Eur J Plant Pathol (2012) 134:381–389 DOI 10.1007/s10658-012-9996-2
Role of hydrogen peroxide and antioxidative enzymes in Pinus tabulaeformis seedlings inoculated with Amanita vaginata and/or Rhizoctonia solani Ru-Qin Zhang & Ming Tang
Accepted: 25 April 2012 / Published online: 20 May 2012 # KNPV 2012
Abstract The ectomycorrhizal fungus Amanita vaginata can control damping off (Rhizoctonia solani) and promote growth of Pinus tabulaeformis seedlings. The aim of this study was to investigate whether reactive oxygen species and antioxidative enzymes play a role in preventing damping off in ectomycorrhizal roots. Two months after P. tabulaeformis roots were inoculated with A. vaginata, the roots were inoculated with R. solani. During the early stages (2–96 h) of R. solani infection, the quantity and localisation of hydrogen peroxide and the activities of superoxide dismutase and catalase were evaluated. A burst of hydrogen peroxide occurred in ectomycorrhizal roots and in nonectomycorrhizal roots when attacked by R. solani. In ectomycorrhizal roots, hydrogen peroxide production peaked 12 h after R. solani inoculation, which coincided with an increase in the activity of superoxide dismutase and catalase, whereas in non-ectomycorrhizal roots,
hydrogen peroxide production peaked 24 h after R. solani inoculation and did not coincide with changes in superoxide dismutase or catalase activity. The imbalanced activities of superoxide dismutase and catalase might cause excessive accumulation of hydrogen peroxide and consequent damage to cell walls. Electron microscopy revealed that there was a positive correlation between hydrogen peroxide levels and the number of amyloplasts, with seedlings inoculated with A. vaginata and/or R. solani showing higher levels. These results indicated that A. vaginata inoculation enhanced damping off resistance and stimulated seedling growth, which may be due to the activation of a burst of hydrogen peroxide and its scavenging enzymes and the production of biochemical substances such as amyloplasts.
R.-Q. Zhang College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China e-mail:
[email protected]
Introduction
R.-Q. Zhang College of Agronomy and Plant Protection, Qingdao Agriculture University, Qingdao, Shandong 266109, China M. Tang (*) College of Forestry, Northwest A&F University, Yangling, Shaanxi 712100, China e-mail:
[email protected]
Keywords Amyloplasts . Antioxidative system . Biological control . Damping off . Ectomycorrhiza
In incompatible host interactions as well as in non-host interactions with fungal pathogens, the plants respond to the fungal pathogen infection by activating defence responses and, hence, disease symptoms do not develop and the plants are termed disease resistant (Mysore and Ryu 2004). However, in arbuscular mycorrhizal (AM) associations, there is increasing evidence that during the early stages of AM establishment, the defence system of the plant is suppressed to enable the symbiont to
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establish (Pozo et al. 2010). Production of reactive oxygen species (ROS) and ROS-scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT) and ascorbic acid oxidase (APX) are some of the defence responses used by plants against pathogens and abiotic stresses (Dixon and Lamb 1990; Wojtaszek 1997; Patykowski 2006; Iannone et al. 2010). Hydrogen peroxide (H2O2) is an important signalling compound involved in the plant’s response to pathogenic microorganisms (Wojtaszek 1997) and mycorrhisation (Fester and Hause 2005), as well as many other plant biology processes (Joo et al. 2001; Foreman et al. 2003; Liszkay et al. 2004). Accumulation of H2O2 during the formation of an AM symbiotic relationship has been reported and several studies have demonstrated an increase of ROS production in AM roots, including the induction of various antioxidative enzyme activities upon mycorrhisation (García-Garrido and Ocampo 2002; Garmendia et al. 2006; Pozo et al. 2010) that are involved in plant resistance to disease (Garmendia et al. 2006) and stress (Abdel Latef 2011). Numerous studies have shown that ECM fungi have dual roles in promoting plant growth and controlling root diseases (Branzanti et al. 1999; MartínPinto et al. 2006; Zhang et al. 2011). Moreover, data have also been reported regarding the antioxidative system involved in mycorrhiza (Salzer et al. 1996; Martin et al. 2001; Baptista et al. 2007). Sebastiana et al. (2009) observed that during the early stages of contact (6 and 12 h) between Castanea sativa and the ECM fungus Pisolithus tinctorius, genes involved in stress and defence are induced in C. sativa, suggesting that the host plant reacts rapidly to the presence of the ECM fungus, eliciting a defence programme similar to that reported for pathogenic interactions. Pinus tabulaeformis (Pt) is an important reforestation and landscaping tree in China. However, damping-off caused by the soil-borne pathogen Rhizoctonia solani is a major hazard for Pt seedlings in nurseries and in areas of reforestation. At present, the disease cannot be controlled by chemical applications because soil-borne diseases are difficult to control and chemical pesticides can pollute soil and groundwater. To date there is limited information about the modes of action responsible for activating the antioxidative system in ECM roots in response to pathogens, and identifying the exact localisation of H2O2 is important to determine its potential biological roles in ECM roots. Therefore, the objectives of this study are to determine
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H2O2 production and localisation, the activity of ROSscavenging enzymes, such as SOD and CAT, in Pt seedlings induced by the ECM fungus Amanita vaginata and/or the pathogen R. solani, and their roles in damping off resistance.
