Effect of Dark Septate Endophytic Fungus Gaeumannomyces Cylindrosporus on Plant Growth, Photosynthesis and Pb Tolerance of Maize

Home , Chaetothyriales, Dark septate endophyte

Pedosphere 27(2): 283–292, 2017 doi:10.1016/S1002-0160(17)60316-3 ISSN 1002-0160/CN 32-1315/P ⃝c 2017 Soil Science Society of China Published by Elsevier B.V. and Science Press

Eﬀect of Dark Septate Endophytic Fungus Gaeumannomyces cylindrosporus on Plant Growth, Photosynthesis and Pb Tolerance of Maize (Zea mays L.)

BAN Yihui1,2, XU Zhouying1, YANG Yurong1, ZHANG Haihan1, CHEN Hui1 and TANG Ming1,3,∗ 1State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Northwest A&F University, Yangling 712100 (China) 2School of Chemistry, Chemical Engineering and Life Sciences, Wuhan University of Technology, Wuhan 430070 (China) 3College of Forestry, Northwest A&F University, Yangling 712100 (China) (Received May 11, 2016; revised January 12, 2017)

ABSTRACT Dark septate endophytic (DSE) fungi are ubiquitous and cosmopolitan, and occur widely in association with plants in heavy metal stress environment. However, little is known about the effect of inoculation with DSE fungi on the host plant under heavy metal stress. In this study, Gaeumannomyces cylindrosporus, which was isolated from Pb-Zn mine tailings in China and had been proven to have high Pb tolerance, was inoculated onto the roots of maize (Zea mays L.) seedlings to study the effect of DSE on plant growth, photosynthesis, and the translocation and accumulation of Pb in plant under stress of different Pb concentrations. The growth indicators (height, basal diameter, root length, and biomass) of maize were detected. Chlorophyll content, photosynthetic characteristics (net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO2 concentration), and chlorophyll fluorescence parameters in leaves of the inoculated and non-inoculated maize were also determined. Inoculation with G. cylindrosporus significantly increased height, basal diameter, root length, and biomass of maize seedlings under Pb stress. Colonization of G. cylindrosporus improved the efficiency of photosynthesis and altered the translocation and accumulation of Pb in the plants. Although inoculation with G. cylindrosporus increased Pb accumulation in host plants in comparison to non-inoculated plants, the translocation factor of Pb in plant body was significantly decreased. The results indicated that Pb was accumulated mainly in the root system of maize and the phytotoxicity of Pb to the aerial part of the plant was alleviated. The improvement of efficiency of photosynthesis and the decrease of translocation factor of Pb, caused by DSE fungal colonization, were efficient strategies to improve Pb tolerance of host plants. Key Words: chlorophyll fluorescence, fungal colonization, growth indicator, heavy metal stress, Pb accumulation, Pb translocation, photosynthetic characteristics

Citation: Ban Y H, Xu Z Y, Yang Y R, Zhang H 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(2): 283–292.

Soil pollution with heavy metals is a pressing issue metal tolerance in plants. worldwide. The continued increase of metal levels in Dark septate endophytic (DSE) fungi, which are soil poses a health risk to humans and animals through one of the groups of endophytic fungi, can colonize food chain. Heavy metals in the soil can lead to toxicity nearly 600 plant species representing about 320 ge- symptoms and inhibit the growth of most plants, espe- nera and 114 families (Jumpponen and Trappe, 1998; cially the nonessential heavy metals, such as Pb, Cd, Mandyam and Jumpponen, 2005). According to the re- Cr, etc. (Nagajyoti et al., 2010). Plants possess a range sults of recent surveys of DSE colonization, the range of potential cellular mechanisms that may be involved of host plant species was obviously enlarged (Zhang et in the detoxification of heavy metals and thus tolerance al., 2013; Massenssini et al., 2014; Gucwa-Przepióra et to metal stress (Hall, 2002). Endophytic fungi not only al., 2016). DSE fungi are ubiquitous in various stress- have the ability to protect against heavy metal toxici- ful environments, especially common in heavy metal- ty but also increase nutrient acquisition of host plants contaminated soils (Likar and Regvar, 2009; Regvar and enhance their metabolic activity to combat stress et al., 2010; Li et al., 2012). Some typical DSE fun- (Selosse et al., 2004; Gadd, 2007). Thus, the alleviation gi isolated from heavy metal-contaminated soils, such of heavy metal toxicity to host plants by endophytic as Exophiala pisciphila McGinnis & Ajello (Li et al., fungi could be an efficient strategy to improve heavy 2011), Gaeumannomyces cylindrosporus Hornby, Slo-

