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Effects of biochar on photosynthesis and antioxidative system of Malus hupehensis Rehd. seedlings under replant conditions
Scientia Horticulturae 175 (2014) 9–15
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Effects of biochar on photosynthesis and antioxidative system of Malus hupehensis Rehd. seedlings under replant conditions Yanfang Wang a,b,1 , Fengbing Pan a,1 , Gongshuai Wang a , Guodong Zhang a , Yanling Wang c , Xuesen Chen a , Zhiquan Mao a,∗ a
State Key Laboratory of Crop Biology/College of Horticultural Science and Engineering, Shandong Agricultural University, Tai’an 271018, Shandong, China College of Chemistry and Material Science, Shandong Agricultural University, Tai’an 271018, Shandong, China c School of Forestry, Shandong Agricultural University, Tai’an 271018, Shandong, China b
a r t i c l e
i n f o
Article history: Received 22 January 2014 Received in revised form 23 May 2014 Accepted 24 May 2014 Keywords: Biochar Malus hupehensis Rehd. Photosynthetic characteristic Antioxidant enzyme activities Replant soil
a b s t r a c t The purpose of this study was to investigate the mechanisms and effects of biochar on the plant growth of Malus hupehensis Rehd. seedlings under replant conditions. Before the M. hupehensis Rehd. seedlings were planted in pots, biochar was added to pots filled with replant soil at four rates: 0, 5, 20, 80 g kg−1 . The growth of seedlings was monitored with plant height and photosynthesis. The antioxidant enzyme activities, lipid peroxidation and osmotic regulation substance contents in seedlings leaves were also measured. The phenolic compounds in the four soil treatments were detected too. The results showed that the addition of biochar significantly decreased the contents of phenolic acids in replant soil through the sorption of biochar. In comparison with the control, biochar applied to replant soil at 80 g kg−1 enhanced the plant height, fresh weight, and photosynthetic parameters. Furthermore, seedlings in soil treated with biochar, particularly at 80 g kg−1 , exhibited higher activity of antioxidant enzymes including superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase. With the addition of biochar, the contents of malondialdehyde, O2 •− and H2 O2 significantly decreased, and the osmotic substances accumulation in leaves also declined. These results suggested that the addition of biochar can alleviate apple replant disease by activating antioxidant enzymes, decreasing lipid peroxidation, and significantly reducing the phenolic acids content of replant soil through the sorption of biochar. © 2014 Published by Elsevier B.V.
1. Introduction Apple replant disease (ARD) is a biological syndrome that occurs on sites previously planted to the same or a closely related tree species. ARD is common in all of the major apple-growing regions of the world (Mazzola and Manici, 2012). The problems are typically expressed as reduced plant growth and development, inhibited root system development, with a subsequent shortened production life and reduced yield (Manici et al., 2003; Mazzola and Manici, 2012). This disease syndrome has been described in China (Zhang et al., 2012), as well as many other parts of the world, including North America (Mazzola, 1998), New Zealand (Kandula et al.,
∗ Corresponding author at: Shandong Agricultural University, Daizong Road No.61, Tai’an 271018, Shandong, China. Tel.: +86 139 53822958/+86 538 8768246. E-mail address: [email protected] (Z. Mao). 1 Yanfang Wang and Fengbing Pan contributed equally to this work. http://dx.doi.org/10.1016/j.scienta.2014.05.029 0304-4238/© 2014 Published by Elsevier B.V.
2010), South Africa (Van Schoor et al., 2009) and Tasmania (Wilson et al., 2004). Accumulated research results attributed ARD to a consortium of abiotic elements and biotic forces (Bai et al., 2009; Tewoldemedhin et al., 2011b). Abiotic factors such as soil structure, nutrition, and the release of allelochemicals through leaching, root exudation, volatilization, and/or decomposition of residues played roles in replant problems (Hofmann et al., 2012; Zhang et al., 2007). Studies have shown that allelochemicals enhance superoxide radical (O2 •− ), H2 O2 and MDA levels and increased membrane leakage in the plant tissues (Ding et al., 2007; Gao et al., 2010). Biotic factors included nematodes, oomycetes bacteria, and fungi species (Mazzola, 1998; Manici et al., 2003; Tewoldemedhin et al., 2011a; Van Schoor et al., 2009). Chemical fungicides have been widely used in controlling ARD. Although fungicides seem to be effective, they are not environmentally friendly. Diverse soil amendments have been used to control the soil disease. The development of biocontrol (Ju et al., 2013; Utkhede and Smith, 2000), applying organic matter to soils (Mazzola and Brown, 2010; Zhang et al., 2012), and resistant cultivars (Laurent et al., 2010) are attractive strategies to manage ARD.
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Table 1 Chemical and physical characteristics of the biochar applied. Value Total C (g kg−1 ) Total N (g kg−1 ) Available N (mg kg−1 ) Available P (mg kg−1 ) Total P (mg kg−1 ) Total Ca (g kg−1 ) Total K (g kg−1 ) Total Mg (g kg−1 ) pH (H2 O) Max water absorption (g g−1 ) Cation exchange capacity (mmolc kg−1 )
741.6 2.1 21.4 52.7 453 5.8 7.6 2.4 8.6 3.8 51.7
K (mg kg−1 ), 28.39; organic matter (mg kg−1 ), 23.1; pH 5.9. The soil was predicted to be sensible to replant disease based on the standard method of ARD bioassays (Schoor et al., 2009). In brief, the replant soil (100 kg) was sterilized in high-pressure steam sterilizer (TOMY SX-500) at 121 ◦ C for 1 h while untreated replant soil (100 kg) was used as the control. Each treatment had 10 clay pots (inner diameter of 25 cm) and each pot was filled with 7 kg soil. Two six-leaf-seedlings were planted in each pot. After 7 weeks, plants were collected, washed with tap water to remove the rootattached soils, and dried with filter paper. The fresh and dry weight of the plant and the height of seedling were measured to assess the presence of ARD in the replant soil. 2.2. Experimental design
Biochar is a coproduct which results from pyrolysis of plant residue, crop straw, wood chips and other wastes under hightemperature and low oxygen conditions (Laird et al., 2009). Studies have shown that biochar amendment in soil can improve plant productivity (Asai et al., 2009; Graber et al., 2010) by improving soil chemical, physical, and biological properties. Biochars of different biomass feedstocks and pyrolysis conditions have different physical and chemical properties (Keiluweit et al., 2010), and have strong adsorption toward the organic compounds (Zhu and Pignatello, 2005). Replant soil have many allelopathic toxins, such as phlorizin, p-hydroxybenzoic acid, cinnamic acid, and phthalic acid (Bai et al., 2009; Ding et al., 2007; Gao et al., 2010). Thus, the adsorption of biochar to allelochemicals may make it useful for mitigating the ARD. Up to date, there has been no study concerning the biochar effect on the photosynthesis of Malus hupehensis Rehd. seedlings under replant conditions. This study investigates the effects of biochar on the concentration of phenolic compounds in replant soil and the photosynthesis and antioxidative system in leaves of M. hupehensis Rehd. seedlings under replant conditions. By testing the effects of biochar on replant soil and subsequent plant growth, we seek to further understand biochar’s effect on the tolerance mechanisms in M. hupehensis Rehd. seedlings and to provide a theoretical and practical means to alleviate ARD. 2. Materials and methods 2.1. Experimental materials The seedlings of M. hupehensis Rehd., the commonly used rootstock for apple trees, were used in this study. The seeds were stratified at 4 ◦ C for 30 days to sprout, and then the sprouts were sown in nursery plates. When the seedlings reached six leaves (about two months), uniform seedlings were planted into pots filled with the different treatment soils. Biochar used in this experiment was produced with pyrolysis of rice husk at 450 ◦ C under anaerobic condition by Xuancheng Jiale Rice Industry Co. Ltd. (Anhui, China). At the time of application, the biochar was screened with a 2 mm sieve. The chemical and physical characteristics of the biochar are listed in Table 1. The biochar’s Cation Exchange Capacity and maximum water absorption were determined according to the method of Chapman (1965) and Vaccari et al. (2011), respectively. Soil used for this experiment was obtained from a 50-year-old apple orchard located in a suburb of Taian city, Shandong province of China. The soil was randomly collected from multiple locations in the orchard (locations were 2 m away from the trunk) at a soil depth of 5–40 cm. Roots and visible plant residues were removed, and the soil samples were air-dried and mixed well. Soil was sandy loam with the following characteristics: NO3 N (mg kg−1 ), 7.8; NH4 N (mg kg−1 ), 5.1; available P (mg kg−1 ), 26.7; available
