- Email: [email protected]
Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress
ARTICLE IN PRESS
JID: SAJB
[m5G;November 22, 2019;5:56]
South African Journal of Botany 000 (2019) 1 9
Contents lists available at ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress Y.M. He, X.M. Fan, G.Q. Zhang, B. Li, T.G. Li, Y.Q. Zu, F.D. Zhan* College of Resources and Environment, Yunnan Agricultural University, Kunming 650201, China
A R T I C L E
I N F O
Article History: Received 29 June 2019 Revised 12 September 2019 Accepted 23 September 2019 Available online xxx Keywords: Endophytic fungi Biomass Photosynthetic physiology Root traits Cadmium content
A B S T R A C T
Arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) are generally colonized in plant roots in metal-polluted environment. A pot experiment including four treatments (AMF inoculation of Funneliformis mosseae, DSE inoculation of Exophiala pisciphila, co-inoculation of AMF + DSE, and non-inoculation) under a high cadmium (Cd) stress (50 mg/kg) was conducted to discuss the ecological functions and interactions of AMF and DSE. The biomass, photosynthetic physiology, root morphology and traits, Cd content of maize were investigated. According to the two-way analysis of variance, AMF and DSE had no synergetic interactions on the maize performance under the high Cd stress. Both AMF inoculation and AMF + DSE co-inoculation, but not DSE inoculation, significantly increased the chlorophyll contents in maize leaves, resulting in stronger photosynthetic physiology, higher biomass, and increased length, surface area, volume, branches, tips number, and mass density (RTD) of maize roots, but decreased specific root length (SRL), specific root area (SRA), specific root branches (SRB), and specific root tips (SRT). However, there was a strong interaction between AMF and DSE on the Cd content in the maize shoots and roots. Single or co-inoculation of AMF and DSE significantly decreased the Cd content in shoots and Cd transfer coefficient of maize. Correlation analysis indicated that the Cd content in shoots was significantly positively correlated with SRL, SRA, SRB and SRT but negatively correlated with RTD. Thus AMF and DSE altered the root traits that contributed to restricting Cd migration from roots to shoots of maize. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Soil cadmium (Cd) pollution intensifies increasingly due to industrial and agricultural production activities, such as mining, smelting, sewage irrigation, and fertilization (Su et al., 2014). Cd is a toxic metal element that is unnecessary for plant growth (Benavides et al., 2005). It causes great toxic effects to the growth characters of crops by generating abundant reactive oxygen species in crops (Anjum et al., 2015), destroying the chloroplast structure, inhibiting photosynthesis of leaves, hindering the growth and water and nutrient absorption and transportation of the plant root system, as well as restricting the plant growth (Gallego et al., 2012; He et al., 2017). The colonization of arbuscular mycorrhizal fungi (AMF) and dark septate endophyte (DSE) is found in plant roots in a Cd-polluted habitat. For instance, AMF and DSE are extensively colonized in the roots of wild plants grown in the lead (Pb) and zinc (Zn) mine areas, where Cd pollution is serious, and often coexist in the roots of the same plant (Liang et al., 2007), showing strong endurance and adaptation to Cd stress (Ban et al., 2012). AMF and DSE are colonized in the roots of oat grass (Arrhenatherum elatius) on the slagheap of the Zn smelting plant (Deram et al., 2008). The AMF infection rate in the roots *Corresponding author. E-mail address: [email protected] (F.D. Zhan).
decreases with the increase of Cd concentration in soils, whereas the DSE infection rate is stable and insensitive to soil Cd concentrations (Deram et al., 2008). The DSE infection rate in the roots of oat grass in Cd-polluted soils is higher than that in non-polluted regions (Deram et al., 2011). AMF can survive in mutualism with more than 80% terrestrial plants and influences the growth character of the host plants. It plays an important role in increasing Cd resistance of the host plants (Garg and Bhandari, 2014). AMF can relieve toxic effects of Cd stress on plants, facilitate root growth, change the morphology of the root system, and enhance Cd absorption and fixation of the roots (Li et al., 2016). AMF improves the physiological activities (e.g. mineral nutrients, photosynthesis, and stress resistance) of host plants (Allah et al., 2015; Zhan et al., 2018), promote the host plant growth, enhance the Cd tolerance of the host plants, and change Cd absorption and accumulation of plants (Zhanet al., 2016). Similarly, the inoculation of DSE can enhance stress resistance of the host plants, increase the photosynthetic pigments content, and improve the photosynthesis effect, thus increasing plant biomass (Ban et al., 2017; He et al., 2017; Zhan et al., 2017). It also facilitates Cd accumulation in plant roots and reduce Cd migration from the roots to the shoots, thus decreasing Cd content in the shoots (He et al., 2017; Li et al., 2011; Wang et al., 2016). Thus, AMF and DSE colonized in the plant
https://doi.org/10.1016/j.sajb.2019.09.018 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB 2
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
roots can facilitate plant growth, improve the leave photosynthesis, and enhance Cd tolerance in plants (Deng and Cao, 2017). In the natural environment, AMF and DSE are often colonized in the roots of the same host plant. Researchers began to pay attention to the influences of AMF and DSE coexistence on the growth characters of plants (Berthelot et al., 2018; Shahabivand et al., 2012). Under Cd stress, either single inoculation or co-inoculation of AMF and DSE can facilitate wheat growth and increased chlorophyll content in leaves. Wheat with single inoculation of DSE achieves the highest biomass and lowest Cd content in the aboveground part (Shahabivand et al., 2012). A single inoculation of AMF increased the biomass of the shoots of ryegrass (Lolium perenne) on soils with composite pollution of Cd/Pb/Zn, whereas single inoculation of DSE and the co-inoculation of AMF and DSE did not substantially affect the biomass of ryegrass. Only the co-inoculation of AMF and DSE remarkably decreased Cd content in the shoots of ryegrass (Berthelot et al., 2018), revealing that the coexistence of AMF and DSE in roots may influence the growth characters and Cd tolerance of the host plants differently. Associated studies are rare, and additional relevant studies must be conducted. In this experiment, one strain of AMF (Funneliformis mosseae) and one strain of DSE (Exophiala pisciphila) were used. A pot experiment, with maize as the host plant under 50 mg/kg Cd stress, was conducted to investigate the effects of AMF and DSE on plant growth, photosynthesis physiology, root morphology and traits, Cd content, and accumulation of maize. The following hypotheses were proposed in this paper: (1) the AMF and DSE colonization in the maize roots can improve the performance of maize under high Cd stress; and (2) changes of Cd content induced by the AMF and DSE colonization were closely related to the root traits of maize. 2. Materials and methods 2.1. Testing samples The testing soils were mountain red earth with pH 7.5. The organic matter content was 13.87 g/kg. The concentrations of total nitrogen (N), phosphorus (P), and potassium (K) were 0.112, 0.32, and 1.98 g/kg, respectively. The contents of alkali-hydrolyzable N, available P and K were 26.18, 1.15, and 33.74 mg/kg, respectively, and the Cd content was 0.275 mg/kg. Soil samples were dried naturally in air in the laboratory and then ground and filtered through a 2-mm sieve. The testing river sands were collected from a construction site. They were dried naturally in air and then screened through the 2-mm sieve. The testing samples in the pot experiment were mixtures of river sand and soil at a proportion of 3:1 (w/w). The soil-sand mixture were autoclaved at 121 ℃ for 2 h. The AMF inoculants used in the experiment were Funneliformis mosseae BGC YN05, which were provided by the Institute of Plant Nutrition and Resources at the Beijing Academy of Agriculture and Forestry. The indoor-dried F. mosseae inoculants contained about 60 70 spores in each gram. The DSE strain used was Exophiala pisciphila was separated from the roots of Arundinella bengalensis (Poaceae), which was grown naturally on the Huize Pb-Zn mine area in Yunnan Province (Zhang et al. 2008). It was stored in the laboratory under 4 °C. The DSE fungus was cultivated onto the potato dextrose agar (PDA) media under 28 °C for two weeks to activate the strain. A native maize cultivar (Huidan No. 4) from a seed market in Dabanqiao Town, Kunming City, Yunnan Province was used in the experiment. Before sowing, full and uniform sized maize seeds were chosen. The maize seeds were immersed in 75% ethanol for 10 min and in 10% sodium hypochlorite for another 10 min, and followed by four rinses with sterile water. After surface sterilization of the maize seeds, they were placed on a sterile filter paper in Petri dishes, added some sterile water to make the filter paper to be wet, and cultured in an incubator at 28 °C for 3 d After germination to about 1 cm, sterile seedlings with uniform growth were chosen for later use.
2.2. Pot experiment First, the maize-DSE consortium was constructed. The 25 cm (height) £ 6.5 cm (diameter) cylinder glass bottles were used, in which 20.0 g perlite and 20 mL Hoagland nutrient solution were added. The set up was autoclaved under 121 °C in 30 min. Eight glass bottles were added with 15 pieces of 2-mm (diameter) DSE colony. After mixing evenly with perlite, two pieces of germinated maize seeds were added and covered with 1-cm thick sterile sands. The bottles were sealed up with sterile polyvinyl alcohol film and cultured for 14 d under 25 °C and light conditions of 1000 8000 Lux for 10 h per day. Some maize seedlings were collected and examined under the microscope to confirm the successful colonization of DSE in the roots. The rest of the glass bottles were not inoculated with DSE colony, and the grown maize seedlings had no DSE colonization. Second, the pot experiment was carried out in a greenhouse. The river sand and soils mixture at the proportion of 3:1 (W/W) was used as the culture media, and the cadmium chloride (CdCl2¢2¢5H2O) solution was added to reach the 50 mg/kg Cd stress level. They were mixed completely and balanced in 2 weeks. Plastic pots, which were 20 cm high and 25 cm wide in diameter, were applied and sterilized using 75% alcohol. Later, 5.0-kg sterilized soil-sand mixture was filled. Four treatments, namely CK (non-inoculation), AMF (F. mosseae inoculation), DSE (E. pisciphila inoculation), and AMF+DSE (co-inoculation of F. mosseae and E. pisciphila) were set. Maize seedlings without DSE colonization and with 50 g killed AMF inoculants was planted in the CK treatment. Maize seedlings without DSE colonization was planted with 50 g AMF inoculants in the AMF treatment. Maize seedlings with DSE colonization and 50 g killed AMF inoculants was planted in the DSE treatment. Maize seedlings with DSE colonization and 50 g AMF inoculants was planted in the AMF+DSE treatment. Each treatment had four pots and one maize seedling was planted per pot. After planting the maize seedlings, 150 mL 50% Hoagland nutrient solution was poured in every 3 d Soil moisture was maintained at a constant through weighting and water supplementation.