∗Corresponding author. E-mail: [email protected]. 284 Y. H. BAN et al. pe, Gutteridge & Sivanesan (Ban et al., 2012), and domized factorial design (2 inoculation treatments × 4 Phialophora/Cadophora complex (Likar and Regvar, Pd concentrations) with 10 replicates. Four plants per 2013), have been proven to have significant tolerance treatment were randomly selected for measurements to heavy metal ions. The high tolerance of DSE fun- of plant biomass and Pb content in plant body, and gi to heavy metals and their great abundance in heavy the leaves and roots of residual plants were used for metal-polluted habitats suggested that DSE fungi may the determinations of Chl concentration, photosynthe- have an important function for host survival in ex- tic characteristics, Chl fluorescence parameters, and treme environments (Likar, 2011). However, little re- DSE colonization. The treatments were either inocula- search has been focused on the effect of inoculation tion or non-inoculation of G. cylindrosporus along with with DSE fungi on host plants under heavy metal the addition of four Pb concentrations (0, 50, 500, and stress in controlled pot culture conditions. Zhang et 1 000 µg g−1) into the substrates. River sand was used al. (2012) demonstrated that inoculation with a DSE as pot culture substrates. After being washed with tap isolate LBF-2 increased the total biomass of Lycium water and air-dried, river sand was passed through a barbarum L. seedlings and the concentration of chloro- 2-mm sieve and then autoclaved for 2 h at 121 ◦C. The phyll (Chl), and also enhanced Chl fluorescence. Howe- basic physico-chemical characteristics of the substrates ver, the study was conducted without heavy metal were as follows: pH (H2O) 7.4, organic matter 0.3 g stress, and it was not clear that the positive effects kg−1, available P 0.31 mg kg−1, alkali-hydro N 0.79 of inoculation with DSE fungi would be enhanced or mg kg−1, and available K 1.32 mg kg−1. Each plas- completely reversed with the addition of heavy me- tic pot (150 mm length × 130 mm width × 150 mm tals. The effect of DSE fungi on the photosynthetic height) was filled with 2 kg culture substrates. The Pb system of host plants may be one important way to al- concentrations in culture substrates were adjusted at ter the sensibility of plant to heavy metals, but so far 50, 500, and 1 000 µg g−1, respectively, by addition of it has not been confirmed. There still exist different Pb(NO3)2 solution. The treatment without Pb(NO3)2 results about the influence of inoculation with DSE on (i.e., 0 µg g−1) was set as the control. Planting was the uptake and accumulation of heavy metals in host carried out after the plastic pots were placed without plants. Li et al. (2011) indicated that Pb accumula- moving for one week to reach Pb equilibrium. tion of maize (Zea mays L.) seedlings colonized by a Maize seeds (cv. Zhengdan 958) were purchased DSE fungus was higher than the non-inoculated con- from Northwest A&F University Seed Co., Shaanxi trols under four concentrations of Pb. However, the Province, China. The seeds were surface-sterilized by results of Likar and Regvar (2013) showed that DSE dipping in 75% (volume:volume) ethanol for 5 min and fungi reduced heavy metal uptake by Salix caprea L. then in 10% (volume:volume) sodium hypochlorite for This difference may result from various factors, such as 10 min under agitation. Sterilized seeds were gently fungus and host plant specificity, cultural conditions, washed by deionized water for several times at room etc. Thus, the relationship between DSE fungi and the temperature, and then placed on the sterile moist filter absorption and translocation of metals in plants de- papers (Xinhua No. 101, China) in Petri dishes for ger- serves further research. mination at 25 ◦C. The germinated seeds were trans- The aim of this work was to study the effect of ino- planted into the plastic pots (2 seeds for each pot) culation with G. cylindrosporus on the growth of maize, at a depth of 2 cm for fungal inoculation. Inoculum the Chl concentration, photosynthetic characteristics, of G. cylindrosporus, as 5-mm plugs excised from an and Chl fluorescence parameters of plant leaves, and edge of an actively growing colony on potato dex- the uptake, translocation, and accumulation of Pb in trose agar (PDA), was inoculated close to the roots plant under different Pb concentrations. of maize seedlings. Fungus-free treatments were mock- inoculated with sterile PDA plugs. The experiment was MATERIALS AND METHODS carried out at 25 ◦C in a greenhouse with a photope- riod of 12 h per day for a culture period of 6 weeks. Experimental design Each pot was irrigated with 100 mL Hoagland’s nutri- G. cylindrosporus isolated from the roots of Ast- ent solution (Hoagland and Arnon, 1950) every week. ragalus adsurgens Pall., which grew naturally on the DSE colonization Pb-Zn mine tailings in Qiandong Mountain of Sha- anxi Province, China, was used as inoculum (Ban et Both non-inoculated and inoculated plant roots al., 2012). The experiment was installed under green- were prepared according to the method of Phillips and house conditions and consisted of a completely ran- Hayman (1970). Root samples were washed several FUNGUS INOCULATION EFFECT ON MAIZE UNDER PB STRESS 285 times with running tap water, cut into 1-cm pieces, (NPQ) was conducted at each saturating pulse, accor- ◦ − ′ ′ and clarified in 10% (weight:volume) KOH at 90 C for ding to the equation of (Fm Fm)/Fm (Bilger and 20–60 min. After that the root samples were bleached Björkman,1991). The rate of linear electron transport with fresh alkaline H2O2 solution, containing 30 mL (ETR) through PSII was calculated as ETR = ΦPSII × 10% (volume:volume) H2O2, 3 mL NH4OH, and 567 PPFD × 0.5 × 0.84, where ΦPSII is the quantum yield mL deionized water, for 10–30 min, acidified with 2% of PSII photochemistry; 0.5 is used because of the sup- (volume:volume) HCl for 5 min, and then soaked in posedly equal distribution of excitation between the 0.05% (weight:volume) trypan blue solution for 12 h at two photosystems; PPFD is the photosynthetic pho- room temperature. All stained roots were transferred ton flux density on the leaf surface; and the factor into acidified glycerol and incubated for 12 h at room 0.84 is the fraction of the incident quanta absorbed temperature. The DSE colonization of roots was deter- by the leaf (Bajkán et al., 2012). Chl was extract- mined using the method modified by McGonigle et al. ed from the second fully expanded leaf using 1:1 (vo- (1990) under a compound-light microscope (Olympus lume:volume) ethanol-acetone solution. A spectropho- BX51, Japan). It was characterized by darkly pigmen- tometer (UVmini-1240, Shimadzu, Japan) was used to ted or blue-stained septate hyphae and microsclerotia, determine the absorbance of Chl a and b in the extracts and was calculated as follows: at 663 and 645 nm, respectively.