The replant soil were mixed with biochar at the rate of 0 (control), 5, 20, and 80 g kg−1 of soil. The biochar and replant soil were mixed well before filling the pots. There were 10 clay pots and each pot was filled with 7 kg of the mixture. Then water was applied to reach about 60% of the soil’s water holding capacity, and the water holding capacity of the soil was tested gravimetrically (Graber et al., 2010). Before planting, the pot was stabilized for 2 weeks and evaporated water was replenished. Seedlings of uniform size with six leaves were transplanted to pots with two seedlings per pot. The pots were arranged in a completely randomized design. Nutrients and water were managed following usual practice, and the N, P, and K contents in the soil were normalized among treatment pots. After the seedling plants grew for 3 months, the photosynthetic rate and plant height were measured and the soil samples were collected, air-dried, passed though a 1.7 mm sieve and stored for the allelochemicals analysis. At the same time, the leaf samples were frozen in liquid nitrogen and stored at −20 ◦ C until analysis. 2.3. Measurement of gas exchange and chlorophyll content All measurements were carried out between 08:30 and 11:30 on September 20, 2013. During the measurements, the relative humidity of the air, the temperature of leaves and the ambient CO2 concentration were about 75%, 28 ◦ C and 320–390 mol mol−1 , respectively. The net photosynthetic rate (Pn), stomatal conductance (Gs), and internal CO2 concentration (Ci) were measured on fully expanded leaves of M. hupehensis Rehd. seedlings using a portable photosynthesis system (Ciras-3, PP Systems, Hitchin, UK). These measurements were made on five randomly selected seedlings (two leaves per seedling) for each treatment and 3 replicates. Total chlorophyll were extracted by grinding leaves (0.2 g) in 80% acetone in the dark and calculated from the absorbance of extract at 663 and 645 nm using the equations of Lichtenthaler (1987), and expressed as mg g−1 FW (fresh weight). 2.4. Measurement of antioxidant enzymes Antioxidant enzymes (SOD, POD, CAT, and APX) were extracted according to the method of Kazemi et al. (2010). Leaf samples (1.0 g) were homogenized in 8 mL of 50 mmol L−1 phosphate buffer (pH 7.8) containing 1% polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 12,500 × g for 20 min at 4 ◦ C, and the supernatant was used for the enzyme activity measurement. All assays were carried out at 2–4 ◦ C. Superoxide dismutase (SOD) activity was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) (Bai et al., 2009), One unit (U) of SOD activity was defined as the amount of enzyme causing 50% of the maximum inhibition of nitroblue tetrazolium (NBT) reduction, and the enzyme activity was expressed as U g−1 FW. Peroxidase (POD) activity
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assay was monitored by changes in absorbance at 470 nm (Omran, 1980). The activity of catalase (CAT) was determined by changes in absorbance at 240 nm (Singh et al., 2010). Ascorbate peroxidase (APX) was measured by monitoring the decrease in absorbance at 290 nm (Nakano and Asada, 1981). One unit (U) of POD, CAT and APX activity was defined as the amount of enzyme causing a change of 0.1 in the absorbance at 470, 240 and 290 nm min−1 , respectively, and the enzyme activity was expressed as U g−1 FW min−1 . 2.5. Measurement of MDA, H2 O2 , and O2 •− Lipid peroxidation in leaf tissue was determined by measuring malondialdehyde (MDA) accumulation using the procedures described by Bai et al. (2009). Leaf samples (0.5 g) were homogenized in 5 mL of 0.1% (w/v) trichloroacetic acid in phosphate buffer (50 mmol L−1 , pH 7.8). The homogenization was centrifuged for 20 min at 12,500 × g and 4 ◦ C. The supernatant (1 mL) was added to 2 mL of 0.67% (w/v) thiobarbituric acid in 10% (w/v) trichloroacetic acid. After incubated in a water bath at 100 ◦ C for 15 min, the mixture was quickly cooled in an ice bath for 15 min, and then centrifuged at 12,000 × g and 4 ◦ C for 10 min. The absorbance of the supernatant was measured at 450, 532, and 600 nm. The content of MDA was determined using the extinction coefficient of 155 mmol−1 cm−1 and expressed as mmol g−1 FW. H2 O2 concentration was measured according to the method described by Patterson et al. (1984). In brief, leaf tissue (0.5 g) was homogenized in 5 mL precooled acetone and centrifuged for 10 min at 2000 × g and 4 ◦ C. Titanium sulfate (0.1%, w/v) and concentrated ammonia (0.2 mL) were added into the supernatant (1 mL). The mixture was allowed to react for10 min at 25 ◦ C and the reaction mixture was centrifuged at 2000 × g and 4 ◦ C for 10 min. Absorbance at 415 nm was measured, and the H2 O2 concentration was calculated according to a standard curve expressed as mol g−1 FW. The rate of O2 •− generation was measured as described by Bai et al. (2009) and expressed as mol h−1 g−1 FW. 2.6. Measurement of proline and soluble sugar contents Proline content was measured according to the method of Kazemi et al. (2010). Leaves tissue (0.5 g) was homogenized with 5 mL of 3% (w/v) aqueous sulfosalicylic acid. After incubated in a water bath at 100 ◦ C for 10 min., the homogenate was centrifuged at 10,000 × g and 4 ◦ C for 5 min. Two milliliters of supernatant were mixed with 2 mL of glacial acetic acid and 2 mL of acid ninhydrin for 40 min at 100 ◦ C. The developed color was extracted in 5 mL toluene and the absorbance was measured at 520 nm against toluene. A standard curve with l-proline was used for the final calculations. Content of proline was expressed as g g−1 FW. The content of soluble sugar was measured according to Irigoyen et al. (1992) and expressed as mg g−1 FW. 2.7. Extraction and analysis of the allelochemicals contents in the soil samples The allelochemicals in soil samples were extracted according to the method of He et al. (2009). The extracts were analyzed by Waters high-performance liquid chromatography (HPLC) equipped with a Waters e2695 pump and a 2998 photodiode array detector (PAD). The standard phenolic compounds (phloroglucinol, gallic acid, p-hydroxybenzoic acid, phthalic acid, vanillic aldehyde and phlorizin) were purchased from Sigma (St. Louis, MO). Solvents were HPLC spectral grade, and all solvents and distilled water were degassed before use. All separations were performed with a Symmetry C18 column (4.6 mm × 250 mm, 5.0 m) and the rate was kept constant at 1.0 mL min−1 . The injection volume was 10 L and
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Table 2 Growth response to sterilization of replant soil tested for apple replant disease. Treatments
Plant height (cm)
Fresh weight (g)
Dry weight (g)
Replant soil Sterilized soil
14.32 ± 0.65b 26.17 ± 1.05a
7.31 ± 0.83b 13.59 ± 0.97a
2.38 ± 0.16b 4.54 ± 0.25a
Data represents the average of three replicates ± SE. Different letters indicate significant difference at p < 0.05.