2.3. Determination of plant growth and fungal colonization Maize height was tested at 60 d after the transplantation, then the maize plant was harvested and divided into the shoots and roots. They were cleaned using distilled water and oven-dried at 75 ℃ for 48 h to determine the weight of the maize plant. The AMF spores in the maize rhizosphere soil were separated through wet sieving, decanting, and sucrose centrifugation (Daniels and Skipper, 1982). The spore quantity was observed and calculated under the microscope. Root samples were collected randomly from each maize plant and dissociated by 10% (W/V) KOH solution for 1 h in 90 ℃ thermostatic water bath; the root samples were washed, dropped a few lactic acid to neutralize the remaining KOH and cut into 0.5 1 cm fragments. Then, 20 fragments were put on a glass slide and dyed with 5% blue ink-acetic acid (Cao et al., 2013). They were observed under the microscope, after the decoloration and infection rates of AMF and DSE were calculated via the crossing method (McGonigle et al., 1990). Table 1 Spore number of AMF and root colonization of AMF and DSE. Treatment
CK DSE AMF AMF+DSE
Spore number of AMF (number/g DW)
AMF colonization rate (%)
13.85§0.98 a 13.2 § 0.94 a
46.15§6.17 a 22.97§4.09 b
DSE colonization rate (%)
25.47§7.24 a 14.35§5.72 b
CK: the control of non-inoculation, DSE: Exophiala pisciphila inoculation, AMF: Funneliformis mosseae inoculation; AMF+DSE: co-inoculation of F. mosseae and E. pisciphila. “ ” indicates the index of the material was not detected. Means followed by the same letter do not differ significantly at p < 0.05 by LSD test.
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
3
Fig. 1. Effects of AMF and DSE on photosynthetic physiology of maize leaves under high Cd stress. Cd: cadmium; CK: the control of non-inoculation, DSE: Exophiala pisciphila inoculation, AMF: Funneliformis mosseae inoculation; AMF+DSE: co-inoculation of F. mosseae and E. pisciphila. The different lowercase letters indicate significant differences among treatments (p<0.05). “ns”, “*” and “**” means p > 0.05, p < 0.05 and p < 0.01, respectively.
2.4. Determination of chlorophyll concentration and photosynthesis in leaves Before the harvest at 60 d after the transplantation, about 0.2 g maize leaves were collected and extracted with 95% ethanol. Later, they were filtered and dissolved to a constant volume of 25 mL in a brown volumetric glass flask. The concentration of chlorophyll a and chlorophyll b were tested via a spectrophotometer (Model 721, Shanghai Precision & Scientific Instrument Co., Ltd. China) at wavelengths 665 nm and 649 nm, respectively. The chlorophyll content (mg/kg) based on fresh weight (FW) was calculated according to the formula by (Li, H.S., 2000). The leaf, which was folded completely, was selected from the maize plants. Photosynthesis indexes, such as net photosynthetic rate (Pn), intercellular carbon dioxide concentration (Ci), transpiration rate (Tr), and stomatal conductance (Gs), were tested on a sunny morning using the LCA-4 photosynthetic tester (Analytical Development Company Limited, ADC, Hoddesdon, England). 2.5. Determination of root traits and cadmium content in maize After the maize roots were cleaned, the roots were scanned using the PERFECTION V700 PHOTO root system scanner. Root morphological
indexes, including root length, root surface area, average diameter (RDI), root volume, root branches number, and root tips number, were analyzed using the WinRHIZO Pro-root system analyzer. The root traits included specific root length (SRL, root length per unit of root mass), specific root area (SRA, root area per unit of root mass), root mass density (RTD, root mass per root volume), specific root branch (SRB, root branch number per unit of root mass), and specific root tip (SRT, root tip number per unit of root mass). Ground dried roots and shoots (0.1 g) were digested with HNO3/ HClO4 (4:1). The Cd concentrations of the plants were tested via the flame atomic absorption spectrophotometry using an atomic absorption spectrometer (TAS-990, Beijing Puxi Instrument Factory, Beijing, China). The appropriate quality control was determined using CdCl2 as the standard solution. The Cd content (mg/kg) based on dry weight (DW) was calculated according to the formula by Bao (2000). Transfer coefficient was calculated as the shoot to root Cd content ratio (Zhan et al., 2018). 2.6. Data processing and statistical analysis Test data were mean of four repetitions and were expressed in mean § standard deviation. The SPSS 22.0 (SPSS Inc, Chicago, IL, USA) and the least significant difference (LSD) method were used to test the difference significance of test data at the level of 0.05. The influences of AMF
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB 4
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
Fig. 2. Effects of AMF and DSE on root morphology of maize under high Cd stress. Cd: cadmium; CK: the control of non-inoculation, DSE: Exophiala pisciphila inoculation, AMF: Funneliformis mosseae inoculation; AMF+DSE: co-inoculation of F. mosseae and E. pisciphila. The different lowercase letters indicate significant differences among treatments (p<0.05). “ns”, “*” and “**” means p > 0.05, p < 0.05 and p < 0.01, respectively.
and DSE on maize growth, photosynthetic physiology, root traits, Cd content and accumulation, as well as the interaction between AMF and DSE were analyzed via the two-way ANOVA (Analysis of Variance). The correlation coefficients between the Cd content in maize shoots with the root traits (n = 16) were analyzed by Pearson correlation. 3. Results 3.1. AMF and DSE colonization in maize roots No significant difference was found on the spore number between the AMF and AMF+DSE treatments. No spores were detected in the CK and DSE treatments. Colonization of hypha was detected in the AMF, DSE, and AMF+DSE treatments. The AMF infection rate of the AMF treatment was significantly higher than that of the AMF+DSE treatment, and the DSE infection rate of DSE treatment was far higher than that of AMF+DSE treatment (Table 1). 3.2. Effects of AMF and DSE on photosynthetic physiology of maize The two-way ANOVA reflected that AMF had significant effects on the chlorophyll content and photosynthetic physiology of maize leaves.
DSE influenced the chlorophyll b content significantly. An extremely significant interaction was found between AMF and DSE on chlorophyll content, and notable interactions were observed on the photosynthetic rate, transpiration rate and intracellular CO2 concentration (Fig. 1). Therefore, the photosynthetic physiology of maize leaves was influenced by the AMF inoculation and AMF+DSE co-inoculation. The contents of chlorophyll a and chlorophyll b in the maize leaves were increased significantly in these two treatments, accompanied by increases in photosynthesis rate, transpiration rate, and stomatal conductance, as well as a sharp reduction of intercellular CO2 concentration in maize leaves (Fig. 1). 3.3. Effects of AMF and DSE on root morphology and traits of maize The two-way ANOVA demonstrated that the DSE did not influence the root morphology and traits of maize significantly, except the specific root length (SRL) and specific root branch (SRB). But the AMF caused significant impacts on root morphology and traits of maize. No interaction between AMF and DSE was found on the root morphology and traits. (Fig. 2 and Fig. 3). The root morphology of maize was changed significantly in the AMF inoculation and AMF+DSE co-inoculation. These two
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
5
Fig. 3. Effects of AMF and DSE on root traits of maize under high Cd stress. Cd: cadmium; CK: the control of non-inoculation, DSE: Exophiala pisciphila inoculation, AMF: Funneliformis mosseae inoculation; AMF+DSE: co-inoculation of F. mosseae and E. pisciphila. Specific root length: root length (cm) per unit of root mass (mg FW); Specific root area: root area (cm2) per unit of root mass (mg FW); Root mass density: root mass (mg FW) per root volume (cm3); Specific root branch: root branch number per unit of root mass (mg FW); Specific root tip: root tip number per unit of root mass (mg FW). The different lowercase letters indicate significant differences among treatments (p<0.05). “ns”, “*” and “**” means p > 0.05, p < 0.05 and p < 0.01, respectively.
treatments significantly increased the root length, root surface area, root volume, root branches number, and root tips number. But the DSE inoculation did not change the root morphology (Fig. 2). Furthermore, all the AMF inoculation, DSE inoculation, and AMF+DSE co-inoculation caused a sharp decrease on the SRL, specific root area (SRA) and SRB. The AMF inoculation and AMF+DSE co-inoculation dramatically dropped the specific root tip (SRT), but increased the root mass density (RTD). And only the AMF inoculation average root diameter (RDI). Thus the AMF inoculation and AMF+DSE co-inoculation influenced the maize root traits significantly (Fig. 3). 3.4. Effects of AMF and DSE on growth and Cd content in maize According to the two-way ANOVA, the effects of DSE on the maize height and biomass were not significant, whereas AMF influenced the maize biomass greatly. No interaction was found between DSE and AMF on the maize height and biomass. However, both the DSE and AMF influenced the Cd content in the maize shoots significantly. There was a strong interaction between AMF and DSE on the Cd content in the maize shoots and roots (Fig. 4).
At 60 d after transplantation, the maize height significantly enhanced by the AMF inoculation. The maize biomass was increased significantly by the AMF inoculation and AMF+DSE co-inoculation. The shoot biomass was increased by 22.8 and 16.4 times, whereas the root biomass was increased by 5.5 and 5.4 times by the AMF inoculation and AMF+DSE co-inoculation, respectively. Additionally, the Cd content in shoots were decreased sharply by the single inoculation and co-inoculation of DSE and AMF by 45 58%. The Cd content of the maize roots was increased significantly by the DSE inoculation and AMF inoculation (Fig. 4). Thus single or dual inoculation of AMF and DSE decreased the Cd transfer coefficient of maize. These results indicated that the AMF and DSE induced the Cd retain in the roots and restricted Cd migration to the shoots of maize. 3.5. Correlation analysis The correlation analysis showed that the Cd content in maize shoots had a very significantly positive correlation (p < 0.01) with SRL, SRA and SRB, a significantly positive correlation (p < 0.05) with SRT, and a significantly negative correlation (p < 0.05) with RTD of the maize roots (Fig. 5). Results indicated a close relation between
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB 6
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
Fig. 4. Effects of AMF and DSE on height, biomass and Cd content in maize under high Cd stress. Cd: cadmium; CK: the control of non-inoculation, DSE: Exophiala pisciphila inoculation, AMF: Funneliformis mosseae inoculation; AMF+DSE: co-inoculation of F. mosseae and E. pisciphila. The different lowercase letters indicate significant differences among treatments (p<0.05). “ns”, “*” and “**” means p > 0.05, p < 0.05 and p < 0.01, respectively.