Measurements of photosynthetic parameters DSE colonization = (n1/n2) × 100% (1) At end of the culture period, photosynthetic pa- where n1 is the number of intersections with fungal rameters (including net photosynthetic rate, stoma- structure and n is the total number of counted inter- 2 tal conductance, intercellular CO2 concentration, and sections. transpiration rate) were determined on fully develo- ped leaves between 8:30–11:30 a.m. using a portable Measurements of Chl fluorescence and concentrations open-flow gas exchange system (LI-6400, LI-COR, Inc., At end of the experiment, Chl fluorescence was USA) according to the manufacturer’s instructions. measured on the second fully expanded leaf at room The system maintained a constant photosynthetically − − ◦ temperature using a portable chlorophyll fluorome- active radiation (1 000 µmol m 2 s 1), 25 C leaf tem- − ter (model Mini-Pam, Heinz Walz, Germany). After perature, 50% relative humidity, and 0.5 dm3 min 1 darkening the leaf for 30 min, the minimum fluores- flow rate of atmosphere. cence (F0) was recorded and the maximum fluorescence Measurements of plant biomass and Pb concentration in the dark adapted state (Fm) was obtained by ap- in plants plication of a saturating light pulse. The plants were illuminated with actinic light (90 µmol L−1 photons Plant height and root length (the length of the lon- m−2 s−1) for 10 min, and then the level of modula- gest root) were measured by precision straight edge, ted fluorescence during a brief interruption of actinic and basal diameter was measured with a vernier cali- ′ illumination in the presence of far-red light (F0), the per (ECV150C, Insize Co., China). Shoots (leaves and maximum fluorescence yield during actinic illumina- stems) and roots were collected, respectively, and the ′ tion (Fm), and the Chl fluorescence yield during ac- roots were thoroughly washed by tap water to remove tinic illumination (Fs) were measured. The yield of the silt. The shoots and roots were then placed in an ◦ variable fluorescence (Fv) was calculated as Fm − F0. oven at 80 C, dried to constant weight, and weighed The maximum quantum yield of photosystem II (PS- after cooling in vacuum desiccators. After that, the II) photochemistry was determined as Fv/Fm, i.e., shoots and roots were ground separately by a mini- (Fm − F0)/Fm. The actual quantum efficiency of PSII vegetation disintegrator (FZ102, Tianjing Test Instru- open centers at light adapted state (Y II) was calcu- ment Co., China) and then digested in a mixture of ′ − ′ lated as (Fm Fs)/Fm (Genty et al., 1989). The co- concentrated HNO3 and HClO4. The Pb content was efficient of photochemical quenching (qP) is a mea- determined with an atomic absorption spectrophoto- surement of the fraction of open centers, calculated meter (Solaar Mk2-M6, Thermo, USA). The transloca- ′ − ′ − ′ as (Fm Fs)/(Fm F0) (Schreiber et al., 1986). The tion factor (TF) of Pb was defined as the ratio of Pb coefficient of non-photochemical quenching (qN) was content in shoot to that in root. calculated as 1 − (F ′ − F ′)/(F − F ) (Bilger and m 0 m 0 Statistical analyses Schreiber, 1987). Calculation of quenching due to non- photochemical dissipation of absorbed light energy The determinations of physiological and biochemi- 286 Y. H. BAN et al. cal parameters were performed with 4 replicates (randomly selected from 10 plants per treatment), and data are presented as means standard errors. All data were tested for normality and analyzed by one- and two-way analyses of variance using SPSS 16.0 software (SPSS Inc., USA). Comparisons between means were carried out using Duncan’s multiple range tests at P < 0.05. Graphical work was carried out using SigmaPlot for Windows version 10.0 software packages.

RESULTS

DSE colonization and plant biomass

No DSE structures were observed in the roots of non-inoculated maize after harvesting. In the inocula- Fig. 1 Dark septate endophytic (DSE) colonization (by Gaeu- ted treatments, typical structures of G. cylindrosporus mannomyces cylindrosporus) in roots of maize under stress of di- were observed under four concentrations of Pb. The fferent Pb concentrations. Vertical bars indicate standard errors of the means (n = 4). Bars with the same letter(s) are not sig- colonization intensity of G. cylindrosporus increased nificantly different at P ≤ 0.05 according to Duncan’s multiple nonlinearly with the increasing Pb concentrations and range test. peaked at 63.6% in the roots of maize grown in the Effect of inoculation with G. cylindrosporus on photo- substrates applied with 1 000 µg g−1 Pb (Fig. 1). Ino- synthesis culation with G. cylindrosporus increased the height, basal diameter, root length, and total biomass of maize Under different Pb concentrations, Chl a content of seedlings under different Pb concentrations stress (Ta- maize seedlings inoculated with G. cylindrosporus was ble I). Under the Pb concentration of 1 000 µg g−1, higher than that of the non-inoculated seedlings (Table the shoot and root biomass of inoculated maize were III). However, the difference was not significant except 1.35 and 2.81 times, respectively, higher than those of under stress of high Pb concentration (1 000 µg g−1), the non-inoculated maize. Inoculation with G. cylin- which was consistent with the results of total Chl con- drosporus significantly alleviated the negative effects of tent. Under Pb concentration of 1 000 µg g−1, the Chl increasing toxicity of heavy metals, and the differences a and Chl (a + b) contents were recorded 66.9% and of growth indicators between the inoculated and non- 59.5% higher in the inoculated plants than in the non- inoculated plants were more significant under high Pb inoculated plants. In addition, Pb concentrations in concentration stress. Based on the results of interac- substrates and inoculation treatments showed no sig- tion between the factors (Table II), both of Pb con- nificant (P > 0.05) effects on Chl a/b value of maize centration and inoculation treatment had significant seedlings (Table II). (P < 0.05) effects on the growth of maize. Effect of inoculation with G. cylindrosporus on Chl

TABLE I Eﬀect of inoculation with Gaeumannomyces cylindrosporus (+ GC) on height, basal diameter, root length, and biomass of maize seedlings under stress of diﬀerent Pb concentrations

Pb concentration Inoculation Height Basal diameter Root length Shoot biomass Root biomass µg g−1 cm g 0 No GC 41.22 ± 1.17a)bcb) 0.35 ± 0.01ab 34.53 ± 1.08c 2.17 ± 0.04bc 2.11 ± 0.10b + GC 43.60 ± 0.21b 0.36 ± 0.01ab 48.24 ± 1.07a 3.81 ± 0.16a 4.05 ± 0.18a 50 No GC 42.87 ± 0.85b 0.36 ± 0.01ab 39.21 ± 2.02b 2.24 ± 0.11b 2.30 ± 0.13b + GC 52.75 ± 1.19a 0.37 ± 0.00a 50.21 ± 0.89a 3.84 ± 0.15a 3.86 ± 0.10a 500 No GC 39.19 ± 1.69bc 0.34 ± 0.02b 30.04 ± 1.67cd 1.50 ± 0.06d 1.23 ± 0.07c + GC 43.90 ± 2.16b 0.35 ± 0.00ab 42.50 ± 1.49b 2.17 ± 0.09bc 2.31 ± 0.04b 1 000 No GC 37.79 ± 1.78c 0.29 ± 0.01c 27.21 ± 1.51d 1.39 ± 0.02d 0.78 ± 0.03d + GC 42.98 ± 2.46b 0.35 ± 0.02ab 30.21 ± 1.64cd 1.88 ± 0.03c 2.19 ± 0.13b a)Means ± standard errors (n = 4). b)Means followed by the same letter(s) within each column are not signiﬁcantly diﬀerent at P < 0.05 according to Duncan’s multiple range test. FUNGUS INOCULATION EFFECT ON MAIZE UNDER PB STRESS 287