the column temperature was maintained at 30 ◦ C. Mobile phase A was acetonitrile, and mobile phase B was distilled water (regulated to pH 2.8 with glacial acetic acid). Both standard and extract compounds were used in the following gradient system: (1) from 0.0 to 35 min, a linear gradient from 5% to 35% A; (2) from 35 to 40 min, isocratic with 35% A; (3) from 40 to 42 min, a linear gradient from 35% to 5% A; (4) from 42 to 52 min, isocratic with 5% A. Detection was performed at 270 nm. The chromatographic data were record and processed with a Waters empower workstation. The concentration of phenolic acids in soil samples was obtained using peak area external standards and was expressed as g kg−1 of dry soil. 2.8. Statistical analysis The experimental data was expressed as means and standard errors (SE) with three biological replications. Statistical calculations were performed with SPSS-19 statistical software. Mean difference comparison among different treatments was done by ANOVA and Duncan’s multiple range test at a 0.05 probability level. 3. Results 3.1. The ARD detection of replant soil Compared with the control treatment, the sterilized soil increased the plant height and fresh and dry weight of apple seedlings by 83%, 86% and 91%, respectively (Table 2). The replant soil depressed the growth of seedling plants which indicated the presence of ARD in the soil. 3.2. Effects of biochar on the seedling plants growth in replant soil 3.2.1. Biomass of seedling plant The biomass of M. hupehensis Rehd. seedlings was determined on September 20, 2013. Compared to the control, biochar additions significantly increased plant height. The plant height was increased by 55%, 41% and 17% with 80, 20 and 5 g kg−1 biochar application (Fig. 1A). The fresh weight of plant seedlings increased as well with biochar addition, up to 100% respect to the control with the 80 g kg−1 biochar dose (Fig. 1B). 3.2.2. Chlorophyll content and photosynthetic rate in leaves of Malus hupehensis Rehd. seedlings Compared to the control (without biochar), Pn (Fig. 2A) and Gs (Fig. 2C) increased remarkably under replant conditions with high biochar application rates. Versus the control, the Pn improved by 19% and 35% biochar applied at rates of 20 and 80 g kg−1 , respectively. Versus the control, the Gs improved by 31% and 47% with biochar applied at rates of 20 and 80 g kg−1 , respectively. There was no significant difference in Pn and Gs between the seedling plants treated with 5 g kg−1 biochar and the control. Treatments with 20 and 80 g kg−1 biochar decreased Ci by 9% and 19%, respectively (Fig. 2B). In addition, the chlorophyll content was significantly improved by the addition of biochar (Fig. 2D), showing an increase by 16.0%, 25.4%, and 31.5% with 5, 20, and 80 g kg−1 biochar addition, respectively.
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Fig. 1. Effects of biochar on the plant height and fresh weight of Malus hupehensis Rehd. seedlings under replant condition. Different letters indicate significant difference between treatments at p < 0.05, according to Duncan’s multiple range test.
3.2.3. Antioxidant enzyme activities in leaves of Malus hupehensis Rehd. seedlings The trend indicates that the application of biochar to replant soil enhanced the antioxidant enzyme activities in the leaves of M. hupehensis Rehd. seedlings under replant condition (Fig. 3). With the highest biochar application rate at 80 g kg−1 , the SOD activity was improved by as much as 12% (Fig. 3A). The POD activity also significantly increased from 18% with the 5 g kg−1 biochar additions to 77% with 80 g kg−1 biochar additions (Fig. 3B). Similar trends in response to biochar additions were observed in CAT and APX activity (Fig. 3C and D). In general, enzyme activities in the leaves increased progressively with the biochar additions in comparison to the control (without biochar). 3.2.4. MDA, H2 O2 , O2 •− , proline and soluble sugar content in leaves of Malus hupehensis Rehd. seedlings MDA concentration in leaves of M. hupehensis Rehd. seedlings decreased significantly in biochar application than that of the control (Table 3). The maximum decrease in MDA concentration was
observed in the treatment with 80 g kg−1 biochar, which was only 53% that of the control. Biochar application also was associated with decreases in H2 O2 content, O2 •− generation rate, proline and soluble sugar concentration in leaves of seedlings. Comparison with the control, the maximum decrease in H2 O2 content, O2 •− generation rate, proline and soluble sugar concentration (to 25%, 71%, 33% and 51%, respectively) was found in the treatment with 80 g kg−1 biochar.
3.2.5. Effect of biochar on the concentration of phenolic acids in replant soil In the replant soil with and without biochar, the phenolic acids were measured by HPLC (Table 4). Specifically, the concentration of phenolic compounds was the most abundant in the control soil, and sharply declined with the addition of biochar. The maximum decreases in the concentrations of phloroglucinol, gallic acid, p-hydroxybenzoic acid, phthalic acid, vanillic aldehyde, and phlorizin – which were 42%, 29%, 17%, 35%, 34%, and 24% that of the
Fig. 2. Effects of biochar on net photosynthetic rate (Pn), stomatal conductance (Gs), internal CO2 concentration (Ci) and total chlorophyll in leaves of Malus hupehensis Rehd. seedlings under replant condition. Different letters indicate significant difference between treatments at p < 0.05, according to Duncan’s multiple range test.
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Fig. 3. Effects of biochar on the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) in leaves of Malus hupehensis Rehd. seedlings under replant condition. Different letters indicate significant difference between treatments at p < 0.05, according to Duncan’s multiple range test.
Table 3 The effect of biochar on MDA, H2 O2 , O2 •− , and proline content in the leaves of M. hupehensis Rehd. seedlings under replant condition. Biochar applied (g kg−1 )
MDA (mmol g−1 FW)
0 5 20 80
11.14 10.71 8.49 5.90
± ± ± ±
0.070a 0.17b 0.13c 0.090d
H2 O2 (mol g−1 FW) 0.61 0.46 0.26 0.15
± ± ± ±
0.013a 0.016b 0.008c 0.009d
O2 •− (mol g−1 FW h−1 ) 0.44 0.39 0.38 0.31
± ± ± ±
0.024a 0.013b 0.010b 0.091c
Proline (g g−1 FW) 200.01 145.71 91.89 65.34
± ± ± ±
3.21a 3.82b 3.09c 7.73d
Soluble sugar (mg g−1 FW) 31.63 27.88 25.64 16.19
± ± ± ±
1.18a 0.42b 1.24b 0.36c
Data represents the average of three replicates ± SE. Different letters indicate significant difference at p < 0.05.