the Cd content in shoots with the root traits of maize. That AMF and DSE altered the root traits contributed to restrict Cd migration from roots to shoots of maize. 4. Discussions Many studies on ecological functions of AMF and DSE in plant roots under the Cd stress have been reported. AMF can improve mineral nutrients, photosynthesis, and resistance physiology of plants, promote growth of the host plants, and strengthen plant resistance to Cd stress (Garg and Bhandari, 2014; Lenoir et al., 2016). In the soil, AMF owns abundant extraradical mycelium, which can expand the absorbing area of roots, promote mineral absorption (e.g. P and S), and improve mineral nutrients of the host plants (Wang et al., 2017). Besides, they increases on the chlorophyll contents, net photosynthetic rate, transpiration rate, and stomatal conductance of leaves but a reduction on intercellular CO2 content. The photosynthetic physiology of leaves and plant biomass are enhanced accordingly (Li et al., 2016; Yang et al., 2015). AMF not only increased the activity of antioxidant enzymes like superoxide dismutase and catalase of the host plant under Cd stress, but also increased the antioxidant (e.g. glutathione, ascorbic acid) contents (Shahabivand et al., 2016; Zhan et al., 2018) and enhanced the scavenging activity of reactive oxygen species of plants and relieved damages of Cd ions to plants (Nath et al., 2016). This experiment revealed that AMF could facilitate the maize growth, increase the chlorophyll content and photosynthesis physiology in leaves, as well as plant height and shoots biomass. Similar to ecological functions of AMF, some research results supported that DSE could improve mineral nutrients, photosynthesis and resistance physiology of plants, promote the growth, and enhance Cd tolerance of host plants under Cd stress (Berthelot et al., 2017; Li et al.,
2011; Likar and Regvar, 2013; Wang et al., 2016). For example, the DSE inoculation increased mineral contents (including N, P, K, Ca, Mg, Fe, Mn and Zn) in tomato plants and improved the mineral nutrition of the host plants (Berthelot et al., 2017; Vergara et al., 2017). The DSE, Gaeumannomyces cylindrosporus, increased the chlorophyll content of maize leaves, enhanced photosynthetic physiology and chlorophyll fluorescence parameters of leaves, and increased plant height and biomass (Ban et al., 2017). The DSE E. pisciphila increased the chlorophyll content and photosynthetic physiology of maize leaves, height, and biomass (He et al., 2017; Li et al., 2011). The DSE increased the activity of antioxidant enzymes and antioxidant content in the host plant (He et al., 2017; Wang et al., 2016; Zhan et al., 2017), promoted the host growth, and relieved Cd biological toxicity to the host plants (Berthelot et al., 2016; Shahabivand et al., 2017). The mechanisms for AMF and DSE in enhancing Cd tolerance of host plants were also related with their effects in changing root traits and restricting Cd migration to the shoots of plants. The AMF and DSE colonized in root cortex of the host plants, which can increase root length, root surface area, root volume, root tips and biomass, and facilitate root growth of the host plants (Ban et al., 2017; Li et al., 2016). As a result, the specific root length (SRL), specific root area (SRA), and specific root tips (SRT) of the roots reduced, but the root mass density (RTD) increased. Similar phenomena were observed in this experiment. Besides, the Cd content in the maize shoots showed an extremely significantly positive correlation with the SRL, SRA, and SRT of the roots, but it had a significant negative correlation with the RTD. These results reflected that large SRL, SRA, and SRT values were conducive to the absorption of cationic mineral nutrients (de la Riva et al. 2018) and also facilitated the absorption of Cd ions. High RTD is beneficial for the roots to store mineral nutrients (Kramer-Walter et al., 2016) and also might contribute to
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
7
Fig. 5. Correlation between root traits with Cd content in maize shoot. Cd: cadmium; specific root length: root length (cm) per unit of root mass (mg FW); specific root area: root area (cm2) per unit of root mass (mg FW); specific root branch: root branch number per unit of root mass (mg FW); specific root tip: root tip number per unit of root mass (mg FW); root mass density: root mass (mg FW) per root volume (cm3).
storing Cd ions in the roots, which supported the current theory of “root economics spectrum (RES)” (Weemstra et al., 2016). Additionally, the AMF and DSE colonization changed the intracellular distribution and chemical morphologies of Cd in roots (Li et al., 2016; Wang et al., 2016) by the metal ion fixation of intraradical mycelium (Zhou et al., 2017) and increasing specific components, such as cellulose and hemicelluloses in the cell wall to bind the Cd ions (Chen et al., 2018). They also facilitated the retention of Cd ions in the plant roots, restricted Cd migration to the shoots from roots, and reduced Cd content in the shoots of plants (He et al., 2017; Hui et al., 2015; Li et al., 2011; Likar and Regvar, 2013; Shahabivand et al., 2017; Zhan et al., 2018). This experiment also confirmed that the AMF, DSE, and AMF+DSE treatments all decreased the transfer coefficient of Cd in maize and reduced the Cd content in the shoots significantly. These results might be one of the important mechanisms of the Cd tolerance enhancement by AMF and DSE in the host plants.
Notably, the AMF and DSE are co-existing in the root cortex of host plants and influence mutually, which might change their colonization rates in the roots. In the roots of Polylepis australis, the colonization rate of AMF was significantly negatively correlated with the colonization rate of DSE (Soteras et al., 2013). By dual inoculating AMF and DSE to ryegrass, the AMF decreased the DSE colonization rate in roots significantly, whereas the DSE did not cause an effect on the AMF colonization rate (Berthelot et al., 2018). This experiment discovered that the co-colonized AMF decreased the colonization rate of DSE, and the co-colonized DSE also decreased the colonization rate of AMF, indicating that they might compete in colonization behaviors in maize roots. However, some studies reported that the colonization rate of DSE in the DSE and AMF + DSE treatments remained the same, indicating the absence of a competitive relationship between the AMF and DSE in colonization in the roots (Saravesi et al., 2014).
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB 8
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
In the ecological functions of regulating growth of host plants, the co-existing AMF and DSE might form competitive, independent, or collaborative relationships. For instance, the mycorrhizal inoculation relieved the adverse impacts of a DSE stain Phialocephala fortinii s.l. Acephala applanata on the growth of Norway spruce and Douglas fir, showing a competitive relationship (Reininger and Sieber, 2012). In heavy metal polluted soils, the co-inoculation of AMF and DSE was more beneficial to the growth of the host wild lettuce than _ the single inoculation of AMF or DSE (Wazny et al., 2018). With respect to the P nutrition, DSE facilitated the mineralization of organic P in soils, and AMF facilitated the P nutrient in the rhizosphere migrating to the host plants. The co-inoculation of AMF and DSE increased the P content of plants significantly, showing a collaborative relationship between the two (Della Monica et al., 2015). In mining waste soils, the growth rate and biomass of Veratrum nigrum reached the maximum with the co-inoculation of AMF and DSE, followed by a single inoculation of AMF. No significant difference was detected between them, indicating that AMF and DSE might be _ independent rather than competitive and collaborative (We˛ zowicz et al., 2017). Nevertheless, the interaction mechanism of AMF and DSE in plant roots remains unknown. Some studies believed that the exudates of DSE mycelium regulated the AMF growth in plant roots and facilitated the extension and branching of AMF mycelium but inhibited the growth of AMF extraradical mycelium (Scervino et al., 2009). Infection of endophytic fungi increased the alkaloid content of the host plants, thus inhibiting AMF growth and colonization and reducing the colonization rate of AMF in the roots (Li et al., 2016). This reflect that some specific compounds play a key role in adjusting the relationship between AMF and DSE. These studies provided a research direction for discussing the relationship, interaction effects and mechanisms between AMF and DSE in plant roots. This subject needs further intensive studies on the physiological and molecular mechanisms on enhancing stress resistance of plant by AMF and DSE in roots. 5. Conclusions The AMF inoculation and AMF+DSE co-inoculation improved the maize performance under a high Cd stress, which was closely related with changes of the photosynthesis physiology and root traits of maize. However, there was no synergistic interaction between AMF and DSE on the maize performance under a high Cd stress. The interaction between the co-colonized AMF and DSE in plant roots needs further studies under conditions of different fungal strains, host plants and environmental factors (such as soil, nutrient, moisture, and temperature). Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Declaration of Competing Interest The authors declare that they have no competing interests. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (41877130, 41701362), the Key Project of Yunnan Agricultural Foundation (2017FG001-014), the Reserve Talents Fund for Young and Middle-Aged Academic and Technological leaders in Yunnan Province (2018HB043), and the Science and Technology Innovation Team of Yunnan Province (2017HC015).
Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j.sajb.2019.09.018.
