- Email: [email protected]
The effects of organic matter on the physiological features of Malus hupehensis seedlings and soil properties under replant conditions
Scientia Horticulturae 146 (2012) 52–58
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
The effects of organic matter on the physiological features of Malus hupehensis seedlings and soil properties under replant conditions Zhaobo Zhang a , Qiang Chen a,b , Chengmiao Yin a , Xiang Shen a , Xuesen Chen a , Haibing Sun a , An’ni Gao a , Zhiquan Mao a,∗ a b
College of Horticultural Science and Engineering/State Key Laboratory of Crop Biology, Shandong Agricultural University, No. 61 Daizong Street, Tai’an, Shandong 271018, China Shandong Institute of Science and Technology Information, Jinan 250101, China
a r t i c l e
i n f o
Article history: Received 10 April 2012 Received in revised form 8 August 2012 Accepted 9 August 2012 Keywords: Malus hupehensis Rehd. Organic matter Physiological features Replant Soil properties
a b s t r a c t Two fermentation methods, aerobic (generating solid compost) and anaerobic fermentation (generating fermented fluid), were adopted to treat the same amount of organic matter to generate solid compost and fermented fluid, respectively. These treatments were used to supplement replant soils in a pot, respectively, where Malus hupehensis Rehd. seedling was replanted. The pilot project was aimed at investigating the influence of organic matter applied to soil on the physiological features of the plant as well as its impact on soil properties. The following four treatments were adopted in the study: new cropping soil, replant soil, replant soil with solid compost, and replant soil with fermented fluid. The growth of seedlings in the four soils was monitored by biomass production and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the plant roots. The activities of soil urease, soil invertase, and soil phosphatase were measured; and the bacterial, fungal, and actinomycetal contents in the soils were also measured. This study showed that the supplementation of organic matter in a fermented liquid form to replant soil significantly enhanced the biomass production of the seedlings compared to the solid compost form. The activities of SOD, POD, and CAT in the roots were increased by both forms of organic matter, with the solid compost form having a stronger effect than the fermented liquid form. Moreover, both forms increased the activities of soil urease, soil invertase, and soil phosphatase, with the fermented liquid form having a stronger impact than the solid compost form. The addition of organic matter in the form of fermented fluid to the replant soil obviously increased the bacteria content in the soil in comparison with the addition of organic matter in the form of solid compost. Compared to the solid compost, the fermented fluid led to a higher increase in the fungal and actinomycetal contents in the soil at the early stage but a lower increase at the late stage. When applied to replant soil, the fermented fluid form of organic matter was superior to the solid compost form of organic matter in preventing apple replant disease (ARD). © 2012 Elsevier B.V. All rights reserved.
1. Introduction Apple replant disease (ARD) causes the inhibition of root system development, stunts tree growth, and reduces yield and quality in replanted apple orchards (Laurent et al., 2008). In replant orchards, the ARD resulting from replanting in the row is more severe than that resulting from replanting between rows (Rumberger et al., 2004; Leinfelder and Merwin, 2006). ARD is often caused by a consortium of biological agents, mainly, including nematodes, bacteria, actinomycete, oomycetes and fungi species (Tewoldemedhin et al., 2011a). van Schoor et al. (2009) thought that Pratylenchus penetrans was the primary nematode species involved in ARD.
∗ Corresponding author. Tel.: +86 538 8241984. E-mail address: [email protected] (Z. Mao). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.08.011
Although several bacterial genera and species have been associated and suggested as being involved in ARD, most bacteria likely impaired plant at inordinately high densities (Tewoldemedhin et al., 2011a). Evidence for the involvement of actinomycetes in ARD is circumstantial (Tewoldemedhin et al., 2011a). However, most researches demonstrated fungal and oomycete genera were the main reason for apple replant disease, i.e. fungal genera: Fusarium (Tewoldemedhin et al., 2011a,b), Rhizoctonia (Tewoldemedhin et al., 2011a,b), Cylindrocarpon (Manici et al., 2003; Tewoldemedhin et al., 2011a,c); oomycete genera: Phytophthora (Tewoldemedhin et al., 2011a,b), Pythium (Tewoldemedhin et al., 2011a,b). Methyl bromide has been widely used in effectively preventing ARD, but it causes serious pollution problems to environment. Hence, it is imperative to find an alternative to replace soil fumigants, such as methyl bromide (Yao et al., 2006). Applying organic matter to soils not only alleviates the adverse effects brought by
Z. Zhang et al. / Scientia Horticulturae 146 (2012) 52–58
many unfavorable factors (Chang et al., 2008) but also increases the quantity and activity of soil microbes (Crecchio and Stotzky, 2001; Bernarda et al., 2012). Irradiation treatment of organic matter compost reduces the capability of the compost to inhibit Fusarium wilt, which indicates that the microbes in the compost have important roles in suppressing pathogens (Yogev et al., 2006; Hagn et al., 2008). In an orchard located in West Virginia in the United States, it has been reported that the application of compost derived from organic matter increases the quantity of beneficial arthropods, improves pest control in the orchard, decreases pathogenic fungi occurrence, and inhibits Monilinia fructicola, thus promoting robust tree growth and maintaining sustainable productivity of the orchard (Brown and Tworkoski, 2004). The application of compost consisting of organic matter can also significantly increase the growth rate of seedlings under replant conditions (van Schoor et al., 2009), which leads to a longer growth period of fruit plants than that of plants treated with methyl bromide (Yao et al., 2006). In addition, the application of organic matter compost leads to a higher usage rate of carbohydrates of i-erythritol and l-rhamnose (Chang et al., 2008) and, therefore, enhances the level of organic carbon in the soil and improves the soil pH (Zaman et al., 2004). Previous studies have demonstrated that the inhibition of Fusarium spp. in apple trees by compost is related to the organic matter composition, maturity level, and processing methods of the compost as well as its application time and amount (Ghorbani et al., 2008). Organic matter fermented fluid is the end product of anaerobic fermentation, and it can increase the quantity of soil microbes (Singh et al., 2007; Terhoeven-Urselmans et al., 2009). As a highquality organic fertilizer, fermented fluid improves fruit yield and quality (Loria et al., 2007; Terhoeven-Urselmans et al., 2009). To date, there is no report that compares the effects on ARD control by applying organic matter fermented fluid and solid compost. In the present study, the common apomictic rootstock, Malus hupehensis Rehd., was used as a subject to investigate the effects of organic matter solid compost and fermented fluid on the physiological features of seedlings and soil properties to provide theoretical and practical implications for the control of ARD.