Materials and methods Inoculation and incubation of Pt seedlings The ECM fungus Amanita vaginata (Bull.: Fr.) Vitt. was isolated from sporocarps harvested from the Huoditang forest region of the Qinling Mountains in China. A. vaginata was selected for these studies because in an earlier study by Zhang et al. (2011) involving five ECM fungi, A. vaginata was shown to have the greatest protective effect (43 %) against damping-off caused by R. solani and to promote growth of Pt seedlings significantly. A. vaginata was subcultured by inoculating a Petri dish of modified Melin Norkrans (MMN) solid medium with a disk of mycelia (5 mm) from a culture that had been stored at 4°C. The cultures were incubated at 25°C in darkness for 20 days. The ECM and pathogen inocula were prepared according to the methods described by Zhang et al. (2011). A whole Petri dish of A. vaginata culture was transferred to a cylindrical polyethylene bag (20×7 cm) containing Cotton Seed Hull solid medium and incubated at 25°C for 30 days. The pathogen inoculum was prepared by first inoculating a 500-ml Erlenmeyer flask containing 100 ml of liquid potato glucose medium with a disc of R. solani mycelia (5 mm) excised from a Petri dish culture that had been stored at 4°C. The flask was incubated at 25°C in the dark for 7 days on a gyratory shaker at 110 rotations per min. 100 ml of liquid culture of R. solani mycelia was then mixed with 1,000 ml of sterilised substrates (vermiculite:sand:garden soil0 3:1:1, v:v:v). Pt seedlings were inoculated and cultivated according to the method of Zhang et al. (2011). The Pt seedlings were surface sterilised in 0.5 % potassium permanganate and rinsed with sterilised water. Pt seedlings with fully developed cotyledons were transferred to an open pot (15×13 cm). Each pot contained 1300 g of substrate, a mixture of sand, garden soil, vermiculite and peat (2:2:1:1, v:v:v:v; sterilised at 121°C, 0.05 Mpa for 2 h), and 200 g of A. vaginata inoculum. For the
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controls, 200 g of sterile Cotton Seed Hull medium was added to the substrate. The seedlings were then covered with about 200 g of the substrate mixture and cultivated in the dark. The pots were transferred to a greenhouse after seedling emergence. Eighty pots were inoculated with the ECM fungus and eighty non-inoculated pots served as the non-ECM controls. Each pot contained six seedlings and were watered once every 2 days with 100 ml of tap water and watered once every 2 weeks with 100 ml of Hoagland nutrient solution. The soil substrate had a pH of 6.0 and contained 28.54 mg kg−1 of soluble nitrogen, 20.87 mg kg−1 of soluble phosphorus, 90.66 mg kg−1 of soluble potassium and 0.895 % organic matter. Using the methods outlined by Bao (2000), the nitrogen, phosphorus, potassium and organic matter levels were determined using proliferation, spectrophotometry, atomic absorption and potassium dichromate, respectively, and the pH value was determined using a PHS-3B acidometer. Two months after the Pt seedlings planting, mycorrhizal colonization was evaluated using a method described by Zhang et al. (2011): 100 short roots were randomly harvested from mycorrhizal or non-mycorrhizal Pt seedlings, and examined with a stereomicroscope. The degree of mycorrhizal formation was expressed as the percentage of root tips colonized. The Pt seedlings were then inoculated with the R. solani inoculum. Forty pots containing ECM seedlings and forty pots containing nonECM seedlings were inoculated with 100 ml of R. solani inoculum by distributing the inoculum evenly over the surface of the soil substrate (treatments AV1 and CK1, respectively); the remaining forty ECM and forty nonECM pots were inoculated with substrate that lacked R. solani (treatments AV0 and CK0, respectively). Quantification and histochemical localisation of H2O2 H2O2 concentration was determined according to Patterson et al. (1984): 0.5 g of fresh root tissue was ground in 5 ml of pre-cooled (−20°C) acetone and quartz sand, and centrifuged for 10 min at 1,500×g at 4°C. The supernatant was used for assaying H2O2. 0.1 ml of titanium chloride (0.1 % w:v) and concentrated ammonia (0.2 ml) were added to the supernatant (1 ml). After reaction for 10 min at 25°C, the reaction mixture was centrifuged for 10 min at 1,500×g. The absorbance at 415 nm was measured and H2O2 concentration was calculated according to the standard curve. The experiment was performed three times with