Table 4 Concentration of six phenolic compounds in four treatment soil. Compounds g kg−1 dry soil
Biochar applied mg kg−1 0
Phloroglucinol Gallic acid p-Hydroxybenzoic acid Phthalic acid Vanillic aldehyde Phlorizin
1041.34 18.42 31.81 689.64 16.98 31.41
5 ± ± ± ± ± ±
193.43a 1.69a 5.37a 26.95a 2.43a 6.93a
20
760.95 10.01 14.28 472.18 12.6 13.45
± ± ± ± ± ±
99.48b 1.92b 1.87b 30.18b 1.15b 1.37b
685.71 7.72 8.29 388.29 9.31 10.50
80 ± ± ± ± ± ±
153.54bc 0.84b 0.95c 21.58c 1.83bc 1.89bc
441.83 5.40 5.33 244.5 5.85 7.42
± ± ± ± ± ±
95.11c 0.52b 0.79c 7.03d 0.98c 1.87c
Data represents the average of three replicates ± SE. Different letters indicate significant difference at p < 0.05.
control, respectively – were observed in the treatment with 80 g kg−1 biochar.
4. Discussion The present results showed that the growth of M. hupehensis Rehd. seedlings in sterilized soils was significantly higher than that of seedlings in the replant soil (Table 2), which suggests that ARD could reduce the growth of M. hupehensis Rehd. seedlings. Biochar has been reported to improve plant growth and yield when it was applied from less than 1 to over 100 t ha−1 (Graber et al., 2010; Major et al., 2010). In this study, the application of 5 g kg−1 biochar is equivalent to an application of 10.1 t ha−1 if calculated for the plough layer of 0.15 m with an average bulk density of 1.35 g cm−3 . The application of biochar to replant soil improved the plant height
and fresh weight of M. hupehensis Rehd. seedlings (Fig. 1), which was consistent with previous report of the beneficial aspects of biochar (Elmer and Pignatello, 2011). Biochar also significantly increased the photosynthetic rate and the chlorophyll content in the leaves of Malus Hupehensis Rehd. seedlings (Fig. 2). In the control plants, the reduced chlorophyll concentrations in the leaves could result from suppressed levels of specific enzymes that are required for the synthesis of pigments, suppressed levels in the uptake of the minerals (e.g. Mg) necessary for chlorophyll synthesis, or the degradation of chlorophyll induced by ROS (Kazemi et al., 2010; Rondon et al., 2007). The application of biochar to replant soil significantly reduced the concentrations of phloroglucinol, gallic acid, p-hydroxybenzoic acid, phthalic acid, vanillic aldehyde, and phlorizin (Table 4). These results could be caused by the sorption of biochar to the phenolic compounds making the phenolic acids ineffective in suppressing neighboring, similar plants (Lehmann et al.,
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2011). In this study, biochar effectively reduced the production of ROS under replant condition (Table 3) to alleviate damage of plant growth and chlorophyll content caused by ROS. In addition, the results suggested that the biochar could trigger the activities of specific enzymes, or cause an increase in the uptake of the necessary minerals (Rondon et al., 2007). Biochar application may also substantially modify soil field capacity (Glaser et al., 2002). For example, the use of biochar in vineyard improved soil water content and then increased plant available water and photosynthetic activity in leaves of Vitis vinifera (L.) (Baronti et al., 2014). Our study supported the results that the addition of biochar increased water availability that is responsible for an increase in Pn and Gs. A similar effect of biochar on soil water availability also has been reported in previous studies (Basso et al., 2013; Kammann et al., 2011). Under normal growth conditions, the production and detoxification of free radicals in tissue cells exist a dynamic equilibrium. Abiotic and biotic stresses can break the equilibrium and cause the formation and accumulation of ROS, including O2 •− , H2 O2 and OH· , which are commonly generated via number of metabolic pathways (Kanazawa et al., 2000). Excessive production of ROS may lead to oxidative stress, DNA and protein damage, membrane permeability, lipid peroxidation, loss of cell function and, ultimately, cell death (Ding et al., 2007). To scavenge ROS and to avoid oxidative stress, plants have antioxidant defense enzymes such as SOD, CAT, POD and APX (Kanazawa et al., 2000). In this study, enhanced O2 •− and H2 O2 (Table 3) suggested that the replant condition induced oxidative stress, while the addition of biochar decreased O2 •− and H2 O2 contents in paralled with the increase in SOD, CAT, POD and APX activities (Fig. 3) in leaves of M. hupehensis Rehd. seedlings. Antioxidant enzyme are important for the removal of excessive ROS in plant cell (Jaleel et al., 2007). Many studies have shown that the application of organic amendments improved the systematic resistance of plants (Ju et al., 2013; Zhang et al., 2012). This study’s results revealed that the application of biochar increased the activities of SOD, CAT, POD and APX in leaves of seedlings. MDA accumulation caused by lipid peroxidation has been reported in response to abiotic and biotic stress (Apel and Hirt, 2004). MDA contents is associated with the O2 •− and H2 O2 production. The level of MDA indicated the oxidative damage of cell membranes during lipid peroxidation. In our study, the O2 •− , H2 O2 and MDA concentration in leaves of M. hupehensis Rehd. seedlings were high in control treatment (Table 3), and the biochar applied to replant soil reduced the concentration of MDA, O2 •− and H2 O2 . It indicated that biochar was useful to enhance plant antioxidant capability and it alleviated damage to plant from the oxidative stress by reducing the concentration of phenolic acids. Studies have shown that plants resist the environmental stress induce production of ROS by increasing their intrinsic defensive system (Alscher et al., 2002; Salin, 1988). Plant subjected to environmental stress could accumulate osmotic substances in cells (Akcay et al., 2010; Kazemi et al., 2010), such as proline, soluble sugar and various betaines which can function as osmoprotectants. As with other studies on the effects of stress on plants (Akcay et al., 2010; Kazemi et al., 2010), we found the accumulation of proline and soluble sugar in M. hupehensis Rehd. seedlings was caused by ARD stress. The accumulation of proline may be due to the mechanisms of osmoregulaion, and the response of plant under ARD stress may be correlated with their antioxidative system (Kazemi et al., 2010). The application of biochar to replant soil probably alleviated the apple replant disease stress probably via the sorption of aromatic acids which had allelopathic effects and were abiotic factors for ARD (Lehmann et al., 2011). Thus, the application of biochar markedly increased antioxidant capability, and decreased membrane permeability and the contents of proline and soluble sugar in the plants.
It is interesting that biochar may alleviate ARD stress in M. hupehensis Rehd. seedlings. The addition of biochar reduced the concentrations of phenolic compounds, and osmotic adjustment substances. Biochar addition significantly alleviated the decline in plant height, chlorophyll content, and Pn. It also increased the activities of antioxidant enzyme and inhibited lipid peroxidation in plant cells. The presented results supported the view that biochar can contribute to protect M. hupehensis Rehd. seedlings against ARD by alleviating the ARD induced oxidative stress. However, further works should be carried out to study the effects of biochar on the soil microbial community and the relationship between the soil microbial and phenolic acids under replant condition.
Acknowledgments The research was supported by the earmarked fund for National Modern Agro-industry Technology Research System (CARS-28), and Program for Changjiang Scholars and Innovative Team in university (IRT1155).