2. Materials and methods 2.1. Experimental materials and treatment The experiment was carried out in 2010 in a 20-year-old Red Fuji apple orchard located in the suburb of Tai’An city, Shandong province of China; and at the fruit tree root system laboratory of the College of Horticultural Sciences and Engineering of Shandong Agricultural University, also located in Tai’An city, respectively. M. hupehensis Rehd., which is a commonly used rootstock for apple trees, was used in this study. The seeds were stratified for 30 days at 4 ◦ C. After shoot tip emergence, the seeds were planted in nursery plates. The seedlings with six leaves were transplanted to a clay pot (with an outside diameter of 29 cm and an inner diameter of 25 cm) that contained 7 kg of replant soil collected from the abovementioned apple orchard. In the 20-year-old Red Fuji apple orchard, its original rootstock was M. robusta, and the soil type was cinnamon soil. The soil was randomly collected from multiple locations in the orchard (locations were 1 m away from the trunk) at a soil depth of 5–40 cm, and the soil samples were mixed well. The organic matter used in this study consisted of chicken feces, sheep manure, cow dung, and wheat straw (2 cm in length) mixed in a ratio of 6:1:1:2 (v/v). The organic matter was routinely composted under natural conditions. The organic matter was fermented by placing the well-mixed organic matter into a white plastic bucket (50 cm in diameter and 120 cm in height) and adding water until the contents were submerged under 30 cm.
53
After the content of the organic matter was mixed with water using a stick, the bucket was sealed with a plastic membrane that contained ventilation holes. The bucket was then buried until 2/3 of the bucket was embedded underground, stirred 5 times with a stick during the whole fermentation process and the fermentation process also proceeded under natural conditions. The compost and fermentation liquid were prepared on May 1, June 15, and July 20. The weight ratio of organic matter (prepared for solid compost and fermented fluid forms) to replant soil (supplemented with solid compost and fermented fluid forms) was 1:25. The ratio originated from growers’ planting experience. Considering the increasing temperature over time, we gradually shortened the organic matter preparation time. The organic matter (compost and fermented fluid forms) was applied to the pots on June 20, July 20, and August 20, respectively. The 4% solid compost of replant soil weight in each pot was supplemented to the surface of soil; subsequently, dug the surface of replant soil repeatedly with a small hoe until the solid compost was mixed even with the soil surface. As for fermented fluid forms, initially mixed the content well in the white plastic bucket with a stick, shared the fermented fluid forms and watered the pilot seedlings in succession; after the fermented fluid forms drying, treated them as the abovementioned compost. Four treatments were set up as follows: T1, newly cropping soil collected from the neighboring wheat field outside the orchard; T2, replant soil collected from the orchard supplemented with solid compost; T3, replant soil collected from the orchard supplemented with fermented fluid from the organic matter; and T4, replant soil collected from the orchard (control). The nutrient and water were managed as usual, and the N, P, and K contents in the soil were normalized among the treatment pots. Two seedlings were planted in each pot, and 30 pots (6 pots were spare to prevent undesirable condition among them, only 24 pots were occupied for each treatment in the experiment in fact) were prepared for each treatment. Each treatment contained 3 biological replications at the same point in time. For the antioxidant enzymes, every biological replication consisted of 4 seedlings, namely 4 seedlings (2 pots) are one set and as a sampling unit. Roots less than 5 cm in length were gathered and mixed well for antioxidant enzymes assay. For the soil enzyme and quantity of soil microbes, the pot soil (∼4 cm radius around each seedling and ∼6 cm in depth) in 2 pots was collected and mixed well as a biological replication. The samples were collected at 9:00 a.m. to 10:00 a.m. at a time. The first sampling was done on June 20 (June) before the application of organic matter on the same day. And the rest samplings were done after 20 days treatment; namely, on July 10 (July), August 10 (August) and September 10 (September), respectively. And the collected samples were prepared for further assay.
2.2. Antioxidant enzymes assay in root The activities of antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)) in root were determined. The SOD activity assay was performed as previously described (Zhang et al., 2009). The SOD activity unit (U) was defined as the amount of enzyme causing half of the maximum inhibition of nitroblue tetrazolium (NBT) reduction, and the enzyme activity was expressed as U g−1 FW. The POD activity assay was performed according to the method developed by Omran (1980). The POD activity was assayed spectrophotometrically by monitoring changes in absorbance at 470 nm. The POD activity unit (U) was defined as the amount of enzyme causing a change of 0.01 in the absorbance at 470 nm per minute, and the enzyme activity was expressed as U g−1 FW. The CAT activity assay was also performed spectrophotometrically by monitoring changes in absorbance at 240 nm (Singh et al., 2010). The CAT activity unit (U) was defined
54
Z. Zhang et al. / Scientia Horticulturae 146 (2012) 52–58
as the amount of enzyme causing a change of 0.1 in the absorbance at 240 nm/min, and the activity was expressed as U g−1 FW.
volumetric flask and diluted to 100 mL, respectively. The absorption was assayed at 570 nm when the color was kept relatively stable and drew the standard curve.