TABLE II lue was improved by colonization of G. cylindrosporus;

F values obtained by two-way analysis of variance to determine however, the differences between the inoculated and the effects of inoculation with Gaeumannomyces cylindrosporus non-inoculated treatments under 50, 500, and 1 000 µg (GC) and Pb concentrations on plant growth indicators g−1 Pb stress were not significant (P > 0.05). Un-

Growth indicatora) Pb GC Pb × GC der four concentrations of Pb, there were no significant differences for the qP values between the inocula- Height 8.54*** 24.25*** 1.95NSb) Basal diameter 8.04** 6.08* 3.60* ted and non-inoculated treatments. The qN value de- Root length 45.20*** 93.95*** 5.42** creased when Pb concentration of substrates was up to Shoot biomass 115.54*** 253.93*** 19.32*** 50 µg g−1, and then increased with the increasing Pb Root biomass 119.07*** 371.99*** 5.35** concentration. There were no significant differences for Chl a content 28.28*** 6.50* 0.46NS Chl b content 173.05*** 61.73*** 1.84NS the NPQ values between the non-inoculated and the Chl a/b value 0.46NS 0.00NS 0.49NS inoculated seedlings under Pb stress of 50, 500, and Total Chl content 46.60*** 11.79** 0.58NS 1 000 µg g−1. The changing trend of ETR with the Pn 73.55*** 101.30*** 2.92NS increasing Pb concentration was consistent with that TR 4.90* 37.48*** 0.29NS Gs 20.18*** 3.30NS 0.23NS of Y (II); however, the differences between the inocula- Ci 68.32*** 378.50*** 34.46*** ted and non-inoculated treatments were significant on- Pb content in root 248.69*** 18.17*** 9.45*** ly under no Pb stress. Pb content in shoot 173.76*** 36.26*** 7.12** Pb content in plant 283.49*** 12.31** 7.64*** Effect of inoculation with G. cylindrosporus on pho- TF of Pb 14.29*** 19.80*** 0.27NS tosynthetic characteristics of maize seedlings under Pb stress is shown in Table IV. The net photosynthetic *, **, ***Significant atP < 0.05, P < 0.01, and P < 0.001, respectively. rate (Pn) was improved under light Pb stress (50 µg a) −1 Chl is the chlorophyll; Pn is the net photosynthetic rate; TR is g ), and reduced obviously under the concentrations the transpiration rate; G is the stomatal conductance; C is the −1 s i of 500 and 1 000 µg g . However, Pn of inoculated intercellular CO2 concentration; TF is the translocation factor. seedlings was 2.14 and 4.11 times higher than that of b)Not significant. the non-inoculated seedlings under the two Pb con- fluorescence of maize seedlings under four different centrations. Transpiration rate (TR) was improved by Pb concentrations is shown in Fig. 2. The value of the colonization of G. cylindrosporus under Pb stress. −1 Fv/Fm decreased with the increasing Pb concen- Under Pb stress levels of 50, 500, and 1 000 µg g , trations both in the inoculated and non-inoculated TR of inoculated seedlings increased by 88.0%, 76.2%, treatments. However, the Fv/Fm values of inoculated and 114.3%, respectively, compared with the uninocu- seedlings under Pb concentrations of 500 and 1 000 µg lated seedlings. Based on the results of interaction be- g−1 were both significantly higher (P < 0.05), between the factors (Table II), inoculation treatment had ing 1.12 and 1.51 times, respectively, that of the non- no significant effects on stomatal conductance (Gs) of inoculated seedlings. The Y (II) value increased firstly maize (P > 0.05), but the effect of Pb stress was ob- and then decreased significantly with the increase of Pb vious. The differences of intercellular CO2 concentra- concentration. The results showed that the Y (II) vation (Ci) between the inoculated and non-inoculated

TABLE III Effect of inoculation with Gaeumannomyces cylindrosporus (+ GC) on the chlorophyll (Chl) content of maize seedlings under stress of different Pb concentrations Pb concentration Inoculation Chl a content Chl b content Chl a/b value Total Chl content µg g−1 mg g−1 FWa) mg g−1 FW 0 No GC 2.048 ± 0.14b)ac) 0.559 ± 0.02b 3.684 ± 0.38a 2.607 ± 0.13a + GC 2.188 ± 0.11a 0.603 ± 0.02ab 3.638 ± 0.26a 2.791 ± 0.10a 50 No GC 2.102 ± 0.08a 0.505 ± 0.02c 4.186 ± 0.27a 2.607 ± 0.07a + GC 2.318 ± 0.14a 0.618 ± 0.01a 3.750 ± 0.19a 2.936 ± 0.15a 500 No GC 1.244 ± 0.14b 0.334 ± 0.02e 3.741 ± 0.44a 1.579 ± 0.14b + GC 1.533 ± 0.20b 0.429 ± 0.01d 3.597 ± 0.55a 1.963 ± 0.19b 1 000 No GC 0.732 ± 0.11c 0.242 ± 0.00f 3.039 ± 0.51a 0.974 ± 0.11c + GC 1.222 ± 0.27b 0.331 ± 0.02e 3.744 ± 0.92a 1.554 ± 0.26b a)Fresh weight. b)Means ± standard errors (n = 4). c)Means followed by the same letter(s) within each column are not significantly different at P < 0.05 according to Duncan’s multiple range test. 288 Y. H. BAN et al. seedlings under four Pb concentrations were signifi- the increasing Pb concentrations. However, inoculation cant (P < 0.05), and Ci of inoculated maize were only with G. cylindrosporus increased total accumulation of 45.3% and 39.0% of that of non-inoculated seedlings, Pb in maize under Pb stress. Under Pb concentration respectively, when the Pb concentration was up to 500 of 1 000 µg g−1, the sum of Pb content in root and shoot and 1 000 µg g−1. of the inoculated seedlings was 1.36 times (P < 0.05) that in the non-inoculated seedlings. More important- Pb uptake and translocation in maize seedlings ly, root colonization by G. cylindrosporus changed the The Pb contents in root and shoot and the tra- translocation of Pb in maize. Under the inoculation nslocation factor of Pb in the inoculated and non- treatment, Pb was accumulated mainly in the roots, inoculated maize seedlings growing in all test sub- and Pb content in shoot was decreased compared with strates are summarized in Table V. Pb was absorbed that in the non-inoculated treatments, thus the ratio and accumulated increasingly by roots and shoots of Pb content in shoot to that in root (i.e., TF) of of both inoculated and non-inoculated seedlings with inoculated seedlings being lower than that of the non-