References Allah, E., Alqarawi, A., Hend, A., 2015. Alleviation of adverse impact of cadmium stress in sunflower (Helianthus annuus L.) by arbuscular mycorrhizal fungi. Pakistan. Journal of Botany 47, 785–795. Anjum, S., Tanveer, M., Hussain, S., Bao, M., Wang, L., Khan, I., et al., 2015. Cadmium toxicity in maize ( Zea mays L.), consequences on antioxidative systems, reactive oxygen species and cadmium accumulation. Environmental Science and Pollution Research 22, 17022–17030. Ban, Y., Xu, Z., Yang, Y., Liu, H., Tang, M., 2012. Colonization characteristics of arbuscular mycorrhizal fungi and dark septate endophytes in different degree Pb-Zn polluted areas. Acta Bot Boreali-Occidentialia Sinica 32, 2336–2343. Ban, Y., Xu, Z., Yang, Y., Zhang, H., Chen, H., Tang, M., 2017. Effect of dark septate endophytic fungus Gaeumannomyces cylindrosporus on plant growth, photosynthesis and Pb tolerance of maize ( Zea mays L.). Pedosphere 27, 283–292. Bao, S., 2000. Soil and Agricultural Chemistry Analysis. China Agriculture Press, Beijing. Benavides, M.P., Gallego, S.M., Tomaro, M.L., 2005. Cadmium toxicity in plants. Brazilian Journal of Plant Physiology 17, 21–34. Berthelot, C., Blaudez, D., Beguiristain, T., Chalot, M., Leyval, C., 2018. Co-inoculation of Lolium perenne with Funneliformis mosseae and the dark septate endophyte Cadophora sp. in a trace element-polluted soil. Mycorrhiza 28, 301–314. Berthelot, C., Blaudez, D., Leyval, C., 2017. Differential growth promotion of poplar and birch inoculated with three dark septate endophytes in two trace element-contaminated soils. International Journal of Phytoremediation 19, 1118–1125. Berthelot, C., Leyval, C., Foulon, J., Chalot, M., Blaudez, D., 2016. Plant growth promotion, metabolite production and metal tolerance of dark septate endophytes isolated from metal-polluted poplar phytomanagement sites. FEMS Microbiology Ecology 92, w144. Cao, M.Q., Wu, Q.S., Zou, Y.N., 2013. An improved ink-acetic acid technique for staining arbuscular mycorrhizas of citrus. International Journal of Agriculture & Biology 15, 386–388. Chen, X., Kang, Y., San So, P., Ng, C., Won, M., 2018. Arbuscular mycorrhizal fungi increase the proportion of cellulose and hemicellulose in the root stele of vetiver grass. Plant and Soil 425, 309–319. Daniels, B.A., Skipper, H.D., 1982. Methods for the recovery and quantitative estimation of propagules from soil. In: Schenck N.C. (eds) Methods and principles of mycorrhizal research. Saint Paul, MN. American Phytopathologic Society 29–35. Della Monica, I., Saparrat, M., Godeas, A., Scervino, J., 2015. The co-existence between dse and amf symbionts affects plant P pools through P mineralization and solubilization processes. Fungal Ecology 17, 10–17. ~o n, T., Pe rez-Ramos, I.M., Navarro-Ferna ndez, C.M., Olmo, M., de la Riva, E.G., Maran Villar, R., 2018. Root traits across environmental gradients in Mediterranean woody communities: are they aligned along the root economics spectrum? Plant and Soil 424, 35–48. Deng, Z., Cao, L., 2017. Fungal endophytes and their interactions with plants in phytoremediation, a review. Chemosphere 168, 1100–1106. Deram, A., Languereau, F., Haluwyn, C., 2011. Mycorrhizal and endophytic fungal colonization in Arrhenatherum elatius L. roots according to the soil contamination in heavy metals. Soil and Sediment Contamination 20, 114–127. Deram, A., Languereau-Leman, F., Howsam, M., Petit, D., Van Haluwyn, C., 2008. Seasonal patterns of cadmium accumulation in Arrhenatherum elatius (Poaceae), influence of mycorrhizal and endophytic fungal colonisation. Soil Biology and Biochemistry 40, 845–848. Gallego, S., Pena, L., Barcia, R., Azpilicueta, C., Iannone, M., Rosales, E.P., et al., 2012. Unravelling cadmium toxicity and tolerance in plants, insight into regulatory mechanisms. Environmental and Experimental Botany 83, 33–46. Garg, N., Bhandari, P., 2014. Cadmium toxicity in crop plants and its alleviation by arbuscular mycorrhizal (AM) fungi: an overview. Plant Biosystems 148, 609–621. He, S., Yang, X., He, Z., Baligar, V., 2017. Morphological and physiological responses of plants to cadmium toxicity: a review. Pedosphere 27, 421–438. He, Y., Yang, Z., Li, M., Jiang, M., Zhan, F., Zu, Y., et al., 2017. Effects of a dark septate endophyte (DSE) on growth, cadmium content and physiology in maize under cadmium stress. Environmental Science and Pollution Research 24, 18494–18504. Hui, F., Liu, J., Gao, Q., Lou, B., 2015. Piriformospora indica confers cadmium tolerance in nicotiana tabacum. Journal of Environmental Sciences 37, 184–191. Kramer-Walter, K., Bellingham, P., Millar, T., Smissen, R., Richardson, S., Laughlin, D., 2016. Root traits are multidimensional, specific root length is independent from root tissue density and the plant economic spectrum. Journal of Ecology 104, 1299–1310. Lenoir, I., Fontaine, J., Sahraoui, A., 2016. Arbuscular mycorrhizal fungal responses to abiotic stresses: a review. Phytochemistry 123, 4–15. Li, H., Luo, N., Zhang, L., Zhao, H., Li, Y., Cai, Q., et al., 2016. Do arbuscular mycorrhizal fungi affect cadmium uptake kinetics, subcellular distribution and chemical forms in rice? Science of the Total Environment 571, 1183–1190. Li, M., Li, H., Wang, K., Shi, L., Liu, J., Zhang, L., 2016. Effect of arbuscular mycorrhizae on the growth, photosynthetic characteristics and cadmium uptake of peanut plant under cadmium stress. Environmental Chemistry 35, 2344–2352.
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9 Li, H.S., 2000. Principles and techniques of plant physiological biochemical experiment. Beijing, Higher Education Press. Li, T., Liu, M.J., Zhang, X.T., Zhang, H.B., Sha, T., Zhao, Z.W., 2011. Improved tolerance of maize (Zea mays L.) to heavy metals by colonization of a dark septate endophyte (DSE) exophiala pisciphila. Science of the Total Environment 409, 1069–1074. Liang, C., Xiao, Y., Zhao, Z., 2007. Arbuscular mycorrhiza and dark septate endophytes in an abandoned lead-zinc mine in huize, Yunnan, China. Chinese Journal of Applied and Environmental Biology 13, 811–817. Likar, M., Regvar, M., 2013. Isolates of dark septate endophytes reduce metal uptake and improve physiology of Salix caprea l. Plant and Soil 370, 593–604. McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L., Swan, J.A., 1990. A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytologist 115, 495–501. Nath, M., Bhatt, D., Prasad, R., Gill, S., Anjum, N., Tuteja, N., 2016. Reactive oxygen species generation-scavenging and signaling during plant-arbuscular mycorrhizal and Piriformospora indica interaction under stress condition. Frontiers in Plant Science 7, 1574. Reininger, V., Sieber, T., 2012. Mycorrhiza reduces adverse effects of dark septate endophytes (DSE) on growth of conifers. PLoS One 7, e42865. Saravesi, K., Ruotsalainen, A., Cahill, J., 2014. Contrasting impacts of defoliation on root colonization by arbuscular mycorrhizal and dark septate endophytic fungi of Medicago sativa. Mycorrhiza 24, 239–245. rgola, M., Fernandez, L., Godeas, A., 2009. Exudates of Scervino, J., Gottlieb, A., Silvani, V., Pe dark septate endophyte (DSE) modulate the development of the arbuscular mycorrhizal fungus (AMF) Gigaspora rosea. Soil Biology and Biochemistry 41, 1753–1756. Shahabivand, S., Maivan, H., Goltapeh, E., Sharifi, M., Aliloo, A., 2012. The effects of root endophyte and arbuscular mycorrhizal fungi on growth and cadmium accumulation in wheat under cadmium toxicity. Plant Physiology and Biochemistry 60, 53–58. Shahabivand, S., Maivan, H., Mahmoudi, E., Soltani, B., Sharifi, M., Aliloo, A., 2016. Antioxidant activity and gene expression associated with cadmium toxicity in wheat affected by mycorrhizal fungus. Zemdirbyste 103, 53–60. Shahabivand, S., Parvaneh, A., Aliloo, A., 2017. Root endophytic fungus Piriformospora indica affected growth, cadmium partitioning and chlorophyll fluorescence of sunflower under cadmium toxicity. Ecotoxicology and Environmental Safety 145, 496–502. Soteras, F., Renison, D., Becerra, A., 2013. Growth response, phosphorus content and root colonization of Polylepis australis bitt. seedlings inoculated with different soil types. New Forest 44, 577–589. Su, C., Jiang, L., Zhang, W., 2014. A review on heavy metal contamination in the soil worldwide, situation, impact and remediation techniques. Environmental Skeptics and Critics 3, 24–38.
9
Vergara, C., Araujo, K.E.C, Urquiaga, S., Schultz, N., Balieiro, F.C., Medeiros, P.S., et al., 2017. Dark septate endophytic fungi help tomato to acquire nutrients from ground plant material. Frontiers in Microbiology 8, 2437. Wang, J., Li, T., Liu, G., Smith, J., Zhao, Z., 2016. Unraveling the role of dark septate endophyte (DSE) colonizing maize (Zea mays) under cadmium stress, physiological, cytological and genic aspects. Scientific Reports 6, 22028. Wang, W., Shi, J., Xie, Q., Jiang, Y., Yu, N., Wang, E., 2017. Nutrient exchange and regulation in arbuscular mycorrhizal symbiosis. Molecular Plant 10, 1147–1158. _ Wazny, R., Rozpadek, ˛ P., J˛edrzejczyk, R., Sliwa, M., Stojakowska, A., Anielska, T., et al., 2018. Does co-inoculation of Lactuca serriola with endophytic and arbuscular mycorrhizal fungi improve plant growth in a polluted environment? Mycorrhiza 28, 235–246. Weemstra, M., Mommer, L., Visser, E.J.W, Van Ruijven, J., Kuyper, T.W., Mohren, G.M., et al., 2016. Towards a multidimensional root trait framework, a tree root review. New Phytologist 211, 1159–1169. _ W˛e zowicz, K., Rozpadek, ˛ P., Turnau, K., 2017. Interactions of arbuscular mycorrhizal and endophytic fungi improve seedling survival and growth in post-mining waste. Mycorrhiza 27, 499–511. Yang, Y., Han, X., Liang, Y., Ghosh, A., Chen, J., Tang, M., 2015. The combined effects of arbuscular mycorrhizal fungi (AMF) and lead (Pb) stress on pb accumulation, plant growth parameters, photosynthesis, and antioxidant enzymes in Robinia pseudoacacia l. PLoS One 10, e145726. Zhan, F., He, Y., Yue, X., Qin, L., Xia, Y., 2016. Effect of mycorrhizal inoculation on plant growth, nutrients and heavy metals uptake by Leucaena leucocephala. Fresenius Environmental Bulletin 25, 1760–1767. Zhan, F., Li, B., Jiang, M., Qin, L., Wang, J., He, Y., et al., 2017. Effects of a root-colonized dark septate endophyte on the glutathione metabolism in maize plants under cadmium stress. Journal of Plant Interactions 12, 421–428. Zhan, F., Li, B., Jiang, M., Yue, X., Xia, Y., Wang, Y., 2018. Arbuscular mycorrhizal fungi enhance antioxidant defense in the leaves and the retention of heavy metals in the roots of maize. Environmental Science and Pollution Research 25, 24338–24347. Zhang, Y., Zhang, Y., Liu, M., Shi, X., Zhao, Z., et al., 2008. Dark septate endophyte (DSE) fungi isolated from metal polluted soils: their taxonomic position, tolerance, and accumulation of heavy metals In Vitro. The Journal of Microbiology 46, 624–632. Zhou, X., Fu, L., Xia, Y., Zheng, L., Chen, C., et al., 2017. Arbuscular mycorrhizal fungi enhance the copper tolerance of Tagetes patula through the sorption and barrier mechanisms of intraradical hyphae. Metallomics 7, 936–948.