2.3. Soil enzyme assays 2.4. Determination of the quantity of soil microbes The soil enzyme assays were performed as previously described by Guan (1986). In detail, the activity of soil urease was assayed by using indophenol blue method with minor changes. 5 g air-dried soil was placed in a 50 mL volumetric flask, added 1 mL toluene, filled the top of the bottle with a stopper tightly and jiggled the volumetric flask for 15 min. Subsequently 5 mL 10% urea solution and 10 mL citrate buffer (pH 6.7) were added to the volumetric flask. The mixture was incubated at 37 ◦ C for 24 h after mixing well carefully. Then distilled water at 38 ◦ C was applied to dilute the mixture to 50 mL (the toluene should float above the scale), mixed them well, and the suspension was filtered. 1 mL filtrate was placed in a 50 mL volumetric flask, diluted with distilled water to 10 mL, followed by adding 4 mL sodium phenolate solution, and immediately joined 3 mL sodium hypochlorite solution. After the mixture was shaken well and placed for 20 min, the mixture was diluted to 50 mL. Its absorption was measured at 578 nm. The abovementioned steps without soil were used for a control. The D-value of absorption (sample minus control) was used to calculate the activity of soil urease. The activity of soil urease was expressed as mg released NH3 -N by 1 g soil at 37 ◦ C per 24 h. The ammonia nitrogen quantity was obtained according to the standard curve. Standard curve: added 0, 1, 3, 5, 7, 9, 11, 13 mL ammonium sulfate solution (0.01 mg mL−1 ) to a 50 mL volumetric flask, respectively. The above flasks were diluted to 20 mL with distilled water. Thereafter 4 mL sodium phenolate solution and 3 mL sodium hypochlorite solution were added. Mixed them well coupling with the adding process. The mixture colored after 20 min, set the volume. The absorption was determined at 578 nm within 1 h. For invertase activity with minor alterations, 5 g of air-dried soil was mixed with 15 mL 8% sucrose and 5 mL phosphate buffer at pH 5.5 and 0.1 mL toluene. The mixture was incubated for 24 h at 37 ◦ C. The suspension was filtered. 1 mL filtrate was transferred to a 50 mL volumetric flask. Added 3 mL 3,5-dinitrosali-cylic acid and heated it in a boiling water bath for 5 min, then the flask was moved to running water and cooled it for 3 min. Afterwards, diluted the mixture with distilled water to 50 mL. The glucose from sucrose hydrolysis catalyzed by invertase reacted with 3,5-dinitrosali-cylic acid. The product 3-aminonitrosalicylic acid was determined the absorption at 508 nm. The activity of invertase was calculated by mg released glucose by 1 g soil per 24 h. A control without soil was used for each sample. Standard curve: added 0, 1, 2, 3, 4, 5, 6, 7 mL glucose solution (0.5 mg mL−1 ) to a 50 mL volumetric flask, respectively. Operated them as the abovementioned method of assaying invertase activity, the standard curve was drawn on the basis of colorimetric absorbance for the vertical axis and glucose concentration as the abscissa. For neutral phosphatase with some modifications, 5 g of airdried soil was incubated at 37 ◦ C for 24 h with 1 mL toluene, 5 mL disodium phenyl phosphate (6.75 g C6 H5 PO4 Na2 ·2H2 O dissolved in 1 L distilled water) and 5 mL citrate buffer (pH 7.0). Afterwards, the mixture was diluted with distilled water at 38 ◦ C to 50 mL and filtered. 1 mL filtrate mixed well with 5 mL (borate buffer pH 9.0), 3 mL 2.5% potassium ferricyanide and 3 mL 0.5% 4-aminoantipyrine. The solution was diluted to 100 mL after changing into pink. The absorption was assayed at 570 nm when the color was kept relatively stable (about 20–30 min). A control without soil was used for each sample. The activity of neutral phosphatase was described as mg released phenol by 1 g soil per 24 h. The phenol content was obtained according to the standard curve. Standard curve: 1, 3, 5, 7, 9, 11 mL phenol solution (0.05 mg mL−1 ) was injected to a 100 mL
The quantity of soil microbes was determined using the agar plate dilution method as previously described by Cheng and Xue (2000). Beef peptone medium was used for the culture of bacteria, and Martin’s medium was used for the culture of fungi. Moreover, Gao 1 agar medium was used for the culture of actinomycetes. 2.5. Statistical analysis The mean separation and analysis of variance were calculated using the Microsoft Excel 2003 and DPS V7.05 software. The experimental treatment consisted of three biological replicates. The data represent mean values ± standard errors (n = 3); different lower case letters a, b, c and d indicate statistical significance at the 0.05 level (Duncan’s new multiple range test, P < 0.05). 3. Results 3.1. Effects of organic matter on the biomass production of M. hupehensis seedlings under replant conditions in September 2010 The biomass of M. hupehensis seedlings was determined on September 10, 2010. Here, we only enumerated the biomass in September. The similar varying trend was observed in July 10 and August 10, but data are not shown to avoid repetition. The height, basal diameter, aboveground fresh biomass weight, and fresh root weight of the seedlings were the highest in treatment T1 (newly cropping soil collected from the neighboring wheat field outside the orchard) followed by those in treatment T3 (replant soil collected from the orchard supplemented with fermented fluid from the organic matter), T2 (replant soil collected from the orchard supplemented with solid compost), and T4 (replant soil collected from the orchard; control), which had the lowest values (Table 1). The height, basal diameter, aboveground fresh biomass weight, and fresh root weight of the seedlings grown in T3 were 0.82, 0.96, 0.79, and 0.83-fold greater than those grown in T1, respectively. However, they were 1.31, 1.13, 1.68, and 1.22-fold greater than those grown in T2, respectively; furthermore, 1.9, 1.56, 2.14, and 1.8-fold greater than those in grown in T4 (control group), respectively. 