Fig. 2 Effect of inoculation with Gaeumannomyces cylindrosporus (+ GC) on the chlorophyll fluorescence kinetic parameters of maize seedlings under stress of different Pb concentrations. Vertical bars indicate standard errors of the means (n = 4). Bars with the same letter(s) are not significantly different at P < 0.05 according to Duncan’s multiple range test. Fv/Fm represents the maximum quantum yield of photosystem II photochemistry, where Fv is the yield of variable fluorescence and Fm is the maximum fluorescence in the dark adapted state; Y (II) is the actual quantum efficiency of photosystem II open centers at light adapted state; qP is the coefficient of photochemical quenching; qN is the coefficient of non-photochemical quenching; NPQ is the quenching due to non-photochemical dissipation of absorbed light energy; ETR is the rate of linear electron transport. FUNGUS INOCULATION EFFECT ON MAIZE UNDER PB STRESS 289

TABLE IV Eﬀect of inoculation with Gaeumannomyces cylindrosporus (+ GC) on photosynthetic characteristicsa) of maize seedlings under stress of diﬀerent Pb concentrations

Pb concentration Inoculation Pn TR Gs Ci −1 −2 −1 −2 −1 −2 −1 −1 µg g µmol CO2 m s mmol H2O m s mol H2O m s µmol CO2 mol 0 No GC 2.34 ± 0.20b)cc) 0.26 ± 0.08cde 0.02 ± 0.01bcd 163.3 ± 6.77cd + GC 4.87 ± 0.29a 0.42 ± 0.04ab 0.02 ± 0.00bc 115.5 ± 13.12ef 50 No GC 3.33 ± 0.26b 0.25 ± 0.05cde 0.03 ± 0.00ab 190.3 ± 7.97c + GC 5.08 ± 0.15a 0.47 ± 0.01a 0.04 ± 0.00a 106.7 ± 3.28f 500 No GC 1.36 ± 0.23d 0.21 ± 0.04de 0.02 ± 0.00de 311.3 ± 2.33b + GC 2.91 ± 0.41bc 0.37 ± 0.02abc 0.02 ± 0.01cde 141.0 ± 10.26de 1 000 No GC 0.36 ± 0.02e 0.14 ± 0.01e 0.01 ± 0.00e 360.0 ± 8.08a + GC 1.48 ± 0.21d 0.30 ± 0.04bcd 0.01 ± 0.00e 140.3 ± 15.82de a) Pn is the net photosynthetic rate; TR is the transpiration rate; Gs is the stomatal conductance; Ci is the intercellular CO2 concentration. b)Means ± standard errors (n = 4). c)Means followed by the same letter(s) within each column are not signiﬁcantly diﬀerent at P < 0.05 according to Duncan’s multiple range test.