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB
[m5G;November 22, 2019;5:56]
South African Journal of Botany 000 (2019) 1 9
Contents lists available at ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress Y.M. He, X.M. Fan, G.Q. Zhang, B. Li, T.G. Li, Y.Q. Zu, F.D. Zhan* College of Resources and Environment, Yunnan Agricultural University, Kunming 650201, China
A R T I C L E
I N F O
Article History: Received 29 June 2019 Revised 12 September 2019 Accepted 23 September 2019 Available online xxx Keywords: Endophytic fungi Biomass Photosynthetic physiology Root traits Cadmium content
A B S T R A C T
Arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) are generally colonized in plant roots in metal-polluted environment. A pot experiment including four treatments (AMF inoculation of Funneliformis mosseae, DSE inoculation of Exophiala pisciphila, co-inoculation of AMF + DSE, and non-inoculation) under a high cadmium (Cd) stress (50 mg/kg) was conducted to discuss the ecological functions and interactions of AMF and DSE. The biomass, photosynthetic physiology, root morphology and traits, Cd content of maize were investigated. According to the two-way analysis of variance, AMF and DSE had no synergetic interactions on the maize performance under the high Cd stress. Both AMF inoculation and AMF + DSE co-inoculation, but not DSE inoculation, significantly increased the chlorophyll contents in maize leaves, resulting in stronger photosynthetic physiology, higher biomass, and increased length, surface area, volume, branches, tips number, and mass density (RTD) of maize roots, but decreased specific root length (SRL), specific root area (SRA), specific root branches (SRB), and specific root tips (SRT). However, there was a strong interaction between AMF and DSE on the Cd content in the maize shoots and roots. Single or co-inoculation of AMF and DSE significantly decreased the Cd content in shoots and Cd transfer coefficient of maize. Correlation analysis indicated that the Cd content in shoots was significantly positively correlated with SRL, SRA, SRB and SRT but negatively correlated with RTD. Thus AMF and DSE altered the root traits that contributed to restricting Cd migration from roots to shoots of maize. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Soil cadmium (Cd) pollution intensifies increasingly due to industrial and agricultural production activities, such as mining, smelting, sewage irrigation, and fertilization (Su et al., 2014). Cd is a toxic metal element that is unnecessary for plant growth (Benavides et al., 2005). It causes great toxic effects to the growth characters of crops by generating abundant reactive oxygen species in crops (Anjum et al., 2015), destroying the chloroplast structure, inhibiting photosynthesis of leaves, hindering the growth and water and nutrient absorption and transportation of the plant root system, as well as restricting the plant growth (Gallego et al., 2012; He et al., 2017). The colonization of arbuscular mycorrhizal fungi (AMF) and dark septate endophyte (DSE) is found in plant roots in a Cd-polluted habitat. For instance, AMF and DSE are extensively colonized in the roots of wild plants grown in the lead (Pb) and zinc (Zn) mine areas, where Cd pollution is serious, and often coexist in the roots of the same plant (Liang et al., 2007), showing strong endurance and adaptation to Cd stress (Ban et al., 2012). AMF and DSE are colonized in the roots of oat grass (Arrhenatherum elatius) on the slagheap of the Zn smelting plant (Deram et al., 2008). The AMF infection rate in the roots *Corresponding author. E-mail address: [email protected] (F.D. Zhan).
decreases with the increase of Cd concentration in soils, whereas the DSE infection rate is stable and insensitive to soil Cd concentrations (Deram et al., 2008). The DSE infection rate in the roots of oat grass in Cd-polluted soils is higher than that in non-polluted regions (Deram et al., 2011). AMF can survive in mutualism with more than 80% terrestrial plants and influences the growth character of the host plants. It plays an important role in increasing Cd resistance of the host plants (Garg and Bhandari, 2014). AMF can relieve toxic effects of Cd stress on plants, facilitate root growth, change the morphology of the root system, and enhance Cd absorption and fixation of the roots (Li et al., 2016). AMF improves the physiological activities (e.g. mineral nutrients, photosynthesis, and stress resistance) of host plants (Allah et al., 2015; Zhan et al., 2018), promote the host plant growth, enhance the Cd tolerance of the host plants, and change Cd absorption and accumulation of plants (Zhanet al., 2016). Similarly, the inoculation of DSE can enhance stress resistance of the host plants, increase the photosynthetic pigments content, and improve the photosynthesis effect, thus increasing plant biomass (Ban et al., 2017; He et al., 2017; Zhan et al., 2017). It also facilitates Cd accumulation in plant roots and reduce Cd migration from the roots to the shoots, thus decreasing Cd content in the shoots (He et al., 2017; Li et al., 2011; Wang et al., 2016). Thus, AMF and DSE colonized in the plant
https://doi.org/10.1016/j.sajb.2019.09.018 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB 2
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
roots can facilitate plant growth, improve the leave photosynthesis, and enhance Cd tolerance in plants (Deng and Cao, 2017). In the natural environment, AMF and DSE are often colonized in the roots of the same host plant. Researchers began to pay attention to the influences of AMF and DSE coexistence on the growth characters of plants (Berthelot et al., 2018; Shahabivand et al., 2012). Under Cd stress, either single inoculation or co-inoculation of AMF and DSE can facilitate wheat growth and increased chlorophyll content in leaves. Wheat with single inoculation of DSE achieves the highest biomass and lowest Cd content in the aboveground part (Shahabivand et al., 2012). A single inoculation of AMF increased the biomass of the shoots of ryegrass (Lolium perenne) on soils with composite pollution of Cd/Pb/Zn, whereas single inoculation of DSE and the co-inoculation of AMF and DSE did not substantially affect the biomass of ryegrass. Only the co-inoculation of AMF and DSE remarkably decreased Cd content in the shoots of ryegrass (Berthelot et al., 2018), revealing that the coexistence of AMF and DSE in roots may influence the growth characters and Cd tolerance of the host plants differently. Associated studies are rare, and additional relevant studies must be conducted. In this experiment, one strain of AMF (Funneliformis mosseae) and one strain of DSE (Exophiala pisciphila) were used. A pot experiment, with maize as the host plant under 50 mg/kg Cd stress, was conducted to investigate the effects of AMF and DSE on plant growth, photosynthesis physiology, root morphology and traits, Cd content, and accumulation of maize. The following hypotheses were proposed in this paper: (1) the AMF and DSE colonization in the maize roots can improve the performance of maize under high Cd stress; and (2) changes of Cd content induced by the AMF and DSE colonization were closely related to the root traits of maize. 2. Materials and methods 2.1. Testing samples The testing soils were mountain red earth with pH 7.5. The organic matter content was 13.87 g/kg. The concentrations of total nitrogen (N), phosphorus (P), and potassium (K) were 0.112, 0.32, and 1.98 g/kg, respectively. The contents of alkali-hydrolyzable N, available P and K were 26.18, 1.15, and 33.74 mg/kg, respectively, and the Cd content was 0.275 mg/kg. Soil samples were dried naturally in air in the laboratory and then ground and filtered through a 2-mm sieve. The testing river sands were collected from a construction site. They were dried naturally in air and then screened through the 2-mm sieve. The testing samples in the pot experiment were mixtures of river sand and soil at a proportion of 3:1 (w/w). The soil-sand mixture were autoclaved at 121 ℃ for 2 h. The AMF inoculants used in the experiment were Funneliformis mosseae BGC YN05, which were provided by the Institute of Plant Nutrition and Resources at the Beijing Academy of Agriculture and Forestry. The indoor-dried F. mosseae inoculants contained about 60 70 spores in each gram. The DSE strain used was Exophiala pisciphila was separated from the roots of Arundinella bengalensis (Poaceae), which was grown naturally on the Huize Pb-Zn mine area in Yunnan Province (Zhang et al. 2008). It was stored in the laboratory under 4 °C. The DSE fungus was cultivated onto the potato dextrose agar (PDA) media under 28 °C for two weeks to activate the strain. A native maize cultivar (Huidan No. 4) from a seed market in Dabanqiao Town, Kunming City, Yunnan Province was used in the experiment. Before sowing, full and uniform sized maize seeds were chosen. The maize seeds were immersed in 75% ethanol for 10 min and in 10% sodium hypochlorite for another 10 min, and followed by four rinses with sterile water. After surface sterilization of the maize seeds, they were placed on a sterile filter paper in Petri dishes, added some sterile water to make the filter paper to be wet, and cultured in an incubator at 28 °C for 3 d After germination to about 1 cm, sterile seedlings with uniform growth were chosen for later use.
2.2. Pot experiment First, the maize-DSE consortium was constructed. The 25 cm (height) £ 6.5 cm (diameter) cylinder glass bottles were used, in which 20.0 g perlite and 20 mL Hoagland nutrient solution were added. The set up was autoclaved under 121 °C in 30 min. Eight glass bottles were added with 15 pieces of 2-mm (diameter) DSE colony. After mixing evenly with perlite, two pieces of germinated maize seeds were added and covered with 1-cm thick sterile sands. The bottles were sealed up with sterile polyvinyl alcohol film and cultured for 14 d under 25 °C and light conditions of 1000 8000 Lux for 10 h per day. Some maize seedlings were collected and examined under the microscope to confirm the successful colonization of DSE in the roots. The rest of the glass bottles were not inoculated with DSE colony, and the grown maize seedlings had no DSE colonization. Second, the pot experiment was carried out in a greenhouse. The river sand and soils mixture at the proportion of 3:1 (W/W) was used as the culture media, and the cadmium chloride (CdCl2¢2¢5H2O) solution was added to reach the 50 mg/kg Cd stress level. They were mixed completely and balanced in 2 weeks. Plastic pots, which were 20 cm high and 25 cm wide in diameter, were applied and sterilized using 75% alcohol. Later, 5.0-kg sterilized soil-sand mixture was filled. Four treatments, namely CK (non-inoculation), AMF (F. mosseae inoculation), DSE (E. pisciphila inoculation), and AMF+DSE (co-inoculation of F. mosseae and E. pisciphila) were set. Maize seedlings without DSE colonization and with 50 g killed AMF inoculants was planted in the CK treatment. Maize seedlings without DSE colonization was planted with 50 g AMF inoculants in the AMF treatment. Maize seedlings with DSE colonization and 50 g killed AMF inoculants was planted in the DSE treatment. Maize seedlings with DSE colonization and 50 g AMF inoculants was planted in the AMF+DSE treatment. Each treatment had four pots and one maize seedling was planted per pot. After planting the maize seedlings, 150 mL 50% Hoagland nutrient solution was poured in every 3 d Soil moisture was maintained at a constant through weighting and water supplementation.