3.2. Influence of organic matter on the activities of SOD, POD, and CAT in the root system of M. hupehensis seedlings grown under replant conditions The application of organic matter to the replant soil enhanced the SOD activity in the root system of M. hupehensis seedlings with the time prolonged, and the enzyme activity significantly varied among the treatments (Fig. 1A). In July, the order of SOD activity in the root system of M. hupehensis seedlings among the treatments was T1 > T2 > T3 > T4, with only limited increases in all the treatments. In August, the SOD activity in the root system of M. hupehensis seedlings grown in T3 was the highest followed by the activity of the seedlings grown in T1 and T2, and all of these increases were significant, T4 was the lowest. In September, the SOD activities in the root system of M. hupehensis seedlings grown in T2 and T3 reached the highest level (approximately 222.5 U g−1 FW, and they were no significant difference), which was much higher than the activities observed in T1 and T4. With organic matter addition to the replant soil, the POD activity in the root system of M. hupehensis seedlings was also enhanced with the extension of time, and it significantly varied among the
Z. Zhang et al. / Scientia Horticulturae 146 (2012) 52–58
55
Table 1 Effects of organic matter on the biomass production of M. hupehensis seedlings under replant conditions in September 2010. Treatment
Plant height (cm)
Basal diameter (cm)
Aboveground fresh biomass weight (g)
Fresh root weight (g)
T1 T2 T3 T4
59.00 ± 0.29 a 37.00 ± 0.76 c 48.50 ± 0.29 b 25.50 ± 1.04 d
0.6030 ± 0.0055 a 0.5100 ± 0.0055 c 0.5780 ± 0.0044 b 0.3700 ± 0.0026 d
13.08 ± 0.05 a 6.16 ± 0.05 c 10.34 ± 0.02 b 4.84 ± 0.03 d
11.67 ± 0.06 a 8.00 ± 0.08 c 9.74 ± 0.03 b 5.41 ± 0.02 d
Values are means of three replicates ± standard error. Different lower case letters within the same column indicate significant difference at 5% level (Duncan’s new multiple range test).
treatments (Fig. 1B). In July, the POD activity in the root system of M. hupehensis seedlings grown in T2 was the highest, even surpassing that in T1, whereas T3 was lower than T1. Afterwards, the activity of T3 increased rapidly, in August, the POD activity in the root system of M. hupehensis seedlings grown in T3 reached its peak value and was 1.06, 1.15, and 1.54-fold greater than that of seedlings grown
in T1, T2, and T4, respectively. In September, the POD activity in the root system of M. hupehensis seedlings grown in T1 was the highest and was 1.2, 1.32, and 2.1-fold greater than that of seedlings grown in T2, T3, and T4, respectively. Compared to that in August, the POD activity of T3 in September decreased; in contrast, the POD activity of T2 in September rose. From July to September, there were no statistical difference in between T1 and T3, as well between T2 and T4. T1 and T3 had also higher CAT activity than T2 and T4. CAT activity in the root system was the highest in August (Fig. 1C). 3.3. Influence of organic matter on the activities of soil urease, soil invertase, and soil phosphatase Compared to those in June, the activities of soil urease except T4 in July ascended; those in August and September were greater than those in June. In July, the activity of soil urease was the highest in T2 followed by T1 and T3, with a significant difference among the treatments. In August, the addition of fermented fluid to the replant soil increased the soil urease activity in T3 to its highest level, with levels 1.21, 1.69, and 1.76-fold greater than the levels in T1, T2, and T4, respectively. In September, the highest activity of soil urease was found in T1, which was slightly higher than the activity found in T3, no obvious difference between them; but the both were significantly higher than the activity found in T2 and T4 (Fig. 2A). The activity of soil invertase in different treatments advanced with the progress of time. The addition of fermented fluid to the replant soil (T3) led to a slightly higher soil invertase activity than that of T4 in July and August, 1.13-fold (significant difference, Fig. 2B) and 1.03-fold (no-significant difference, Fig. 2B), respectively; but to a markedly higher soil invertase activity than that of T4 in September, up to 1.99-fold (significant difference, Fig. 2B). The activity of soil invertase in T1 was higher than that of T3, with levels 1.15, 1.26, and 1.61-fold greater than the levels in T3 in June, July, and August, respectively. In September, the activity of soil invertase was the highest in T3 followed by T1, and the activities in the both of these treatments were significantly higher than the activities in T2 and T4 (Fig. 2B). The activity of soil phosphatase varied significantly among the treatments from July to September, with the order of T1 > T3 > T2 > T4, and the enzyme activity in successive month gradually increased over time compared to the former month. For all treatments, the activity of soil phosphatase reached a maximum level in September, with levels 1.22 (T1), 1.0 (T2), 1.32 (T3), and 1.21-fold (T4) greater than the levels in August (Fig. 2C). 3.4. Influence of organic matter on the quantity of bacteria, fungi, and actinomycetes in the soil
Fig. 1. Effects of organic matter on the activities of (A), superoxide dismutase (SOD); (B), peroxidase (POD); and (C), catalase (CAT) in the root system of M. hupehensis seedlings grown under four treatments condition on June, July, August and September.