TABLE V Effect of inoculation with Gaeumannomyces cylindrosporus (+ GC) on Pb uptake and translocation in maize seedlings under stress of different Pb concentrations Pb concentration Inoculation Pb content Translocation factor Root Shoot Root + shoot µg g−1 mg kg−1 DWa) 0 No GC 1.80 ± 0.08b)dc) 0.62 ± 0.12e 2.42 ± 0.17e 0.34 ± 0.06a + GC 1.70 ± 0.01d 0.41 ± 0.03e 2.11 ± 0.03e 0.24 ± 0.02bc 50 No GC 24.30 ± 1.01d 7.20 ± 0.55cd 31.50 ± 1.52d 0.30 ± 0.01ab + GC 26.25 ± 1.07d 5.50 ± 0.87d 31.75 ± 1.09d 0.21 ± 0.04bc 500 No GC 65.64 ± 6.62c 14.35 ± 1.09b 79.99 ± 6.07c 0.23 ± 0.04bc + GC 85.48 ± 10.44c 8.34 ± 0.41c 93.82 ± 10.47c 0.10 ± 0.01d 1 000 No GC 162.36 ± 12.38b 23.60 ± 1.36a 185.96 ± 12.42b 0.15 ± 0.01cd + GC 235.82 ± 13.79a 16.43 ± 1.41b 252.25 ± 14.71a 0.07 ± 0.01d a)Dry weight. b)Means ± standard errors (n = 4). c)Means followed by the same letter(s) within each column are not significantly different at P < 0.05 according to Duncan’s multiple range test. inoculated seedlings under four different concentra- site in Southwest China and found that E. pisciphila tions of Pb (Table V). This indicated that inocula- H93 had high tolerance to Cd. In the present study, tion with G. cylindrosporus decreased Pb translocation G. cylindrosporus used in the inoculation experiment from roots to shoots and that the Pb toxicity to maize was isolated from the roots of A. adsurgens growing at was also alleviated with the decrease of Pb content in a metal-enriched site. After determination of the sen- shoots. sitivity of the isolate to heavy metal ions, G. cylindrosporus was found to have high ability to resist DISCUSSION Pb stress, considering the EC50 (the effective concen- Recently, numerous surveys have proved that many tration of heavy metal that inhibits 50% of mycelial dominant plant species in heavy metal-contaminated growth) and MICs (the minimum inhibitory concen- land are widely associated with DSE fungi (Deram et trations) of Pb ions were up to 1.83 and 4.5 mg mL−1, al., 2008; Likar and Regvar, 2009; Li et al., 2012; Ban respectively (Ban et al., 2012). et al., 2015). High tolerance of DSE fungi to heavy In the present study, maize seedlings were success- metal pollution and their great abundance in stress fully inoculated with G. cylindrosporus. Colonization habitats suggest that DSE fungi might have important intensity of maize roots by G. cylindrosporus increased ecological functions. The DSE fungi can be readily iso- with the increase of Pb concentrations, indicating that lated from heavy metal-contaminated sites. Zhang et G. cylindrosporus had a high inherent tolerance or a al. (2008) isolated 3 DSE strains from a waste smelter low sensitivity to Pb. Arbuscular mycorrhizal fungi are 290 Y. H. BAN et al. known to have beneficial effects on the growth and pho- ctors; thus, it was considered to be a reliable indi- tosynthesis of host plants under heavy metals tress cator of stress (Figueroa et al., 1997). In this study, (Shahabivand et al., 2012; Rozp¸adek et al., 2014), Fv/Fm in the leaves of inoculated plants was found to whereas the knowledge of how these plants respond be significantly higher than that in the leaves of non- to inoculation with DSE fungi is presently limited. In inoculated plants. It might indicate that inoculation the present study, we found that the biomass and ot- of G. cylindrosporus could improve the resistence of her growth indicators of the inoculated maize increased maize to Pb stress and that the toxicity of heavy metal compared with those of the non-inoculated one un- to plant body was decreased obviously. qP and Y (II) der stress of different Pb concentrations. Host plant have been used as photochemical quenching parame- growth caused by inoculation with DSE fungi may be ters for monitoring the general trends in the energy a result of improved nutrient absorption (Jumpponen dissipation adjustments in PSII (Maxwell and Johnson, and Trappe, 1998; Narisawa et al., 2007). In this stu- 2000). The increases of qP and Y (II) in the inocula- dy, inoculation with G. cylindrosporus increased the ted maize suggested that inoculation with G. cylindro- ratio of root to shoot biomass compared with the sporus improved the efficiency of PSII photochemi- non-inoculated plants. It indicated that the inoculated stry. The parameter qN reflects activation of the non- plants invested more biomass on root production. The photochemical processes during the light period, most- infection of DSE fungi could change root morphology ly leading to the non-radiative energy dissipation to and structures and promote the development of late- heat (Björkmanand Demmig-Adams, 1995; Roháˇcek, ral and adventitious roots (Wu et al., 2010), followed 2002). qN will increase when the environmental con- by the improvement of root activity and the enhance- ditions become worse. For this study, qN of non-ino- ment of its ability to absorb nutrients and water. Se- culated seedlings was higher than that of inoculated cretion and secondary metabolites of DSE fungi may seedlings under different Pb concentrations, indicating also alter rhizosphere microenvironment and directly that inoculation by G. cylindrosporus could protect influence the growth of roots. Bartholdy et al. (2001) plant body from heavy metal toxicity, and the effects found that Phialocephala fortinii could synthesize hy- were significant. NPQ is an important parameter that droxamate siderophore, which increased the uptake of measures a change in the efficiency of heat dissipation Fe(III) by roots. Other researchers suggested that the relative to the dark-adapted state (Maxwell and John- increased synthesis of phytohormones (Oelmüller et son, 2000; Sheng et al., 2008). In the present study, al., 2009; Andrade-Linares et al., 2011) and enzymatic NPQ of inoculated maize was lower than that of non- degradation of main organic nutrients (Mandyam and inoculated maize, which indicated that the inoculated Jumpponen, 2005; Newsham, 2011) may contribute to plants have higher ability to protect leaves from light- the increase of root biomass. induced damage. Heavy metals have negative impacts on the physi- Arbuscular mycorrhizal fungi are known to have ology of plants, which can result in a decrease in the significant effects on the uptake and accumulation of concentrations of photosynthetic pigments, and da- heavy metals of host plants. However, the knowledge of mage the process of photosynthesis (Pajević et al., the effects of DSE inoculation on this field is present- 2009; Boriˇsev et al., 2012). In this study, inoculation ly limited. In this study, there was a decline in TF of G. cylindrosporus increased the contents of Chl a with increasing concentrations of Pb in the inocula- and total Chl in the leaves of maize under Pb stress. ted and non-inoculated plants. It was believed that Similar results were observed by Likar and Regvar most plants had the ability to transfer heavy metal ions (2013). However, the combination of Pb ions in roots from root