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Effects of biochar on photosynthesis and antioxidative system of Malus hupehensis Rehd. seedlings under replant conditions Yanfang Wang a,b,1 , Fengbing Pan a,1 , Gongshuai Wang a , Guodong Zhang a , Yanling Wang c , Xuesen Chen a , Zhiquan Mao a,∗ a
State Key Laboratory of Crop Biology/College of Horticultural Science and Engineering, Shandong Agricultural University, Tai’an 271018, Shandong, China College of Chemistry and Material Science, Shandong Agricultural University, Tai’an 271018, Shandong, China c School of Forestry, Shandong Agricultural University, Tai’an 271018, Shandong, China b
a r t i c l e
i n f o
Article history: Received 22 January 2014 Received in revised form 23 May 2014 Accepted 24 May 2014 Keywords: Biochar Malus hupehensis Rehd. Photosynthetic characteristic Antioxidant enzyme activities Replant soil
a b s t r a c t The purpose of this study was to investigate the mechanisms and effects of biochar on the plant growth of Malus hupehensis Rehd. seedlings under replant conditions. Before the M. hupehensis Rehd. seedlings were planted in pots, biochar was added to pots filled with replant soil at four rates: 0, 5, 20, 80 g kg−1 . The growth of seedlings was monitored with plant height and photosynthesis. The antioxidant enzyme activities, lipid peroxidation and osmotic regulation substance contents in seedlings leaves were also measured. The phenolic compounds in the four soil treatments were detected too. The results showed that the addition of biochar significantly decreased the contents of phenolic acids in replant soil through the sorption of biochar. In comparison with the control, biochar applied to replant soil at 80 g kg−1 enhanced the plant height, fresh weight, and photosynthetic parameters. Furthermore, seedlings in soil treated with biochar, particularly at 80 g kg−1 , exhibited higher activity of antioxidant enzymes including superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase. With the addition of biochar, the contents of malondialdehyde, O2 •− and H2 O2 significantly decreased, and the osmotic substances accumulation in leaves also declined. These results suggested that the addition of biochar can alleviate apple replant disease by activating antioxidant enzymes, decreasing lipid peroxidation, and significantly reducing the phenolic acids content of replant soil through the sorption of biochar. © 2014 Published by Elsevier B.V.
1. Introduction Apple replant disease (ARD) is a biological syndrome that occurs on sites previously planted to the same or a closely related tree species. ARD is common in all of the major apple-growing regions of the world (Mazzola and Manici, 2012). The problems are typically expressed as reduced plant growth and development, inhibited root system development, with a subsequent shortened production life and reduced yield (Manici et al., 2003; Mazzola and Manici, 2012). This disease syndrome has been described in China (Zhang et al., 2012), as well as many other parts of the world, including North America (Mazzola, 1998), New Zealand (Kandula et al.,
∗ Corresponding author at: Shandong Agricultural University, Daizong Road No.61, Tai’an 271018, Shandong, China. Tel.: +86 139 53822958/+86 538 8768246. E-mail address: [email protected] (Z. Mao). 1 Yanfang Wang and Fengbing Pan contributed equally to this work. http://dx.doi.org/10.1016/j.scienta.2014.05.029 0304-4238/© 2014 Published by Elsevier B.V.
2010), South Africa (Van Schoor et al., 2009) and Tasmania (Wilson et al., 2004). Accumulated research results attributed ARD to a consortium of abiotic elements and biotic forces (Bai et al., 2009; Tewoldemedhin et al., 2011b). Abiotic factors such as soil structure, nutrition, and the release of allelochemicals through leaching, root exudation, volatilization, and/or decomposition of residues played roles in replant problems (Hofmann et al., 2012; Zhang et al., 2007). Studies have shown that allelochemicals enhance superoxide radical (O2 •− ), H2 O2 and MDA levels and increased membrane leakage in the plant tissues (Ding et al., 2007; Gao et al., 2010). Biotic factors included nematodes, oomycetes bacteria, and fungi species (Mazzola, 1998; Manici et al., 2003; Tewoldemedhin et al., 2011a; Van Schoor et al., 2009). Chemical fungicides have been widely used in controlling ARD. Although fungicides seem to be effective, they are not environmentally friendly. Diverse soil amendments have been used to control the soil disease. The development of biocontrol (Ju et al., 2013; Utkhede and Smith, 2000), applying organic matter to soils (Mazzola and Brown, 2010; Zhang et al., 2012), and resistant cultivars (Laurent et al., 2010) are attractive strategies to manage ARD.
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Table 1 Chemical and physical characteristics of the biochar applied. Value Total C (g kg−1 ) Total N (g kg−1 ) Available N (mg kg−1 ) Available P (mg kg−1 ) Total P (mg kg−1 ) Total Ca (g kg−1 ) Total K (g kg−1 ) Total Mg (g kg−1 ) pH (H2 O) Max water absorption (g g−1 ) Cation exchange capacity (mmolc kg−1 )
741.6 2.1 21.4 52.7 453 5.8 7.6 2.4 8.6 3.8 51.7
K (mg kg−1 ), 28.39; organic matter (mg kg−1 ), 23.1; pH 5.9. The soil was predicted to be sensible to replant disease based on the standard method of ARD bioassays (Schoor et al., 2009). In brief, the replant soil (100 kg) was sterilized in high-pressure steam sterilizer (TOMY SX-500) at 121 ◦ C for 1 h while untreated replant soil (100 kg) was used as the control. Each treatment had 10 clay pots (inner diameter of 25 cm) and each pot was filled with 7 kg soil. Two six-leaf-seedlings were planted in each pot. After 7 weeks, plants were collected, washed with tap water to remove the rootattached soils, and dried with filter paper. The fresh and dry weight of the plant and the height of seedling were measured to assess the presence of ARD in the replant soil. 2.2. Experimental design
Biochar is a coproduct which results from pyrolysis of plant residue, crop straw, wood chips and other wastes under hightemperature and low oxygen conditions (Laird et al., 2009). Studies have shown that biochar amendment in soil can improve plant productivity (Asai et al., 2009; Graber et al., 2010) by improving soil chemical, physical, and biological properties. Biochars of different biomass feedstocks and pyrolysis conditions have different physical and chemical properties (Keiluweit et al., 2010), and have strong adsorption toward the organic compounds (Zhu and Pignatello, 2005). Replant soil have many allelopathic toxins, such as phlorizin, p-hydroxybenzoic acid, cinnamic acid, and phthalic acid (Bai et al., 2009; Ding et al., 2007; Gao et al., 2010). Thus, the adsorption of biochar to allelochemicals may make it useful for mitigating the ARD. Up to date, there has been no study concerning the biochar effect on the photosynthesis of Malus hupehensis Rehd. seedlings under replant conditions. This study investigates the effects of biochar on the concentration of phenolic compounds in replant soil and the photosynthesis and antioxidative system in leaves of M. hupehensis Rehd. seedlings under replant conditions. By testing the effects of biochar on replant soil and subsequent plant growth, we seek to further understand biochar’s effect on the tolerance mechanisms in M. hupehensis Rehd. seedlings and to provide a theoretical and practical means to alleviate ARD. 2. Materials and methods 2.1. Experimental materials The seedlings of M. hupehensis Rehd., the commonly used rootstock for apple trees, were used in this study. The seeds were stratified at 4 ◦ C for 30 days to sprout, and then the sprouts were sown in nursery plates. When the seedlings reached six leaves (about two months), uniform seedlings were planted into pots filled with the different treatment soils. Biochar used in this experiment was produced with pyrolysis of rice husk at 450 ◦ C under anaerobic condition by Xuancheng Jiale Rice Industry Co. Ltd. (Anhui, China). At the time of application, the biochar was screened with a 2 mm sieve. The chemical and physical characteristics of the biochar are listed in Table 1. The biochar’s Cation Exchange Capacity and maximum water absorption were determined according to the method of Chapman (1965) and Vaccari et al. (2011), respectively. Soil used for this experiment was obtained from a 50-year-old apple orchard located in a suburb of Taian city, Shandong province of China. The soil was randomly collected from multiple locations in the orchard (locations were 2 m away from the trunk) at a soil depth of 5–40 cm. Roots and visible plant residues were removed, and the soil samples were air-dried and mixed well. Soil was sandy loam with the following characteristics: NO3 N (mg kg−1 ), 7.8; NH4 N (mg kg−1 ), 5.1; available P (mg kg−1 ), 26.7; available