The application of organic matter to the replant soil significantly changed the quantity of bacteria in the soil, which was consistent with the change in the activity of soil phosphatase (Fig. 2C). Namely, from July to September, the number of soil bacteria was in the order of T1 > T3 > T2 > T4, with increasing over time. In September, the
56
Z. Zhang et al. / Scientia Horticulturae 146 (2012) 52–58
Fig. 2. Effects of organic matter on the activities of (A), soil urease; (B), soil invertase; and (C), soil phosphatase during the process of different treatments on June, July, August and September.
bacterial quantity in all the treatments peaked, and the number of bacteria in T1 was 1.96, 1.52, and 3.26-fold greater than the number of bacteria in T2, T3, and T4, respectively (Fig. 3A). The addition of organic matter to the replant soil also increased the quantity of fungi in the soil. In July, the number of fungi in T2 and T3 was 1.48 and 1.7-fold greater than that in T4, respectively; also higher than that in T1, but T1 was higher than T4. In August, the number of fungi in T1 was slightly higher than that of T3, with the fungal number being 1.31, 1.05, and 1.95-fold greater than the fungal number in T2, T3, and T4, respectively. In addition, the number of fungi in T3 was remarkably higher than that of T2. In September, the number of fungi in T1 was still the highest, with the fungal number being 1.12, 1.19, and 1.26-fold greater than the fungal number in T2, T3, and T4, respectively; while T2 surpassed T3 (Fig. 3B). From June to September, the number of actinomycetes in all treatments increased, and the content of actinomycetes in T1 was significantly higher than that of T2, T3, and T4. The addition of either fermented fluid (T3) or compost (T2) to the replant soil significantly enhanced the content of actinomycetes in the soil as compared to
Fig. 3. Effects of organic matter on the quantities of (A), bacteria; (B), fungi; and (C), actinomycetes in the soils with different treatments on June, July, August and September.
T4 (Fig. 3 C). In July, the content of actinomycetes in T3 was slightly higher than that of T2, with a ratio of 1.05; on the contrary, in both August and September, the actinomycetes content in T2 was higher than that in T3, with ratios of 1.14 and 1.06, respectively. However, there were no significant differences between T2 and T3 in September (Fig. 3 C). 4. Discussion In this study, we showed that the application of fermented fluid to replant soil increased the biomass production of M. hupehensis seedlings. It has also been previously reported that the application of fermented fluid from organic matter increases crop yield (Khalil et al., 2000). Thus, the fermented fluid played a role in alleviating ARD. This study demonstrated that the addition of fermented fluid or compost to replant soil increased the activities of SOD, POD, and CAT in the roots of M. hupehensis seedlings compared to the activities in the roots of seedlings grown in replant soil without
Z. Zhang et al. / Scientia Horticulturae 146 (2012) 52–58
supplements. Antioxidant enzymes, such as SOD, POD, and CAT, are important for the removal of excessive radicals in plant tissues (Chaparzadeh et al., 2004; Uzildaya et al., 2012). Numerous studies have demonstrated that the application of organic matter improves the systematic resistance in plants (Vallad et al., 2003; Wu et al., 2008). The compost protects plants from oxidative damage, and this protection is performed via significant increase in the activities of the antioxidant enzymes (Tartoura and Youssef, 2010). Their research results coincide with the conclusion that compost components, i.e. organic matters, humic substances, biotic agents, are effectively contributed in the physiological metabolism to counteract oxidative stress induced by Cd2+ contaminated soil (Tartoura and Youssef, 2010). The present result showed that the fermented fluid from organic matter induced higher activities of SOD, POD, and CAT in the roots of M. hupehensis seedlings than the solid compost, and perhaps the fermented fluid was more helpful to activate a stronger antioxidant capability in the plant, thus mitigating the damages from environmental stress to the plant. Soil enzymes are derived from plant residues, animal carcasses, and soil microbes and they are key indicators for topsoil health ˇ (Snajdr and Baldrian, 2006). In the present study, the addition of organic matter in the form of fermented fluid or compost to replant soil increased the activities of soil urease, soil invertase, and soil phosphatase, which may have resulted from increased soil humus content, enhanced viability of soil microbes, and accelerated carbon and nutritional cycling in the soil (Casucci et al., 2003; Tian ˇ et al., 2010; Stursová and Baldrian, 2011). The application of maize residues to soil without earthworms significantly enhances the five soil enzyme activities (soil protease, urease, invertase, alkaline phosphatase and dehydrogenase) compared with the control treatment during rice and wheat cultivation (Tao et al., 2009). The abovementioned result demonstrates organic matter can promote the soil enzymes. Applications of compost and compost extracts are identified as promising, practical tools for managing ARD, especially under the marginal production conditions of South African apple producing regions (van Schoor et al., 2009). Furthermore, van Schoor et al. (2009) also verifies that compost and compost extracts still significantly increase seedling growth parameters for several of the ARD soils tested; suggesting that they can ameliorate the effects of ARD, in addition to supplying nutrients. Thus, the compost contributed to alleviating ARD. Moreover, we found the fermented fluid led to higher soil enzyme activities than the compost, which might be due to the difference in nutrient availability ˇ in the two supplements, as supported by the study of Snajdr and Baldrian (2006); and they conclude that nutrient availability affects the activities of soil enzymes. Soil microbes have a close