2.3. Determination of plant growth and fungal colonization Maize height was tested at 60 d after the transplantation, then the maize plant was harvested and divided into the shoots and roots. They were cleaned using distilled water and oven-dried at 75 ℃ for 48 h to determine the weight of the maize plant. The AMF spores in the maize rhizosphere soil were separated through wet sieving, decanting, and sucrose centrifugation (Daniels and Skipper, 1982). The spore quantity was observed and calculated under the microscope. Root samples were collected randomly from each maize plant and dissociated by 10% (W/V) KOH solution for 1 h in 90 ℃ thermostatic water bath; the root samples were washed, dropped a few lactic acid to neutralize the remaining KOH and cut into 0.5 1 cm fragments. Then, 20 fragments were put on a glass slide and dyed with 5% blue ink-acetic acid (Cao et al., 2013). They were observed under the microscope, after the decoloration and infection rates of AMF and DSE were calculated via the crossing method (McGonigle et al., 1990). Table 1 Spore number of AMF and root colonization of AMF and DSE. Treatment
CK DSE AMF AMF+DSE
Spore number of AMF (number/g DW)
AMF colonization rate (%)
13.85§0.98 a 13.2 § 0.94 a
46.15§6.17 a 22.97§4.09 b
DSE colonization rate (%)
25.47§7.24 a 14.35§5.72 b
CK: the control of non-inoculation, DSE: Exophiala pisciphila inoculation, AMF: Funneliformis mosseae inoculation; AMF+DSE: co-inoculation of F. mosseae and E. pisciphila. “ ” indicates the index of the material was not detected. Means followed by the same letter do not differ significantly at p < 0.05 by LSD test.
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
3
Fig. 1. Effects of AMF and DSE on photosynthetic physiology of maize leaves under high Cd stress. Cd: cadmium; CK: the control of non-inoculation, DSE: Exophiala pisciphila inoculation, AMF: Funneliformis mosseae inoculation; AMF+DSE: co-inoculation of F. mosseae and E. pisciphila. The different lowercase letters indicate significant differences among treatments (p<0.05). “ns”, “*” and “**” means p > 0.05, p < 0.05 and p < 0.01, respectively.
2.4. Determination of chlorophyll concentration and photosynthesis in leaves Before the harvest at 60 d after the transplantation, about 0.2 g maize leaves were collected and extracted with 95% ethanol. Later, they were filtered and dissolved to a constant volume of 25 mL in a brown volumetric glass flask. The concentration of chlorophyll a and chlorophyll b were tested via a spectrophotometer (Model 721, Shanghai Precision & Scientific Instrument Co., Ltd. China) at wavelengths 665 nm and 649 nm, respectively. The chlorophyll content (mg/kg) based on fresh weight (FW) was calculated according to the formula by (Li, H.S., 2000). The leaf, which was folded completely, was selected from the maize plants. Photosynthesis indexes, such as net photosynthetic rate (Pn), intercellular carbon dioxide concentration (Ci), transpiration rate (Tr), and stomatal conductance (Gs), were tested on a sunny morning using the LCA-4 photosynthetic tester (Analytical Development Company Limited, ADC, Hoddesdon, England). 2.5. Determination of root traits and cadmium content in maize After the maize roots were cleaned, the roots were scanned using the PERFECTION V700 PHOTO root system scanner. Root morphological
indexes, including root length, root surface area, average diameter (RDI), root volume, root branches number, and root tips number, were analyzed using the WinRHIZO Pro-root system analyzer. The root traits included specific root length (SRL, root length per unit of root mass), specific root area (SRA, root area per unit of root mass), root mass density (RTD, root mass per root volume), specific root branch (SRB, root branch number per unit of root mass), and specific root tip (SRT, root tip number per unit of root mass). Ground dried roots and shoots (0.1 g) were digested with HNO3/ HClO4 (4:1). The Cd concentrations of the plants were tested via the flame atomic absorption spectrophotometry using an atomic absorption spectrometer (TAS-990, Beijing Puxi Instrument Factory, Beijing, China). The appropriate quality control was determined using CdCl2 as the standard solution. The Cd content (mg/kg) based on dry weight (DW) was calculated according to the formula by Bao (2000). Transfer coefficient was calculated as the shoot to root Cd content ratio (Zhan et al., 2018). 2.6. Data processing and statistical analysis Test data were mean of four repetitions and were expressed in mean § standard deviation. The SPSS 22.0 (SPSS Inc, Chicago, IL, USA) and the least significant difference (LSD) method were used to test the difference significance of test data at the level of 0.05. The influences of AMF
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB 4
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
Fig. 2. Effects of AMF and DSE on root morphology of maize under high Cd stress. Cd: cadmium; CK: the control of non-inoculation, DSE: Exophiala pisciphila inoculation, AMF: Funneliformis mosseae inoculation; AMF+DSE: co-inoculation of F. mosseae and E. pisciphila. The different lowercase letters indicate significant differences among treatments (p<0.05). “ns”, “*” and “**” means p > 0.05, p < 0.05 and p < 0.01, respectively.
and DSE on maize growth, photosynthetic physiology, root traits, Cd content and accumulation, as well as the interaction between AMF and DSE were analyzed via the two-way ANOVA (Analysis of Variance). The correlation coefficients between the Cd content in maize shoots with the root traits (n = 16) were analyzed by Pearson correlation. 3. Results 3.1. AMF and DSE colonization in maize roots No significant difference was found on the spore number between the AMF and AMF+DSE treatments. No spores were detected in the CK and DSE treatments. Colonization of hypha was detected in the AMF, DSE, and AMF+DSE treatments. The AMF infection rate of the AMF treatment was significantly higher than that of the AMF+DSE treatment, and the DSE infection rate of DSE treatment was far higher than that of AMF+DSE treatment (Table 1). 3.2. Effects of AMF and DSE on photosynthetic physiology of maize The two-way ANOVA reflected that AMF had significant effects on the chlorophyll content and photosynthetic physiology of maize leaves.
DSE influenced the chlorophyll b content significantly. An extremely significant interaction was found between AMF and DSE on chlorophyll content, and notable interactions were observed on the photosynthetic rate, transpiration rate and intracellular CO2 concentration (Fig. 1). Therefore, the photosynthetic physiology of maize leaves was influenced by the AMF inoculation and AMF+DSE co-inoculation. The contents of chlorophyll a and chlorophyll b in the maize leaves were increased significantly in these two treatments, accompanied by increases in photosynthesis rate, transpiration rate, and stomatal conductance, as well as a sharp reduction of intercellular CO2 concentration in maize leaves (Fig. 1). 3.3. Effects of AMF and DSE on root morphology and traits of maize The two-way ANOVA demonstrated that the DSE did not influence the root morphology and traits of maize significantly, except the specific root length (SRL) and specific root branch (SRB). But the AMF caused significant impacts on root morphology and traits of maize. No interaction between AMF and DSE was found on the root morphology and traits. (Fig. 2 and Fig. 3). The root morphology of maize was changed significantly in the AMF inoculation and AMF+DSE co-inoculation. These two
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
5
Fig. 3. Effects of AMF and DSE on root traits of maize under high Cd stress. Cd: cadmium; CK: the control of non-inoculation, DSE: Exophiala pisciphila inoculation, AMF: Funneliformis mosseae inoculation; AMF+DSE: co-inoculation of F. mosseae and E. pisciphila. Specific root length: root length (cm) per unit of root mass (mg FW); Specific root area: root area (cm2) per unit of root mass (mg FW); Root mass density: root mass (mg FW) per root volume (cm3); Specific root branch: root branch number per unit of root mass (mg FW); Specific root tip: root tip number per unit of root mass (mg FW). The different lowercase letters indicate significant differences among treatments (p<0.05). “ns”, “*” and “**” means p > 0.05, p < 0.05 and p < 0.01, respectively.
treatments significantly increased the root length, root surface area, root volume, root branches number, and root tips number. But the DSE inoculation did not change the root morphology (Fig. 2). Furthermore, all the AMF inoculation, DSE inoculation, and AMF+DSE co-inoculation caused a sharp decrease on the SRL, specific root area (SRA) and SRB. The AMF inoculation and AMF+DSE co-inoculation dramatically dropped the specific root tip (SRT), but increased the root mass density (RTD). And only the AMF inoculation average root diameter (RDI). Thus the AMF inoculation and AMF+DSE co-inoculation influenced the maize root traits significantly (Fig. 3). 3.4. Effects of AMF and DSE on growth and Cd content in maize According to the two-way ANOVA, the effects of DSE on the maize height and biomass were not significant, whereas AMF influenced the maize biomass greatly. No interaction was found between DSE and AMF on the maize height and biomass. However, both the DSE and AMF influenced the Cd content in the maize shoots significantly. There was a strong interaction between AMF and DSE on the Cd content in the maize shoots and roots (Fig. 4).
At 60 d after transplantation, the maize height significantly enhanced by the AMF inoculation. The maize biomass was increased significantly by the AMF inoculation and AMF+DSE co-inoculation. The shoot biomass was increased by 22.8 and 16.4 times, whereas the root biomass was increased by 5.5 and 5.4 times by the AMF inoculation and AMF+DSE co-inoculation, respectively. Additionally, the Cd content in shoots were decreased sharply by the single inoculation and co-inoculation of DSE and AMF by 45 58%. The Cd content of the maize roots was increased significantly by the DSE inoculation and AMF inoculation (Fig. 4). Thus single or dual inoculation of AMF and DSE decreased the Cd transfer coefficient of maize. These results indicated that the AMF and DSE induced the Cd retain in the roots and restricted Cd migration to the shoots of maize. 3.5. Correlation analysis The correlation analysis showed that the Cd content in maize shoots had a very significantly positive correlation (p < 0.01) with SRL, SRA and SRB, a significantly positive correlation (p < 0.05) with SRT, and a significantly negative correlation (p < 0.05) with RTD of the maize roots (Fig. 5). Results indicated a close relation between
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB 6
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
Fig. 4. Effects of AMF and DSE on height, biomass and Cd content in maize under high Cd stress. Cd: cadmium; CK: the control of non-inoculation, DSE: Exophiala pisciphila inoculation, AMF: Funneliformis mosseae inoculation; AMF+DSE: co-inoculation of F. mosseae and E. pisciphila. The different lowercase letters indicate significant differences among treatments (p<0.05). “ns”, “*” and “**” means p > 0.05, p < 0.05 and p < 0.01, respectively.