to shoot; however, the translocation efficien- by DSE was the key mechanism for improving Pb to- cy of heavy metal was decreased with the increasing lerance of maize, in comparison with the protection of concentrations of heavy metals. Heavy metals have Chl, according to the results of Table V. negative impacts on the physiology of plants, which As so far, no research focused on the effect of can result in decreased transpiration and photosynthe- inoculation with DSE fungi on Chl fluorescence para- sis (Pajević et al., 2009; Boriˇsev et al., 2012). Some meters of host plants under heavy metal stress. Thus, researchers suggested that heavy metal translocation it is a first report that DSE fungi could mitigate the was driven by transpiration (Kuzkovina et al., 2004; toxic influence of heavy metal on PSII reaction cen- Likar and Regvar, 2013), so the decreased transpira- ter. Fv/Fm is a measure of the capacity of PSII prima- tion that caused by the toxicity of heavy metals may ry photochemistry, which itself is particularly sensi- lead to the decline of TF. In this study, the TF of tive to a variety of environmental stress-inducing fa- Pb in the inoculated plants was significantly decreased FUNGUS INOCULATION EFFECT ON MAIZE UNDER PB STRESS 291 compared to that of the non-inoculated plants. It was nken P. 2011. Effects of dark septate endophytes on tomato considered that the effect of inoculation with a DSE plant performance. Mycorrhiza. 21: 413–422. fungus on the uptake and translocation of heavy me- BajkánS, Várkonyi Z, Lehoczki E. 2012. Comparative study on energy partitioning in photosystem II of two Arabidopsis tha- tals in plants was probably due to the natural prop- liana mutants with reduced non-photochemical quenching erties and basic physiological properties of the DSE capacity. Acta Physiol Plant. 34: 1027–1034. fungus itself. Melanin in DSE hyphae was deemed to Ban Y, Tang M, Chen H, Xu Z, Zhang H, Yang Y. 2012. The response of dark septate endophytes (DSE) to heavy metals be the most important component of cell wall, and pos- in pure culture. PLoS ONE. 7: e47968. sessed many potential sites for metal binding. There- Ban Y, Xu Z, Zhang H, Chen H, Tang M. 2015. Soil chemistry fore, it has high capacities for metal biosorption, with properties, translocation of heavy metals, and mycorrhizal the majority of metal remaining within the wall struc- fungi associated with six plant species growing on lead-zinc mine tailings. Ann Microbiol. 65: 503–515. ture (Gadd, 2007; Li et al., 2012). Inoculation of DSE Bartholdy B A, Berreck M, Haselwandter K. 2001. Hydroxa- fungi can increase root dry weight and the number of mate siderophore synthesis by Phialocephala fortinii, a typi- hair root tips (Wu et al., 2010), so the metal absorption cal dark septate fungal root endophyte. Biometals. 14: 33– of roots became more sufficient and easy with the in- 42. Bilger W, BjörkmanO. 1991. Temperature dependence of violax- creases of surface area and root biomass. In addition, anthin de-epoxidation and non-photochemical fluorescence chelation of metals in soil by the extracellular secre- quenching in intact leaves of Gossypium hirsutum L. and tion of endophytic fungi or the exudates of inoculated Malva parviflora L. Planta. 184: 226–234. Bilger W, Schreiber U. 1987. Energy-dependent quenching of roots, and intracellular sequestration of metals were dark-level chlorophyll fluorescence in intact leaves. In Govin- also considered as important mechanisms for reducing djee, Barber J, Cramer W A, Goedheer J H C, Lavorel J, the mobility of heavy metals (Mandyam and Jumppo- Marcelle R, Zilinskas B A (eds.) Excitation Energy and Elec- nen, 2005; Saraswat and Rai, 2011; Leonhardt et al., tron Transfer in Photosynthesis. Springer, Netherlands. pp. 157–162. 2014). BjörkmanO, Demmig-Adams B. 1995. Regulation of photosynthetic light energy capture, conversion, and dissipation in CONCLUSIONS leaves of higher plants. In Schulze E D, Caldwell M M (eds.) Ecophysiology of Photosynthesis. Springer, Berlin, Heidel- Maize seedlings inoculated with G. cylindrosporus berg. pp. 17–47. had higher growth performance, compared with the BoriˇsevM, PajevićS, NikolićN, Borivoj K, Zupunskiˇ M, Kebert non-inoculated seedlings, under stress of different Pb M, PilipovićA, OrlovićS. 2012. Response of Salix alba L. to heavy metals and diesel fuel contamination. Afr J Biotech- concentrations. According to the measured results of nol. 11: 14313–14319. Chl content, photosynthetic characteristics, and Chl Deram A, Languereau-Leman F, Howsam M, Petit D, Haluwyn fluorescence parameters in leaves of maize under dif- C V. 2008. Seasonal patterns of cadmium accumulation in Arrhenatherum elatius (Poaceae): Influence of mycorrhizal ferent Pb concentrations, colonization of G. cylindro- and endophytic fungal colonisation. Soil Biol Biochem. 40: sporus could effectively decrease the damage of pho- 845–848. tosynthetic system caused by Pb stress. Colonization Figueroa M E, Fernández-BacoL, Luque T, Davy A J. 1997. of G. cylindrosporus could also decrease Pb accumula- Chlorophyll fluorescence, stress and survival in populations of Mediterranean grassland species. J Veg Sci. 8: 881–888. tion in the leaves of host plants under Pb stress, which Gadd G M. 2007. Geomycology: biogeochemical transformations mainly caused by the Pb biosorption of hyphae and the of rocks, minerals, metals and radionuclides by fungi, biowea- physiological change of host plants, resulting in the im- thering and bioremediation. Mycol Res. 111: 3–49. provement of Pb tolerance of host plants. Genty B, Briantais J M, Baker N R. 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys ACKNOWLEDGEMENT Acta. 990: 87–92. Gucwa-PrzepióraE, Chmura D, Sokolowska K. 2016. AM and This research was supported by the National Na- DSE colonization of invasive plants in urban habitat: a stu- tural Science Foundation of China (Nos. 41671268, 31- dy of Upper Silesia (southern Poland). J Plant Res. 129: 270639, and 31400435), the Program for Changjiang 603–614. Scholars and Innovative Research Team in University Hall J L. 2002. Cellular mechanisms for heavy metal detoxification and tolerance. J Exp Bot. 53: 1–11. of China (No. IRT1035), the Hubei Provincial Natural Hoagland D R, Arnon D I. 1950. The water-culture method for Science Foundation of China (No. 2015CFB596), and growing plants without soil. In California Agricultural Ex- the Shaanxi Provincial Science and Technology Inno- periment Station Circular 347. University of California, Cali- fornia Agricultural Experiment Station, Berkeley. pp. 1–32. vation Project Plan of China (No. 2016KTCL02-07). Jumpponen A R I, Trappe J M. 1998. Dark septate endophytes: A review of facultative biotrophic root-colonizing fungi. New REFERENCES Phytol. 140: 295–310. Kuzkovina Y A, Knee M, Quigley M F. 2004. Cadmium and cop- Andrade-Linares D R, Grosch R, Restrepo S, Krumbein A, Fra- per uptake and translocation in five willow (Salix L.) species. 292 Y. H. BAN et al.