relationship with the stability and function of the soil system (Singh et al., 2007; TerhoevenUrselmans et al., 2009). In aged apple orchards, the structure of bacterial and fungal communities in the rhizosphere soil has changed over time (Rumberger et al., 2007). The addition of organic matter to the replant soil dramatically changed the structure of soil microbe communities, including increased quantities of bacteria, fungi, and actinomycetes; this change may have been due to richer energetic contents supplied by the organic matter to the soil microbes in the replant soil (Patra et al., 1995). Laurent et al. (2008) consider that there is a positive correlation between biomass production of replanted apple seedlings and the amount of soil bacteria and soil fungi. Consistent with the present study, Tewoldemedhin et al. (2011a) also advocate that actinomycetes may not be responsible for the occurrence of ARD. The alleviation of ARD observed in the present study may have resulted from the increase of beneficial fungi. Soil fertility, abundance, and diversity of fungi have been reported to relieve ARD (Laurent et al., 2008). In the present study, the addition of organic matter compost or fermented fluid to replant soil improved M. hupehensis seedling biomass
57
production and could create a more comfortable and friendly soil environment. The study of organic matter application in the form of fermented fluid to replant soil provided a new approach for the prevention of ARD. The effects of two types of organic matter supplements (fermented fluid and compost) on ARD control were compared in this study. However, the mechanism behind the difference, especially the mechanism responsible for the better ARD control with fermented fluid, remains to be addressed in future studies. Acknowledgments The research was supported by the earmarked fund for National Modern Agro-industry Technology Research System (CARS–28) and the Key Innovation Project for Agricultural Application Technology of Shandong Province. References Bernard, E., Larkin, R.P., Tavantzis, S., Erich, M.S., Alyokhin, A., Sewell, G., Lannan, A., Gross, S.D., 2012. Compost rapeseed rotation, and biocontrol agents significantly impact soil microbial communities in organic and conventional potato production systems. Appl. Soil Ecol. 52, 29–41. Brown, M.W., Tworkoski, T., 2004. Pest management benefits of compost mulch in apple orchards. Agr. Ecosyst. Environ. 103, 465–472. Casucci, C., Okeke, B.C., Frankenberger, W.T.J., 2003. Effects of mercury on microbial biomass and enzyme activities in soil. Biol. Trace Elem. Res. 94, 179–191. Chang, C.Y., Chao, C.C., Chao, W.L., 2008. Community structure and functional diversity of indigenous fluorescent Pseudomonas of long-term swine compost applied maize rhizosphere. Soil Biol. Biochem. 40, 495–504. Chaparzadeh, N., D’Amico, M.L., Khavari-Nejad, R.A., Izzo, R., Navari-Izzo, F., 2004. Antioxidative responses of Calendula officinalis under salinity conditions. Plant Physiol. Biochem. 42, 695–701. Cheng, L.J., Xue, Q.H., 2000. Microbiology Experiment Technology. World Publishing Corporation, Xi’an, China, pp. 80–83 (in Chinese). Crecchio, C., Stotzky, G., 2001. Biodegradation and insecticidal activity of the toxin from Bacillus thuringiensis subsp kurstaki bound on complexes of montmorillonite-humic acids-A1 hydroxypolymers. Soil Biol. Biochem. 33, 573–581. Ghorbani, R., Koocheki, A., Jahan, M., Asadi, G.A., 2008. Impact of organic amendments and compost extracts on tomato production and storability in agroecological systems. Agron. Sustain. Dev. 28, 307–311. Guan, S.Y., 1986. Soil Enzyme and its Research Method. Agricultural Press, Beijing, pp. 274–340 (in Chinese). Hagn, A., Engel, M., Kleikamp, B., Munch, J.C., Schloter, M., Bruns, C., 2008. Microbial community shifts in Pythium ultimum-inoculated suppressive substrates. Biol. Fertil. Soils 44, 481–490. Khalil, M.E.A., Badran, N.M., El-Emam, M.A.A., 2000. Effect of different organic manures on growth and nutritional status of corn. Egypt J. Soil Sci. 40, 245–263. Laurent, A.S., Merwin, I.A., Thies, J.E., 2008. Long-term orchard groundcover management systems affect soil microbial communities and apple replant disease severity. Plant Soil 304, 209–225. Leinfelder, M.M., Merwin, I.A., 2006. Rootstock selection, preplant soil treatments, and tree planting positions as factors in managing apple replant disease. HortScience 41, 394–401. Loria, E.R., Sawyer, J.E., Barker, D.W., Lundvall, J.P., Lorimor, J.C., 2007. Use of anaerobically digested swine manure as a nitrogen source in corn production. Agron. J. 99, 1119–1129. Manici, L.M., Ciavatta, C., Kelderer, M., Erschbaumer, G., 2003. Replant problems in South Tyrol: role of fungal pathogens and microbial population in conventional and organic apple orchards. Plant Soil 256, 315–324. Omran, R.G., 1980. Peroxide levels and the activities of catalase peroxidase, and indoleacetic acid oxidase during and after chilling cucumber seedlings. Plant Physiol. 65, 407–408. Patra, D.D., Chand, S., Anwar, M., 1995. Seasonal changes in microbial biomass in soils cropped with palmarosa (Cymbopogon martinii L.) and Japanese mint (Mentha arvensis L.) in subtropical India. Biol. Fertil. Soils 19, 193–196. Rumberger, A., Merwin, I.A., Thies, J.E., 2007. Microbial community development in the rhizosphere of apple trees at a replant site. Soil Biol. Biochem. 39, 1645–1654. Rumberger, A., Yao, S., Merwin, I.A., Nelson, E.B., Thies, J.E., 2004. Rootstock genotype and orchard replant position rather than soil fumigation or compost amendment determine tree growth and rhizosphere bacterial community composition in an apple replant soil. Plant Soil 264, 247–260. Singh, B.K., Sharma, S.R., Singh, B., 2010. Antioxidant enzymes in cabbage: variability and inheritance of superoxide dismutase, peroxidase and catalase. Sci. Hortic. 124, 9–13. Singh, K.P., Suman, A., Singh, P.N., Srivastava, T.K., 2007. Improving quality of sugarcane-growing soils by organic amendments under subtropical climatic conditions of India. Biol. Fertil. Soils 44, 367–376.