the Cd content in shoots with the root traits of maize. That AMF and DSE altered the root traits contributed to restrict Cd migration from roots to shoots of maize. 4. Discussions Many studies on ecological functions of AMF and DSE in plant roots under the Cd stress have been reported. AMF can improve mineral nutrients, photosynthesis, and resistance physiology of plants, promote growth of the host plants, and strengthen plant resistance to Cd stress (Garg and Bhandari, 2014; Lenoir et al., 2016). In the soil, AMF owns abundant extraradical mycelium, which can expand the absorbing area of roots, promote mineral absorption (e.g. P and S), and improve mineral nutrients of the host plants (Wang et al., 2017). Besides, they increases on the chlorophyll contents, net photosynthetic rate, transpiration rate, and stomatal conductance of leaves but a reduction on intercellular CO2 content. The photosynthetic physiology of leaves and plant biomass are enhanced accordingly (Li et al., 2016; Yang et al., 2015). AMF not only increased the activity of antioxidant enzymes like superoxide dismutase and catalase of the host plant under Cd stress, but also increased the antioxidant (e.g. glutathione, ascorbic acid) contents (Shahabivand et al., 2016; Zhan et al., 2018) and enhanced the scavenging activity of reactive oxygen species of plants and relieved damages of Cd ions to plants (Nath et al., 2016). This experiment revealed that AMF could facilitate the maize growth, increase the chlorophyll content and photosynthesis physiology in leaves, as well as plant height and shoots biomass. Similar to ecological functions of AMF, some research results supported that DSE could improve mineral nutrients, photosynthesis and resistance physiology of plants, promote the growth, and enhance Cd tolerance of host plants under Cd stress (Berthelot et al., 2017; Li et al.,
2011; Likar and Regvar, 2013; Wang et al., 2016). For example, the DSE inoculation increased mineral contents (including N, P, K, Ca, Mg, Fe, Mn and Zn) in tomato plants and improved the mineral nutrition of the host plants (Berthelot et al., 2017; Vergara et al., 2017). The DSE, Gaeumannomyces cylindrosporus, increased the chlorophyll content of maize leaves, enhanced photosynthetic physiology and chlorophyll fluorescence parameters of leaves, and increased plant height and biomass (Ban et al., 2017). The DSE E. pisciphila increased the chlorophyll content and photosynthetic physiology of maize leaves, height, and biomass (He et al., 2017; Li et al., 2011). The DSE increased the activity of antioxidant enzymes and antioxidant content in the host plant (He et al., 2017; Wang et al., 2016; Zhan et al., 2017), promoted the host growth, and relieved Cd biological toxicity to the host plants (Berthelot et al., 2016; Shahabivand et al., 2017). The mechanisms for AMF and DSE in enhancing Cd tolerance of host plants were also related with their effects in changing root traits and restricting Cd migration to the shoots of plants. The AMF and DSE colonized in root cortex of the host plants, which can increase root length, root surface area, root volume, root tips and biomass, and facilitate root growth of the host plants (Ban et al., 2017; Li et al., 2016). As a result, the specific root length (SRL), specific root area (SRA), and specific root tips (SRT) of the roots reduced, but the root mass density (RTD) increased. Similar phenomena were observed in this experiment. Besides, the Cd content in the maize shoots showed an extremely significantly positive correlation with the SRL, SRA, and SRT of the roots, but it had a significant negative correlation with the RTD. These results reflected that large SRL, SRA, and SRT values were conducive to the absorption of cationic mineral nutrients (de la Riva et al. 2018) and also facilitated the absorption of Cd ions. High RTD is beneficial for the roots to store mineral nutrients (Kramer-Walter et al., 2016) and also might contribute to
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
7
Fig. 5. Correlation between root traits with Cd content in maize shoot. Cd: cadmium; specific root length: root length (cm) per unit of root mass (mg FW); specific root area: root area (cm2) per unit of root mass (mg FW); specific root branch: root branch number per unit of root mass (mg FW); specific root tip: root tip number per unit of root mass (mg FW); root mass density: root mass (mg FW) per root volume (cm3).
storing Cd ions in the roots, which supported the current theory of “root economics spectrum (RES)” (Weemstra et al., 2016). Additionally, the AMF and DSE colonization changed the intracellular distribution and chemical morphologies of Cd in roots (Li et al., 2016; Wang et al., 2016) by the metal ion fixation of intraradical mycelium (Zhou et al., 2017) and increasing specific components, such as cellulose and hemicelluloses in the cell wall to bind the Cd ions (Chen et al., 2018). They also facilitated the retention of Cd ions in the plant roots, restricted Cd migration to the shoots from roots, and reduced Cd content in the shoots of plants (He et al., 2017; Hui et al., 2015; Li et al., 2011; Likar and Regvar, 2013; Shahabivand et al., 2017; Zhan et al., 2018). This experiment also confirmed that the AMF, DSE, and AMF+DSE treatments all decreased the transfer coefficient of Cd in maize and reduced the Cd content in the shoots significantly. These results might be one of the important mechanisms of the Cd tolerance enhancement by AMF and DSE in the host plants.
Notably, the AMF and DSE are co-existing in the root cortex of host plants and influence mutually, which might change their colonization rates in the roots. In the roots of Polylepis australis, the colonization rate of AMF was significantly negatively correlated with the colonization rate of DSE (Soteras et al., 2013). By dual inoculating AMF and DSE to ryegrass, the AMF decreased the DSE colonization rate in roots significantly, whereas the DSE did not cause an effect on the AMF colonization rate (Berthelot et al., 2018). This experiment discovered that the co-colonized AMF decreased the colonization rate of DSE, and the co-colonized DSE also decreased the colonization rate of AMF, indicating that they might compete in colonization behaviors in maize roots. However, some studies reported that the colonization rate of DSE in the DSE and AMF + DSE treatments remained the same, indicating the absence of a competitive relationship between the AMF and DSE in colonization in the roots (Saravesi et al., 2014).
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB 8
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9
In the ecological functions of regulating growth of host plants, the co-existing AMF and DSE might form competitive, independent, or collaborative relationships. For instance, the mycorrhizal inoculation relieved the adverse impacts of a DSE stain Phialocephala fortinii s.l. Acephala applanata on the growth of Norway spruce and Douglas fir, showing a competitive relationship (Reininger and Sieber, 2012). In heavy metal polluted soils, the co-inoculation of AMF and DSE was more beneficial to the growth of the host wild lettuce than _ the single inoculation of AMF or DSE (Wazny et al., 2018). With respect to the P nutrition, DSE facilitated the mineralization of organic P in soils, and AMF facilitated the P nutrient in the rhizosphere migrating to the host plants. The co-inoculation of AMF and DSE increased the P content of plants significantly, showing a collaborative relationship between the two (Della Monica et al., 2015). In mining waste soils, the growth rate and biomass of Veratrum nigrum reached the maximum with the co-inoculation of AMF and DSE, followed by a single inoculation of AMF. No significant difference was detected between them, indicating that AMF and DSE might be _ independent rather than competitive and collaborative (We˛ zowicz et al., 2017). Nevertheless, the interaction mechanism of AMF and DSE in plant roots remains unknown. Some studies believed that the exudates of DSE mycelium regulated the AMF growth in plant roots and facilitated the extension and branching of AMF mycelium but inhibited the growth of AMF extraradical mycelium (Scervino et al., 2009). Infection of endophytic fungi increased the alkaloid content of the host plants, thus inhibiting AMF growth and colonization and reducing the colonization rate of AMF in the roots (Li et al., 2016). This reflect that some specific compounds play a key role in adjusting the relationship between AMF and DSE. These studies provided a research direction for discussing the relationship, interaction effects and mechanisms between AMF and DSE in plant roots. This subject needs further intensive studies on the physiological and molecular mechanisms on enhancing stress resistance of plant by AMF and DSE in roots. 5. Conclusions The AMF inoculation and AMF+DSE co-inoculation improved the maize performance under a high Cd stress, which was closely related with changes of the photosynthesis physiology and root traits of maize. However, there was no synergistic interaction between AMF and DSE on the maize performance under a high Cd stress. The interaction between the co-colonized AMF and DSE in plant roots needs further studies under conditions of different fungal strains, host plants and environmental factors (such as soil, nutrient, moisture, and temperature). Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Declaration of Competing Interest The authors declare that they have no competing interests. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (41877130, 41701362), the Key Project of Yunnan Agricultural Foundation (2017FG001-014), the Reserve Talents Fund for Young and Middle-Aged Academic and Technological leaders in Yunnan Province (2018HB043), and the Science and Technology Innovation Team of Yunnan Province (2017HC015).
Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j.sajb.2019.09.018.