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three replicates each time. Mean±standard deviation (SD, n09) was shown. To prepare samples for examination using electron microscopy, the seedlings (10 plants) were harvested at random 48 h after R. solani inoculation and treated with cerium (III) chloride (CeCl3), which is a marker for H2O2, according to a method modified from Bestwick et al. (1997) and Pellinen et al. (1999). 0.4 cm pieces of the base of the stem were cut and stained with 5 mM CeCl3 [in 50 mM 3-(N-morpholino) propanesulphonic acid (MOPS, pH 7.2)] by incubating the samples for 1 h at room temperature. Control samples were incubated in water for 1 h at room temperature. The penetration of the mixture was improved by vacuum pumping for 0.5 h. The samples were pre-fixed in 1.25 % (v:v) glutardialdehyde and 1.25 % paraformaldehyde in 50 mM sodium cacodylate buffer (pH 7.2) for 1 h at room temperature. After rinsing twice with 50 mM sodium cacodylate buffer, the samples were post-fixed with buffer containing 1 % osmium tetroxide for 45 min, rinsed again, and dehydrated in a graded ethanol series (30–100 %, v:v). Ethanol was substituted with Epon Araldite for 24 h at room temperature. Polymerisation was conducted at 60°C for 48 h. The tissue was sliced and the ultrathin sections were visualised with a JEM-1230 electron microscope and photographed. Extraction of antioxidative enzymes and activity assays Twenty plants were randomly collected in each hour (2, 6, 12, 24, 48, 72 and 96 h after inoculation with R. solani) in each treatment. Plants were dug out gently with a tweezer and cleaned with tap water. The fresh root tissue was homogenised immediately in liquid nitrogen, and then 1.0 g of powdered homogenate was resuspended in 10 ml of 50 mM phosphate buffer (pH 7.8), 0.1 % (v:v) 2-mercaptoethanol, 1 mM ethylenediamine tetraacetic acid disodium (EDTA-Na2) and 1 % (w:v) polyvinyl-polypyrrolidone at 4°C. The homogenates were centrifuged at 13,000×g for 15 min at 4°C, and the supernatants were used to determine the quantify and activity of SOD, and CAT. Protein was quantified using the Coomassie Brilliant Blue G250 microassay using bovinum serum albumin (BSA) as a standard. Both SOD and CAT activity was assayed according to the modified method described by Gao (2000). The reaction mixture consisted of 100 mM phosphate buffer (pH 7.8), 0.2 mM EDTA, 19.8 mM L-methionine, 57 μM p-nitrotetrazolium blue
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Statistical analysis The significant differences between different treatments were analysed using one way analysis of variance (ANOVA) at P00.01 or P00.05 with SAS (version 8.0) statistical software.
Results Influence of inoculation on Pt seedlings As reported in a previous study (Zhang et al. 2011), Pt seedlings inoculated with A. vaginata (8.34 cm) were significantly taller than the control seedlings (6.22 cm). Colonization rate of ECM roots (57 %) was higher than that of control roots (0 %). Disease symptoms were observed 72 h after Pt seedlings were inoculated with R. solani. The pathogen caused extensive water-soaked lesions at the base of stem, which were followed by wilt and damping off of the plant. Compared with non-ECM seedlings the mortality of ECM seedlings was lower and the protective effect against damping off was 43 %. Qualification and localisation of H2O2 in Pt seedling roots To ascertain the involvement of H2O2 during the early stages of R. solani infection of Pt roots H2O2 was quantified. As depicted in Fig. 1, one way analysis of
CK0
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Hydrogen peroxide content (µmol/gFW)
(NBT), 0.9 μM riboflavin and 50 μl plant protein extract. After incubation at 30°C for 6 min under continuous light, absorbance was read at 560 nm. One unit (U) of SOD activity was defined as the amount of enzyme inhibiting 50 % of NBT photochemical reduction under the above assay conditions. CAT activity was assayed by measuring the decrease in the H2O2 concentration at 240 nm. 0.6 ml of 30 % H2O2 was diluted to 100 ml with a 50 mM phosphate buffer (pH 7.0) and used as a CAT enzyme reaction substrate. The reactions were initiated by adding 50 μl of extract. The decrease in absorbance was evaluated at 240 nm at 25°C. One unit (U) of CAT activity was defined as the amount of enzyme necessary to decompose 1 μmol H2O2 in 1 min under the above assay conditions. Three replicates were performed in each hour and 20 plants were included in three replicates. The experiment was performed three times. Mean±standard deviation (SD, n09) was shown.
Eur J Plant Pathol (2012) 134:381–389 a
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Time after inoculation with Rhizoctonia solani (hours)
Fig. 1 Content of H2O2 in the roots of Pinus tabulaeformis seedlings inoculated with Amanita vaginata and/or Rhizoctonia solani. Lower case letters in the figure indicate significant differences (P0.05) in H2O2 content between non-ECM roots that were not inoculated with R. solani (CK0) and A. vaginata roots that were not inoculated with R. solani (AV0), or between non-ECM roots that were inoculated with R. solani (CK1) and A. vaginatacolonised roots inoculated with R. solani (AV1). However, there were significant differences in H2O2 content between AV1 and AV0, and between CK1 and CK0 (P