58
Z. Zhang et al. / Scientia Horticulturae 146 (2012) 52–58
ˇ Snajdr, J., Baldrian, P., 2006. Production of lignocellulose-degrading enzymes and changes in soil bacterial communities during the growth of Pleurotus ostreatus in soil with different carbon content. Folia. Microbiol. 51, 579–590. ˇ Stursová, M., Baldrian, P., 2011. Effects of soil properties and management on the activity of soil organic matter transforming enzymes and the quantification of soil-bound and free activity. Plant Soil 338, 99–110. Tao, J., Griffiths, B., Zhang, S., Chen, X., Liu, M., Hu, F., Li, H., 2009. Effects of earthworms on soil enzyme activity in an organic residue amended rice–wheat rotation agro-ecosystem. Appl. Soil Ecol. 42, 221–226. Tartoura, K.A.H., Youssef, S.A.G., 2010. Effect of compost on the antioxidant defense systems of cucumber (Cucumis sativus L.) against cadmium toxicity. Ann. Agric. Sci. (Cairo) 55, 191–203. Terhoeven-Urselmans, T., Scheller, E., Raubuch, M., Ludwig, B., Joergensen, R.G., 2009. CO2 evolution and N mineralization after biogas slurry application in the field and its yield effects on spring barley. Appl. Soil Ecol. 42, 297–302. Tewoldemedhin, Y.T., Mazzola, M., Labuschagne, I., McLeod, A., 2011a. A multiphasic approach reveals that apple replant disease is caused by multiple biological agents with some agents acting synergistically. Soil Biol. Biochem. 43, 1917–1927. Tewoldemedhin, Y.T., Mazzola, M., Botha, W.J., Spies, C.F.J., McLeod, A., 2011b. Characterization of fungi (Fusarium and Rhizoctonia) and oomycetes (Phytophthora and Pythium) associated with apple orchards in South Africa. Eur. J. Plant Pathol. 130, 215–229. Tewoldemedhin, Y.T., Mazzola, M., Mostert, L., McLeod, A., 2011c. Cylindrocarpon species associated with apple tree roots in South Africa and their quantification using real-time PCR. Eur. J. Plant Pathol. 129, 637–651. Tian, L., Dell, E., Shi, W., 2010. Chemical composition of dissolved organic matter in agroecosystems: correlations with soil enzyme activity and carbon and nitrogen mineralization. Appl. Soil Ecol. 46, 426–435.
Uzilday, B., Turkan, I., Sekmen, A.H., Ozgur, R., Karakaya, H.C., 2012. Comparison of ROS formation and antioxidant enzymes in Cleome gynandra (C4 ) and Cleome spinosa (C3 ) under drought stress. Plant Sci. 182, 59–70. Vallad, G.E., Cooperband, L., Goodman, R.M., 2003. Plant foliar disease suppression mediated by composted forms of paper mill residuals exhibits molecular features of induced resistance. Physiol. Mol. Plant Pathol. 63, 65–77. van Schoor, L., Denman, S., Cook, N.C., 2009. Characterisation of apple replant disease under South African conditions and potential biological management strategies. Sci. Hortic. 119, 153–162. Wu, H.S., Yang, X.N., Fan, J.Q., Miao, W.G., Ling, N., Xu, Y.C., Huang, Q.W., Shen, Q.R., 2008. Suppression of Fusarium wilt of watermelon by a bio-organic fertilizer containing combinations of antagonistic microorganisms. BioControl 54, 287–300. Yao, S.R., Merwin, I.A., Abawi, G.A., Thies, J.E., 2006. Soil fumigation and compost amendment alter soil microbial community composition but do not improve tree growth or yield in an apple replant site. Soil Biol. Biochem. 38, 578–599. Yogev, A., Raviv, M., Hadar, Y., Cohen, R., Katan, J., 2006. Plant waste-based composts suppressive to diseases caused by pathogenic Fusarium oxysporum. Eur. J. Plant Pathol. 116, 267–278. Zaman, M., Matsushima, M., Chang, S.X., Inubushi, K., Nguyen, L., Goto, S., Kaneko, F., Yoneyama, T., 2004. Nitrogen mineralization, N2 O production and soil microbiological properties as affected by long-term applications of sewage sludge composts. Biol. Fertil. Soils 40, 101–109. Zhang, Y.K., Han, X.J., Chen, X.L., Jin, H., Cui, X.M., 2009. Exogenous nitric oxide on antioxidative system and ATPase activities from tomato seedlings under copper stress. Sci. Hortic. 123, 217–223.