The replant soil were mixed with biochar at the rate of 0 (control), 5, 20, and 80 g kg−1 of soil. The biochar and replant soil were mixed well before filling the pots. There were 10 clay pots and each pot was filled with 7 kg of the mixture. Then water was applied to reach about 60% of the soil’s water holding capacity, and the water holding capacity of the soil was tested gravimetrically (Graber et al., 2010). Before planting, the pot was stabilized for 2 weeks and evaporated water was replenished. Seedlings of uniform size with six leaves were transplanted to pots with two seedlings per pot. The pots were arranged in a completely randomized design. Nutrients and water were managed following usual practice, and the N, P, and K contents in the soil were normalized among treatment pots. After the seedling plants grew for 3 months, the photosynthetic rate and plant height were measured and the soil samples were collected, air-dried, passed though a 1.7 mm sieve and stored for the allelochemicals analysis. At the same time, the leaf samples were frozen in liquid nitrogen and stored at −20 ◦ C until analysis. 2.3. Measurement of gas exchange and chlorophyll content All measurements were carried out between 08:30 and 11:30 on September 20, 2013. During the measurements, the relative humidity of the air, the temperature of leaves and the ambient CO2 concentration were about 75%, 28 ◦ C and 320–390 mol mol−1 , respectively. The net photosynthetic rate (Pn), stomatal conductance (Gs), and internal CO2 concentration (Ci) were measured on fully expanded leaves of M. hupehensis Rehd. seedlings using a portable photosynthesis system (Ciras-3, PP Systems, Hitchin, UK). These measurements were made on five randomly selected seedlings (two leaves per seedling) for each treatment and 3 replicates. Total chlorophyll were extracted by grinding leaves (0.2 g) in 80% acetone in the dark and calculated from the absorbance of extract at 663 and 645 nm using the equations of Lichtenthaler (1987), and expressed as mg g−1 FW (fresh weight). 2.4. Measurement of antioxidant enzymes Antioxidant enzymes (SOD, POD, CAT, and APX) were extracted according to the method of Kazemi et al. (2010). Leaf samples (1.0 g) were homogenized in 8 mL of 50 mmol L−1 phosphate buffer (pH 7.8) containing 1% polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 12,500 × g for 20 min at 4 ◦ C, and the supernatant was used for the enzyme activity measurement. All assays were carried out at 2–4 ◦ C. Superoxide dismutase (SOD) activity was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) (Bai et al., 2009), One unit (U) of SOD activity was defined as the amount of enzyme causing 50% of the maximum inhibition of nitroblue tetrazolium (NBT) reduction, and the enzyme activity was expressed as U g−1 FW. Peroxidase (POD) activity
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assay was monitored by changes in absorbance at 470 nm (Omran, 1980). The activity of catalase (CAT) was determined by changes in absorbance at 240 nm (Singh et al., 2010). Ascorbate peroxidase (APX) was measured by monitoring the decrease in absorbance at 290 nm (Nakano and Asada, 1981). One unit (U) of POD, CAT and APX activity was defined as the amount of enzyme causing a change of 0.1 in the absorbance at 470, 240 and 290 nm min−1 , respectively, and the enzyme activity was expressed as U g−1 FW min−1 . 2.5. Measurement of MDA, H2 O2 , and O2 •− Lipid peroxidation in leaf tissue was determined by measuring malondialdehyde (MDA) accumulation using the procedures described by Bai et al. (2009). Leaf samples (0.5 g) were homogenized in 5 mL of 0.1% (w/v) trichloroacetic acid in phosphate buffer (50 mmol L−1 , pH 7.8). The homogenization was centrifuged for 20 min at 12,500 × g and 4 ◦ C. The supernatant (1 mL) was added to 2 mL of 0.67% (w/v) thiobarbituric acid in 10% (w/v) trichloroacetic acid. After incubated in a water bath at 100 ◦ C for 15 min, the mixture was quickly cooled in an ice bath for 15 min, and then centrifuged at 12,000 × g and 4 ◦ C for 10 min. The absorbance of the supernatant was measured at 450, 532, and 600 nm. The content of MDA was determined using the extinction coefficient of 155 mmol−1 cm−1 and expressed as mmol g−1 FW. H2 O2 concentration was measured according to the method described by Patterson et al. (1984). In brief, leaf tissue (0.5 g) was homogenized in 5 mL precooled acetone and centrifuged for 10 min at 2000 × g and 4 ◦ C. Titanium sulfate (0.1%, w/v) and concentrated ammonia (0.2 mL) were added into the supernatant (1 mL). The mixture was allowed to react for10 min at 25 ◦ C and the reaction mixture was centrifuged at 2000 × g and 4 ◦ C for 10 min. Absorbance at 415 nm was measured, and the H2 O2 concentration was calculated according to a standard curve expressed as mol g−1 FW. The rate of O2 •− generation was measured as described by Bai et al. (2009) and expressed as mol h−1 g−1 FW. 2.6. Measurement of proline and soluble sugar contents Proline content was measured according to the method of Kazemi et al. (2010). Leaves tissue (0.5 g) was homogenized with 5 mL of 3% (w/v) aqueous sulfosalicylic acid. After incubated in a water bath at 100 ◦ C for 10 min., the homogenate was centrifuged at 10,000 × g and 4 ◦ C for 5 min. Two milliliters of supernatant were mixed with 2 mL of glacial acetic acid and 2 mL of acid ninhydrin for 40 min at 100 ◦ C. The developed color was extracted in 5 mL toluene and the absorbance was measured at 520 nm against toluene. A standard curve with l-proline was used for the final calculations. Content of proline was expressed as g g−1 FW. The content of soluble sugar was measured according to Irigoyen et al. (1992) and expressed as mg g−1 FW. 2.7. Extraction and analysis of the allelochemicals contents in the soil samples The allelochemicals in soil samples were extracted according to the method of He et al. (2009). The extracts were analyzed by Waters high-performance liquid chromatography (HPLC) equipped with a Waters e2695 pump and a 2998 photodiode array detector (PAD). The standard phenolic compounds (phloroglucinol, gallic acid, p-hydroxybenzoic acid, phthalic acid, vanillic aldehyde and phlorizin) were purchased from Sigma (St. Louis, MO). Solvents were HPLC spectral grade, and all solvents and distilled water were degassed before use. All separations were performed with a Symmetry C18 column (4.6 mm × 250 mm, 5.0 m) and the rate was kept constant at 1.0 mL min−1 . The injection volume was 10 L and
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Table 2 Growth response to sterilization of replant soil tested for apple replant disease. Treatments
Plant height (cm)
Fresh weight (g)
Dry weight (g)
Replant soil Sterilized soil
14.32 ± 0.65b 26.17 ± 1.05a
7.31 ± 0.83b 13.59 ± 0.97a
2.38 ± 0.16b 4.54 ± 0.25a
Data represents the average of three replicates ± SE. Different letters indicate significant difference at p < 0.05.
