showed a significant increase, suggesting that NR and NOS in soil were the primary factors responsible for the NO biosynthesis, resulting in a continuous rise in NO content in the CG group. An anoxic environment leads to excessive electron accumulation in complexes I and III in the mitochondrial respiratory chain47, leading to an increase in the NADH/NAD+ ratio48. At the same time, mitochondria also produce a large amount of ATP in response to stress49, which collaboratively establishes a necessary condition for O2·− production. Under such conditions, NO can activate the electron carrier protein in mitochondria, enhancing its interaction with O2 and forming O2·−. This process reduces the mitochondrial O2 consumption rate by competitively binding to cytochrome C oxidase. It also impacts the production of O2·− in vivo by changing the local O2 concentration50,51.

In addition, NADPH oxidase is another significant producer of ROS in plants and can be activated by exogenous NO52. Figure 3 showed a significantly increased activity of NADPH oxidase in the BG group compared with the AG group. The decreased activity of NADPH oxidase in the CG group (P < 0.05) may result from irreversible cell membrane damage to the cell caused by increased stress. This is evidenced by the decrease in the levels of ROS and the significant increase in MDA contents in the CG group compared with the BG group in Fig. 1. In addition, the results also indicated that the ROS levels and antioxidant enzyme activities were elevated following the administration of exogenous NO treatment, suggesting that NO is the source of ROS53–55.

GRS is characterized by the presence of irregular rust-like patches on the root epidermis. Figure 5 showed a P. ginseng root with GRS. The main root of P. ginseng had a large patch, and the lateral roots adjacent to the main root patch also produced GRS. These patches are distributed in a centralized manner on the soil, indicating that the soil is the initial source of ROS. In summary, P. ginseng can produce a significant amount of ROS when exposed to exogenous NO stress. The NO is produced by activated NR and NOS in acidic soil.

Fig. 5.

Fig. 5

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Patches characterization of GRS.

Formation of GRS

Microorganisms can convert most nitrogen from un-decayed plant residues in soil into nitrate ions through nitrification56. In a water-saturated soil, H2O2 transports nitrate ions and base ions (Ca2+, Mg2+, K+) on the colloid from the surface to the deeper soil layers. This results in an increase in H+ in the topsoil and a decrease in soil pH42. Under this condition, Al3+ persists in a stable state, resulting in the root surface of GRS being rich in Al2Si3O3, Al3Cl (CH3COO)5·1.5CH3COOH7. NADPH oxidase is located in the cell membrane, where it generates O2·− with a very short half-life and is not capable of being transported over long distances. Hypoxia occurs when soil becomes saturated, causing the iron ions in the soil around the root to exist as Fe2+ instead of Fe3+ in acidic environments. These Fe2+ can be oxidized to red Fe3+ by ROS from NADPH oxidase. This Fe3+ is then deposited on the cell wall, leading to the formation of a red-rust color epidermis. In addition, in the presence of Fe2+, the Fenton reaction can produce the extremely destructive and more toxic ·OH (Fe2+ + H2O2 → ·OH + OH− + Fe3+)57. The presence of C14H10Fe2NS2 in the epidermis indicates a similarity in the C:N ratio to that of cell membrane fatty acids, suggesting it is a product of disrupted cell membrane fatty acid7.

The above results showed that ROS is the essential factor response for GRS, and NO in soil is the origin of ROS. An aqueous solution of SNP can produce NO. When P. ginseng roots were treated with this solution, they exhibited irregular red to reddish brown patches, indicating that GRS was caused by excessive NO in soil. Results are shown in Fig. 4.

Conclusion

In conclusion, the saturated water and un-decomposed branches in the soil resulted in a decrease in O2 level and an increase in acidity. This, in turn, led to an increase in s-NR and s-NOS activities, resulting in the production of a large amount of NO. Consequently, P. ginseng roots experience Q2a burst of ROS. The soluble Fe2+ near the root was oxidized to red Fe3+ and deposited on the epidermis of the P. ginseng roots, with a red-rust color of GRS. Therefore, NO is the primary factor producing GRS.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant Number: 20012210001) and the National Key Research and Development Program of China (Grant Number: 2021YFD1600901).

Author contributions

P.C.Y. was responsible for Material preparation, data collection and analysis and wrote the main manuscript text. W.Z., L.Y.W., and W.F.L. carried out the figure/table preparation and interpretation of data. X.B.L., Y.Y.,X.W.S. and Z.P.M. contributed to the study design, provided the technical support, collected and organized data and provided new ways to solve problems. X.C.M. participated in the conceptualization and design of the experiment, interpretation of data, and revision and approval of the final version of the manuscript. All authors reviewed and approved the final manuscript.

Data availability

The data supporting this study's findings are available from the corresponding author upon request. Source data are provided in this paper.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Data Availability Statement

The data supporting this study's findings are available from the corresponding author upon request. Source data are provided in this paper.

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