References Allah, E., Alqarawi, A., Hend, A., 2015. Alleviation of adverse impact of cadmium stress in sunflower (Helianthus annuus L.) by arbuscular mycorrhizal fungi. Pakistan. Journal of Botany 47, 785–795. Anjum, S., Tanveer, M., Hussain, S., Bao, M., Wang, L., Khan, I., et al., 2015. Cadmium toxicity in maize ( Zea mays L.), consequences on antioxidative systems, reactive oxygen species and cadmium accumulation. Environmental Science and Pollution Research 22, 17022–17030. Ban, Y., Xu, Z., Yang, Y., Liu, H., Tang, M., 2012. Colonization characteristics of arbuscular mycorrhizal fungi and dark septate endophytes in different degree Pb-Zn polluted areas. Acta Bot Boreali-Occidentialia Sinica 32, 2336–2343. Ban, Y., Xu, Z., Yang, Y., Zhang, H., Chen, H., Tang, M., 2017. Effect of dark septate endophytic fungus Gaeumannomyces cylindrosporus on plant growth, photosynthesis and Pb tolerance of maize ( Zea mays L.). Pedosphere 27, 283–292. Bao, S., 2000. Soil and Agricultural Chemistry Analysis. China Agriculture Press, Beijing. Benavides, M.P., Gallego, S.M., Tomaro, M.L., 2005. Cadmium toxicity in plants. Brazilian Journal of Plant Physiology 17, 21–34. Berthelot, C., Blaudez, D., Beguiristain, T., Chalot, M., Leyval, C., 2018. Co-inoculation of Lolium perenne with Funneliformis mosseae and the dark septate endophyte Cadophora sp. in a trace element-polluted soil. Mycorrhiza 28, 301–314. Berthelot, C., Blaudez, D., Leyval, C., 2017. Differential growth promotion of poplar and birch inoculated with three dark septate endophytes in two trace element-contaminated soils. International Journal of Phytoremediation 19, 1118–1125. Berthelot, C., Leyval, C., Foulon, J., Chalot, M., Blaudez, D., 2016. Plant growth promotion, metabolite production and metal tolerance of dark septate endophytes isolated from metal-polluted poplar phytomanagement sites. FEMS Microbiology Ecology 92, w144. Cao, M.Q., Wu, Q.S., Zou, Y.N., 2013. An improved ink-acetic acid technique for staining arbuscular mycorrhizas of citrus. International Journal of Agriculture & Biology 15, 386–388. Chen, X., Kang, Y., San So, P., Ng, C., Won, M., 2018. Arbuscular mycorrhizal fungi increase the proportion of cellulose and hemicellulose in the root stele of vetiver grass. Plant and Soil 425, 309–319. Daniels, B.A., Skipper, H.D., 1982. Methods for the recovery and quantitative estimation of propagules from soil. In: Schenck N.C. (eds) Methods and principles of mycorrhizal research. Saint Paul, MN. American Phytopathologic Society 29–35. Della Monica, I., Saparrat, M., Godeas, A., Scervino, J., 2015. The co-existence between dse and amf symbionts affects plant P pools through P mineralization and solubilization processes. Fungal Ecology 17, 10–17. ~o n, T., Pe rez-Ramos, I.M., Navarro-Ferna ndez, C.M., Olmo, M., de la Riva, E.G., Maran Villar, R., 2018. Root traits across environmental gradients in Mediterranean woody communities: are they aligned along the root economics spectrum? Plant and Soil 424, 35–48. Deng, Z., Cao, L., 2017. Fungal endophytes and their interactions with plants in phytoremediation, a review. Chemosphere 168, 1100–1106. Deram, A., Languereau, F., Haluwyn, C., 2011. Mycorrhizal and endophytic fungal colonization in Arrhenatherum elatius L. roots according to the soil contamination in heavy metals. Soil and Sediment Contamination 20, 114–127. Deram, A., Languereau-Leman, F., Howsam, M., Petit, D., Van Haluwyn, C., 2008. Seasonal patterns of cadmium accumulation in Arrhenatherum elatius (Poaceae), influence of mycorrhizal and endophytic fungal colonisation. Soil Biology and Biochemistry 40, 845–848. Gallego, S., Pena, L., Barcia, R., Azpilicueta, C., Iannone, M., Rosales, E.P., et al., 2012. Unravelling cadmium toxicity and tolerance in plants, insight into regulatory mechanisms. Environmental and Experimental Botany 83, 33–46. Garg, N., Bhandari, P., 2014. Cadmium toxicity in crop plants and its alleviation by arbuscular mycorrhizal (AM) fungi: an overview. Plant Biosystems 148, 609–621. He, S., Yang, X., He, Z., Baligar, V., 2017. Morphological and physiological responses of plants to cadmium toxicity: a review. Pedosphere 27, 421–438. He, Y., Yang, Z., Li, M., Jiang, M., Zhan, F., Zu, Y., et al., 2017. Effects of a dark septate endophyte (DSE) on growth, cadmium content and physiology in maize under cadmium stress. Environmental Science and Pollution Research 24, 18494–18504. Hui, F., Liu, J., Gao, Q., Lou, B., 2015. Piriformospora indica confers cadmium tolerance in nicotiana tabacum. Journal of Environmental Sciences 37, 184–191. Kramer-Walter, K., Bellingham, P., Millar, T., Smissen, R., Richardson, S., Laughlin, D., 2016. Root traits are multidimensional, specific root length is independent from root tissue density and the plant economic spectrum. Journal of Ecology 104, 1299–1310. Lenoir, I., Fontaine, J., Sahraoui, A., 2016. Arbuscular mycorrhizal fungal responses to abiotic stresses: a review. Phytochemistry 123, 4–15. Li, H., Luo, N., Zhang, L., Zhao, H., Li, Y., Cai, Q., et al., 2016. Do arbuscular mycorrhizal fungi affect cadmium uptake kinetics, subcellular distribution and chemical forms in rice? Science of the Total Environment 571, 1183–1190. Li, M., Li, H., Wang, K., Shi, L., Liu, J., Zhang, L., 2016. Effect of arbuscular mycorrhizae on the growth, photosynthetic characteristics and cadmium uptake of peanut plant under cadmium stress. Environmental Chemistry 35, 2344–2352.
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018
JID: SAJB
ARTICLE IN PRESS
[m5G;November 22, 2019;5:56]
Y.M. He et al. / South African Journal of Botany 00 (2019) 1 9 Li, H.S., 2000. Principles and techniques of plant physiological biochemical experiment. Beijing, Higher Education Press. Li, T., Liu, M.J., Zhang, X.T., Zhang, H.B., Sha, T., Zhao, Z.W., 2011. Improved tolerance of maize (Zea mays L.) to heavy metals by colonization of a dark septate endophyte (DSE) exophiala pisciphila. Science of the Total Environment 409, 1069–1074. Liang, C., Xiao, Y., Zhao, Z., 2007. Arbuscular mycorrhiza and dark septate endophytes in an abandoned lead-zinc mine in huize, Yunnan, China. Chinese Journal of Applied and Environmental Biology 13, 811–817. Likar, M., Regvar, M., 2013. Isolates of dark septate endophytes reduce metal uptake and improve physiology of Salix caprea l. Plant and Soil 370, 593–604. McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L., Swan, J.A., 1990. A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytologist 115, 495–501. Nath, M., Bhatt, D., Prasad, R., Gill, S., Anjum, N., Tuteja, N., 2016. Reactive oxygen species generation-scavenging and signaling during plant-arbuscular mycorrhizal and Piriformospora indica interaction under stress condition. Frontiers in Plant Science 7, 1574. Reininger, V., Sieber, T., 2012. Mycorrhiza reduces adverse effects of dark septate endophytes (DSE) on growth of conifers. PLoS One 7, e42865. Saravesi, K., Ruotsalainen, A., Cahill, J., 2014. Contrasting impacts of defoliation on root colonization by arbuscular mycorrhizal and dark septate endophytic fungi of Medicago sativa. Mycorrhiza 24, 239–245. rgola, M., Fernandez, L., Godeas, A., 2009. Exudates of Scervino, J., Gottlieb, A., Silvani, V., Pe dark septate endophyte (DSE) modulate the development of the arbuscular mycorrhizal fungus (AMF) Gigaspora rosea. Soil Biology and Biochemistry 41, 1753–1756. Shahabivand, S., Maivan, H., Goltapeh, E., Sharifi, M., Aliloo, A., 2012. The effects of root endophyte and arbuscular mycorrhizal fungi on growth and cadmium accumulation in wheat under cadmium toxicity. Plant Physiology and Biochemistry 60, 53–58. Shahabivand, S., Maivan, H., Mahmoudi, E., Soltani, B., Sharifi, M., Aliloo, A., 2016. Antioxidant activity and gene expression associated with cadmium toxicity in wheat affected by mycorrhizal fungus. Zemdirbyste 103, 53–60. Shahabivand, S., Parvaneh, A., Aliloo, A., 2017. Root endophytic fungus Piriformospora indica affected growth, cadmium partitioning and chlorophyll fluorescence of sunflower under cadmium toxicity. Ecotoxicology and Environmental Safety 145, 496–502. Soteras, F., Renison, D., Becerra, A., 2013. Growth response, phosphorus content and root colonization of Polylepis australis bitt. seedlings inoculated with different soil types. New Forest 44, 577–589. Su, C., Jiang, L., Zhang, W., 2014. A review on heavy metal contamination in the soil worldwide, situation, impact and remediation techniques. Environmental Skeptics and Critics 3, 24–38.
9
Vergara, C., Araujo, K.E.C, Urquiaga, S., Schultz, N., Balieiro, F.C., Medeiros, P.S., et al., 2017. Dark septate endophytic fungi help tomato to acquire nutrients from ground plant material. Frontiers in Microbiology 8, 2437. Wang, J., Li, T., Liu, G., Smith, J., Zhao, Z., 2016. Unraveling the role of dark septate endophyte (DSE) colonizing maize (Zea mays) under cadmium stress, physiological, cytological and genic aspects. Scientific Reports 6, 22028. Wang, W., Shi, J., Xie, Q., Jiang, Y., Yu, N., Wang, E., 2017. Nutrient exchange and regulation in arbuscular mycorrhizal symbiosis. Molecular Plant 10, 1147–1158. _ Wazny, R., Rozpadek, ˛ P., J˛edrzejczyk, R., Sliwa, M., Stojakowska, A., Anielska, T., et al., 2018. Does co-inoculation of Lactuca serriola with endophytic and arbuscular mycorrhizal fungi improve plant growth in a polluted environment? Mycorrhiza 28, 235–246. Weemstra, M., Mommer, L., Visser, E.J.W, Van Ruijven, J., Kuyper, T.W., Mohren, G.M., et al., 2016. Towards a multidimensional root trait framework, a tree root review. New Phytologist 211, 1159–1169. _ W˛e zowicz, K., Rozpadek, ˛ P., Turnau, K., 2017. Interactions of arbuscular mycorrhizal and endophytic fungi improve seedling survival and growth in post-mining waste. Mycorrhiza 27, 499–511. Yang, Y., Han, X., Liang, Y., Ghosh, A., Chen, J., Tang, M., 2015. The combined effects of arbuscular mycorrhizal fungi (AMF) and lead (Pb) stress on pb accumulation, plant growth parameters, photosynthesis, and antioxidant enzymes in Robinia pseudoacacia l. PLoS One 10, e145726. Zhan, F., He, Y., Yue, X., Qin, L., Xia, Y., 2016. Effect of mycorrhizal inoculation on plant growth, nutrients and heavy metals uptake by Leucaena leucocephala. Fresenius Environmental Bulletin 25, 1760–1767. Zhan, F., Li, B., Jiang, M., Qin, L., Wang, J., He, Y., et al., 2017. Effects of a root-colonized dark septate endophyte on the glutathione metabolism in maize plants under cadmium stress. Journal of Plant Interactions 12, 421–428. Zhan, F., Li, B., Jiang, M., Yue, X., Xia, Y., Wang, Y., 2018. Arbuscular mycorrhizal fungi enhance antioxidant defense in the leaves and the retention of heavy metals in the roots of maize. Environmental Science and Pollution Research 25, 24338–24347. Zhang, Y., Zhang, Y., Liu, M., Shi, X., Zhao, Z., et al., 2008. Dark septate endophyte (DSE) fungi isolated from metal polluted soils: their taxonomic position, tolerance, and accumulation of heavy metals In Vitro. The Journal of Microbiology 46, 624–632. Zhou, X., Fu, L., Xia, Y., Zheng, L., Chen, C., et al., 2017. Arbuscular mycorrhizal fungi enhance the copper tolerance of Tagetes patula through the sorption and barrier mechanisms of intraradical hyphae. Metallomics 7, 936–948.
Please cite this article as: Y.M. He et al., Effects of arbuscular mycorrhizal fungi and dark septate endophytes on maize performance and root traits under a high cadmium stress, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.09.018