Int J Phytoremediat. 6: 269–287. and willow (Salix spp.) clones in relation to photosynthesis. Leonhardt T, SáckýJ, Simekˇ P, Santr˚uˇcekJ,ˇ Kotrba P. 2014. Arch Biol Sci. 61: 239–247. Metallothionein-like peptides involved in sequestration of Zn Phillips J M, Hayman D S. 1970. Improved procedures for clea- in the Zn-accumulating ectomycorrhizal fungus Russula at- ring roots and staining parasitic and vesicular-arbuscular my- ropurpurea. Metallomics. 6: 1693–1701. corrhizal fungi for rapid assessment of infection. T Brit Mycol Li H Y, Li D W, He C M, Zhou Z P, Mei T, Xu H M. 2012. Diver- Soc. 55: 158–161. sity and heavy metal tolerance of endophytic fungi from six Regvar M, Likar M, Piltaver A, KugoniˇcN, Smith J E. 2010. dominant plant species in a Pb-Zn mine wasteland in China. Fungal community structure under goat willows (Salix caprea Fungal Ecol. 5: 309–315. L.) growing at metal polluted site: the potential of screening Li T, Liu M J, Zhang X T, Zhang H B, Sha T, Zhao Z W. 2011. in a model phytostabilisation study. Plant Soil. 330: 345– Improved tolerance of maize (Zea mays L.) to heavy metals 356. by colonization of a dark septate endophyte (DSE) Exophiala RoháˇcekK. 2002. Chlorophyll fluorescence parameters: the defi- pisciphila. Sci Total Environ. 409: 1069–1074. nitions, photosynthetic meaning, and mutual relationships. Likar M. 2011. Dark septate endophytes and mycorrhizal fungi Photosynthetica. 40: 13–29. of trees affected by pollution. In Maria P A, Frank A C (eds.) Rozp¸adekP, W¸e˙zowicz K, Stojakowska A, Malarz J, Surówka Endophytes of Forest Trees. Springer, Netherlands. pp. 189– E, SobczykL,Anielskaa T, Wa˙znyd R, Miszalskic Z, Turnau 201. K. 2014. Mycorrhizal fungi modulate phytochemical produc- Likar M, Regvar M. 2009. Application of temporal temperature tion and antioxidant activity of Cichorium intybus L. (Aste- gradient gel electrophoresis for characterisation of fungal en- raceae) under metal toxicity. Chemosphere. 112: 217–224. dophyte communities of Salix caprea L. in a heavy metal Saraswat S, Rai J P N. 2011. Mechanism of metal tolerance polluted soil. Sci Total Environ. 407: 6179–6187. and detoxification in mycorrhizal fungi. In Khan M S, Zai- di A, Goel R, Musarrat J (eds.) Biomanagement of Metal- Likar M, Regvar M. 2013. Isolates of dark septate endophytes Contaminated Soils. Springer, Netherlands. pp. 225–240. reduce metal uptake and improve physiology of Salix caprea Schreiber U, Schliwa U, Bilger W. 1986. Continuous recording L. Plant Soil. 370: 593–604. of photochemical and non-photochemical chlorophyll fluores- Mandyam K, Jumpponen A. 2005. Seeking the elusive function cence quenching with a new type of modulation fluorometer. of the root-colonising dark septate endophytic fungi. Stud Photosynth Res. 10: 51–62. Mycol. 53: 173–189. Selosse M A, Baudoin E, Vandenkoornhuyse P. 2004. Symbiotic Massenssini A M, Bonduki V H A, TótolaM R, Ferreira F A, microorganisms, a key for ecological success and protection Costa M D. 2014. Arbuscular mycorrhizal associations and of plants. CR Biol. 327: 639–648. occurrence of dark septate endophytes in the roots of Brazi- Shahabivand S, Maivan H Z, Goltapeh E M, Sharifi M, Aliloo A lian weed plants. Mycorrhiza. 24: 153–159. A. 2012. The effects of root endophyte and arbuscular myco- Maxwell K, Johnson G N. 2000. Chlorophyll fluorescence—a pra- rrhizal fungi on growth and cadmium accumulation in wheat ctical guide. J Exp Bot. 51: 659–668. under cadmium toxicity. Plant Physiol Biochem. 60: 53–58. McGonigle T P, Miller M H, Evans D G, Fairchild G L, Swan Sheng M, Tang M, Chen H, Yang B, Zhang F, Huang Y. 2008. J A. 1990. A new method which gives an objective measure Influence of arbuscular mycorrhizae on photosynthesis and of colonization of roots by vesicular—arbuscular mycorrhizal water status of maize plants under salt stress. Mycorrhiza. fungi. New Phytol. 115: 495–501. 18: 287–296. Nagajyoti P C, Lee K D, Sreekanth T V M. 2010. Heavy metals, Wu L Q, Lv Y L, Meng Z X, Chen J, Guo S X. 2010. The promot- occurrence and toxicity for plants: a review. Environ Chem ing role of an isolate of dark-septate fungus on its host plant Lett. 8: 199–216. Saussurea involucrata Kar. et Kir. Mycorrhiza. 20: 127–135. Narisawa K, Hambleton S, Currah R S. 2007. Heteroconium Zhang H H, Tang M, Chen H, Wang Y J. 2012. Effects of a dark- chaetospira, a dark septate root endophyte allied to the Her- septate endophytic isolate LBF-2 on the medicinal plant Ly- potrichiellaceae (Chaetothyriales) obtained from some forest cium barbarum L. J Microbiol. 50: 91–96. soil samples in Canada using bait plants. Mycoscience. 48: Zhang Y, Li T, Zhao Z W. 2013. Colonization characteristics and 274–281. composition of dark septate endophytes (DSE) in a lead and Newsham K K. 2011. A meta-analysis of plant responses to dark zinc slag heap in Southwest China. Soil Sediment Contam. septate root endophytes. New Phytol. 190: 783–793. 22: 532–545. OelmüllerR, Sherameti I, Tripathi S, Varma A. 2009. Pirifor- Zhang Y, Zhang Y, Liu M, Shi X, Zhao Z. 2008. Dark septate en- mospora indica, a cultivable root endophyte with multiple dophyte (DSE) fungi isolated from metal polluted soils: their biotechnological applications. Symbioses. 49: 1–17. taxonomic position, tolerance, and accumulation of heavy PajevićS, BoriˇsevM, NikolićN, KrstićB, PilipovićA, Orlović metals in vitro. J Microbiol. 46: 624–632. S. 2009. Phytoremediation capacity of poplar (Populus spp.)