the column temperature was maintained at 30 ◦ C. Mobile phase A was acetonitrile, and mobile phase B was distilled water (regulated to pH 2.8 with glacial acetic acid). Both standard and extract compounds were used in the following gradient system: (1) from 0.0 to 35 min, a linear gradient from 5% to 35% A; (2) from 35 to 40 min, isocratic with 35% A; (3) from 40 to 42 min, a linear gradient from 35% to 5% A; (4) from 42 to 52 min, isocratic with 5% A. Detection was performed at 270 nm. The chromatographic data were record and processed with a Waters empower workstation. The concentration of phenolic acids in soil samples was obtained using peak area external standards and was expressed as g kg−1 of dry soil. 2.8. Statistical analysis The experimental data was expressed as means and standard errors (SE) with three biological replications. Statistical calculations were performed with SPSS-19 statistical software. Mean difference comparison among different treatments was done by ANOVA and Duncan’s multiple range test at a 0.05 probability level. 3. Results 3.1. The ARD detection of replant soil Compared with the control treatment, the sterilized soil increased the plant height and fresh and dry weight of apple seedlings by 83%, 86% and 91%, respectively (Table 2). The replant soil depressed the growth of seedling plants which indicated the presence of ARD in the soil. 3.2. Effects of biochar on the seedling plants growth in replant soil 3.2.1. Biomass of seedling plant The biomass of M. hupehensis Rehd. seedlings was determined on September 20, 2013. Compared to the control, biochar additions significantly increased plant height. The plant height was increased by 55%, 41% and 17% with 80, 20 and 5 g kg−1 biochar application (Fig. 1A). The fresh weight of plant seedlings increased as well with biochar addition, up to 100% respect to the control with the 80 g kg−1 biochar dose (Fig. 1B). 3.2.2. Chlorophyll content and photosynthetic rate in leaves of Malus hupehensis Rehd. seedlings Compared to the control (without biochar), Pn (Fig. 2A) and Gs (Fig. 2C) increased remarkably under replant conditions with high biochar application rates. Versus the control, the Pn improved by 19% and 35% biochar applied at rates of 20 and 80 g kg−1 , respectively. Versus the control, the Gs improved by 31% and 47% with biochar applied at rates of 20 and 80 g kg−1 , respectively. There was no significant difference in Pn and Gs between the seedling plants treated with 5 g kg−1 biochar and the control. Treatments with 20 and 80 g kg−1 biochar decreased Ci by 9% and 19%, respectively (Fig. 2B). In addition, the chlorophyll content was significantly improved by the addition of biochar (Fig. 2D), showing an increase by 16.0%, 25.4%, and 31.5% with 5, 20, and 80 g kg−1 biochar addition, respectively.
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Fig. 1. Effects of biochar on the plant height and fresh weight of Malus hupehensis Rehd. seedlings under replant condition. Different letters indicate significant difference between treatments at p < 0.05, according to Duncan’s multiple range test.
3.2.3. Antioxidant enzyme activities in leaves of Malus hupehensis Rehd. seedlings The trend indicates that the application of biochar to replant soil enhanced the antioxidant enzyme activities in the leaves of M. hupehensis Rehd. seedlings under replant condition (Fig. 3). With the highest biochar application rate at 80 g kg−1 , the SOD activity was improved by as much as 12% (Fig. 3A). The POD activity also significantly increased from 18% with the 5 g kg−1 biochar additions to 77% with 80 g kg−1 biochar additions (Fig. 3B). Similar trends in response to biochar additions were observed in CAT and APX activity (Fig. 3C and D). In general, enzyme activities in the leaves increased progressively with the biochar additions in comparison to the control (without biochar). 3.2.4. MDA, H2 O2 , O2 •− , proline and soluble sugar content in leaves of Malus hupehensis Rehd. seedlings MDA concentration in leaves of M. hupehensis Rehd. seedlings decreased significantly in biochar application than that of the control (Table 3). The maximum decrease in MDA concentration was
observed in the treatment with 80 g kg−1 biochar, which was only 53% that of the control. Biochar application also was associated with decreases in H2 O2 content, O2 •− generation rate, proline and soluble sugar concentration in leaves of seedlings. Comparison with the control, the maximum decrease in H2 O2 content, O2 •− generation rate, proline and soluble sugar concentration (to 25%, 71%, 33% and 51%, respectively) was found in the treatment with 80 g kg−1 biochar.
3.2.5. Effect of biochar on the concentration of phenolic acids in replant soil In the replant soil with and without biochar, the phenolic acids were measured by HPLC (Table 4). Specifically, the concentration of phenolic compounds was the most abundant in the control soil, and sharply declined with the addition of biochar. The maximum decreases in the concentrations of phloroglucinol, gallic acid, p-hydroxybenzoic acid, phthalic acid, vanillic aldehyde, and phlorizin – which were 42%, 29%, 17%, 35%, 34%, and 24% that of the
Fig. 2. Effects of biochar on net photosynthetic rate (Pn), stomatal conductance (Gs), internal CO2 concentration (Ci) and total chlorophyll in leaves of Malus hupehensis Rehd. seedlings under replant condition. Different letters indicate significant difference between treatments at p < 0.05, according to Duncan’s multiple range test.
Y. Wang et al. / Scientia Horticulturae 175 (2014) 9–15
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Fig. 3. Effects of biochar on the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) in leaves of Malus hupehensis Rehd. seedlings under replant condition. Different letters indicate significant difference between treatments at p < 0.05, according to Duncan’s multiple range test.
Table 3 The effect of biochar on MDA, H2 O2 , O2 •− , and proline content in the leaves of M. hupehensis Rehd. seedlings under replant condition. Biochar applied (g kg−1 )
MDA (mmol g−1 FW)
0 5 20 80
11.14 10.71 8.49 5.90
± ± ± ±
0.070a 0.17b 0.13c 0.090d
H2 O2 (mol g−1 FW) 0.61 0.46 0.26 0.15
± ± ± ±
0.013a 0.016b 0.008c 0.009d
O2 •− (mol g−1 FW h−1 ) 0.44 0.39 0.38 0.31
± ± ± ±
0.024a 0.013b 0.010b 0.091c
Proline (g g−1 FW) 200.01 145.71 91.89 65.34
± ± ± ±
3.21a 3.82b 3.09c 7.73d
Soluble sugar (mg g−1 FW) 31.63 27.88 25.64 16.19
± ± ± ±
1.18a 0.42b 1.24b 0.36c
Data represents the average of three replicates ± SE. Different letters indicate significant difference at p < 0.05.
Table 4 Concentration of six phenolic compounds in four treatment soil. Compounds g kg−1 dry soil
Biochar applied mg kg−1 0
Phloroglucinol Gallic acid p-Hydroxybenzoic acid Phthalic acid Vanillic aldehyde Phlorizin
1041.34 18.42 31.81 689.64 16.98 31.41
5 ± ± ± ± ± ±
193.43a 1.69a 5.37a 26.95a 2.43a 6.93a
20
760.95 10.01 14.28 472.18 12.6 13.45
± ± ± ± ± ±
99.48b 1.92b 1.87b 30.18b 1.15b 1.37b
685.71 7.72 8.29 388.29 9.31 10.50
80 ± ± ± ± ± ±
153.54bc 0.84b 0.95c 21.58c 1.83bc 1.89bc
441.83 5.40 5.33 244.5 5.85 7.42
± ± ± ± ± ±
95.11c 0.52b 0.79c 7.03d 0.98c 1.87c
Data represents the average of three replicates ± SE. Different letters indicate significant difference at p < 0.05.
control, respectively – were observed in the treatment with 80 g kg−1 biochar.
4. Discussion The present results showed that the growth of M. hupehensis Rehd. seedlings in sterilized soils was significantly higher than that of seedlings in the replant soil (Table 2), which suggests that ARD could reduce the growth of M. hupehensis Rehd. seedlings. Biochar has been reported to improve plant growth and yield when it was applied from less than 1 to over 100 t ha−1 (Graber et al., 2010; Major et al., 2010). In this study, the application of 5 g kg−1 biochar is equivalent to an application of 10.1 t ha−1 if calculated for the plough layer of 0.15 m with an average bulk density of 1.35 g cm−3 . The application of biochar to replant soil improved the plant height
and fresh weight of M. hupehensis Rehd. seedlings (Fig. 1), which was consistent with previous report of the beneficial aspects of biochar (Elmer and Pignatello, 2011). Biochar also significantly increased the photosynthetic rate and the chlorophyll content in the leaves of Malus Hupehensis Rehd. seedlings (Fig. 2). In the control plants, the reduced chlorophyll concentrations in the leaves could result from suppressed levels of specific enzymes that are required for the synthesis of pigments, suppressed levels in the uptake of the minerals (e.g. Mg) necessary for chlorophyll synthesis, or the degradation of chlorophyll induced by ROS (Kazemi et al., 2010; Rondon et al., 2007). The application of biochar to replant soil significantly reduced the concentrations of phloroglucinol, gallic acid, p-hydroxybenzoic acid, phthalic acid, vanillic aldehyde, and phlorizin (Table 4). These results could be caused by the sorption of biochar to the phenolic compounds making the phenolic acids ineffective in suppressing neighboring, similar plants (Lehmann et al.,
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Y. Wang et al. / Scientia Horticulturae 175 (2014) 9–15
2011). In this study, biochar effectively reduced the production of ROS under replant condition (Table 3) to alleviate damage of plant growth and chlorophyll content caused by ROS. In addition, the results suggested that the biochar could trigger the activities of specific enzymes, or cause an increase in the uptake of the necessary minerals (Rondon et al., 2007). Biochar application may also substantially modify soil field capacity (Glaser et al., 2002). For example, the use of biochar in vineyard improved soil water content and then increased plant available water and photosynthetic activity in leaves of Vitis vinifera (L.) (Baronti et al., 2014). Our study supported the results that the addition of biochar increased water availability that is responsible for an increase in Pn and Gs. A similar effect of biochar on soil water availability also has been reported in previous studies (Basso et al., 2013; Kammann et al., 2011). Under normal growth conditions, the production and detoxification of free radicals in tissue cells exist a dynamic equilibrium. Abiotic and biotic stresses can break the equilibrium and cause the formation and accumulation of ROS, including O2 •− , H2 O2 and OH· , which are commonly generated via number of metabolic pathways (Kanazawa et al., 2000). Excessive production of ROS may lead to oxidative stress, DNA and protein damage, membrane permeability, lipid peroxidation, loss of cell function and, ultimately, cell death (Ding et al., 2007). To scavenge ROS and to avoid oxidative stress, plants have antioxidant defense enzymes such as SOD, CAT, POD and APX (Kanazawa et al., 2000). In this study, enhanced O2 •− and H2 O2 (Table 3) suggested that the replant condition induced oxidative stress, while the addition of biochar decreased O2 •− and H2 O2 contents in paralled with the increase in SOD, CAT, POD and APX activities (Fig. 3) in leaves of M. hupehensis Rehd. seedlings. Antioxidant enzyme are important for the removal of excessive ROS in plant cell (Jaleel et al., 2007). Many studies have shown that the application of organic amendments improved the systematic resistance of plants (Ju et al., 2013; Zhang et al., 2012). This study’s results revealed that the application of biochar increased the activities of SOD, CAT, POD and APX in leaves of seedlings. MDA accumulation caused by lipid peroxidation has been reported in response to abiotic and biotic stress (Apel and Hirt, 2004). MDA contents is associated with the O2 •− and H2 O2 production. The level of MDA indicated the oxidative damage of cell membranes during lipid peroxidation. In our study, the O2 •− , H2 O2 and MDA concentration in leaves of M. hupehensis Rehd. seedlings were high in control treatment (Table 3), and the biochar applied to replant soil reduced the concentration of MDA, O2 •− and H2 O2 . It indicated that biochar was useful to enhance plant antioxidant capability and it alleviated damage to plant from the oxidative stress by reducing the concentration of phenolic acids. Studies have shown that plants resist the environmental stress induce production of ROS by increasing their intrinsic defensive system (Alscher et al., 2002; Salin, 1988). Plant subjected to environmental stress could accumulate osmotic substances in cells (Akcay et al., 2010; Kazemi et al., 2010), such as proline, soluble sugar and various betaines which can function as osmoprotectants. As with other studies on the effects of stress on plants (Akcay et al., 2010; Kazemi et al., 2010), we found the accumulation of proline and soluble sugar in M. hupehensis Rehd. seedlings was caused by ARD stress. The accumulation of proline may be due to the mechanisms of osmoregulaion, and the response of plant under ARD stress may be correlated with their antioxidative system (Kazemi et al., 2010). The application of biochar to replant soil probably alleviated the apple replant disease stress probably via the sorption of aromatic acids which had allelopathic effects and were abiotic factors for ARD (Lehmann et al., 2011). Thus, the application of biochar markedly increased antioxidant capability, and decreased membrane permeability and the contents of proline and soluble sugar in the plants.
It is interesting that biochar may alleviate ARD stress in M. hupehensis Rehd. seedlings. The addition of biochar reduced the concentrations of phenolic compounds, and osmotic adjustment substances. Biochar addition significantly alleviated the decline in plant height, chlorophyll content, and Pn. It also increased the activities of antioxidant enzyme and inhibited lipid peroxidation in plant cells. The presented results supported the view that biochar can contribute to protect M. hupehensis Rehd. seedlings against ARD by alleviating the ARD induced oxidative stress. However, further works should be carried out to study the effects of biochar on the soil microbial community and the relationship between the soil microbial and phenolic acids under replant condition.
Acknowledgments The research was supported by the earmarked fund for National Modern Agro-industry Technology Research System (CARS-28), and Program for Changjiang Scholars and Innovative Team in university (IRT1155).
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