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
The effects of organic matter on the physiological features of Malus hupehensis seedlings and soil properties under replant conditions Zhaobo Zhang a , Qiang Chen a,b , Chengmiao Yin a , Xiang Shen a , Xuesen Chen a , Haibing Sun a , An’ni Gao a , Zhiquan Mao a,∗ a b
College of Horticultural Science and Engineering/State Key Laboratory of Crop Biology, Shandong Agricultural University, No. 61 Daizong Street, Tai’an, Shandong 271018, China Shandong Institute of Science and Technology Information, Jinan 250101, China
a r t i c l e
i n f o
Article history: Received 10 April 2012 Received in revised form 8 August 2012 Accepted 9 August 2012 Keywords: Malus hupehensis Rehd. Organic matter Physiological features Replant Soil properties
a b s t r a c t Two fermentation methods, aerobic (generating solid compost) and anaerobic fermentation (generating fermented fluid), were adopted to treat the same amount of organic matter to generate solid compost and fermented fluid, respectively. These treatments were used to supplement replant soils in a pot, respectively, where Malus hupehensis Rehd. seedling was replanted. The pilot project was aimed at investigating the influence of organic matter applied to soil on the physiological features of the plant as well as its impact on soil properties. The following four treatments were adopted in the study: new cropping soil, replant soil, replant soil with solid compost, and replant soil with fermented fluid. The growth of seedlings in the four soils was monitored by biomass production and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the plant roots. The activities of soil urease, soil invertase, and soil phosphatase were measured; and the bacterial, fungal, and actinomycetal contents in the soils were also measured. This study showed that the supplementation of organic matter in a fermented liquid form to replant soil significantly enhanced the biomass production of the seedlings compared to the solid compost form. The activities of SOD, POD, and CAT in the roots were increased by both forms of organic matter, with the solid compost form having a stronger effect than the fermented liquid form. Moreover, both forms increased the activities of soil urease, soil invertase, and soil phosphatase, with the fermented liquid form having a stronger impact than the solid compost form. The addition of organic matter in the form of fermented fluid to the replant soil obviously increased the bacteria content in the soil in comparison with the addition of organic matter in the form of solid compost. Compared to the solid compost, the fermented fluid led to a higher increase in the fungal and actinomycetal contents in the soil at the early stage but a lower increase at the late stage. When applied to replant soil, the fermented fluid form of organic matter was superior to the solid compost form of organic matter in preventing apple replant disease (ARD). © 2012 Elsevier B.V. All rights reserved.
1. Introduction Apple replant disease (ARD) causes the inhibition of root system development, stunts tree growth, and reduces yield and quality in replanted apple orchards (Laurent et al., 2008). In replant orchards, the ARD resulting from replanting in the row is more severe than that resulting from replanting between rows (Rumberger et al., 2004; Leinfelder and Merwin, 2006). ARD is often caused by a consortium of biological agents, mainly, including nematodes, bacteria, actinomycete, oomycetes and fungi species (Tewoldemedhin et al., 2011a). van Schoor et al. (2009) thought that Pratylenchus penetrans was the primary nematode species involved in ARD.
∗ Corresponding author. Tel.: +86 538 8241984. E-mail address: [email protected] (Z. Mao). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.08.011
Although several bacterial genera and species have been associated and suggested as being involved in ARD, most bacteria likely impaired plant at inordinately high densities (Tewoldemedhin et al., 2011a). Evidence for the involvement of actinomycetes in ARD is circumstantial (Tewoldemedhin et al., 2011a). However, most researches demonstrated fungal and oomycete genera were the main reason for apple replant disease, i.e. fungal genera: Fusarium (Tewoldemedhin et al., 2011a,b), Rhizoctonia (Tewoldemedhin et al., 2011a,b), Cylindrocarpon (Manici et al., 2003; Tewoldemedhin et al., 2011a,c); oomycete genera: Phytophthora (Tewoldemedhin et al., 2011a,b), Pythium (Tewoldemedhin et al., 2011a,b). Methyl bromide has been widely used in effectively preventing ARD, but it causes serious pollution problems to environment. Hence, it is imperative to find an alternative to replace soil fumigants, such as methyl bromide (Yao et al., 2006). Applying organic matter to soils not only alleviates the adverse effects brought by
Z. Zhang et al. / Scientia Horticulturae 146 (2012) 52–58
many unfavorable factors (Chang et al., 2008) but also increases the quantity and activity of soil microbes (Crecchio and Stotzky, 2001; Bernarda et al., 2012). Irradiation treatment of organic matter compost reduces the capability of the compost to inhibit Fusarium wilt, which indicates that the microbes in the compost have important roles in suppressing pathogens (Yogev et al., 2006; Hagn et al., 2008). In an orchard located in West Virginia in the United States, it has been reported that the application of compost derived from organic matter increases the quantity of beneficial arthropods, improves pest control in the orchard, decreases pathogenic fungi occurrence, and inhibits Monilinia fructicola, thus promoting robust tree growth and maintaining sustainable productivity of the orchard (Brown and Tworkoski, 2004). The application of compost consisting of organic matter can also significantly increase the growth rate of seedlings under replant conditions (van Schoor et al., 2009), which leads to a longer growth period of fruit plants than that of plants treated with methyl bromide (Yao et al., 2006). In addition, the application of organic matter compost leads to a higher usage rate of carbohydrates of i-erythritol and l-rhamnose (Chang et al., 2008) and, therefore, enhances the level of organic carbon in the soil and improves the soil pH (Zaman et al., 2004). Previous studies have demonstrated that the inhibition of Fusarium spp. in apple trees by compost is related to the organic matter composition, maturity level, and processing methods of the compost as well as its application time and amount (Ghorbani et al., 2008). Organic matter fermented fluid is the end product of anaerobic fermentation, and it can increase the quantity of soil microbes (Singh et al., 2007; Terhoeven-Urselmans et al., 2009). As a highquality organic fertilizer, fermented fluid improves fruit yield and quality (Loria et al., 2007; Terhoeven-Urselmans et al., 2009). To date, there is no report that compares the effects on ARD control by applying organic matter fermented fluid and solid compost. In the present study, the common apomictic rootstock, Malus hupehensis Rehd., was used as a subject to investigate the effects of organic matter solid compost and fermented fluid on the physiological features of seedlings and soil properties to provide theoretical and practical implications for the control of ARD.
2. Materials and methods 2.1. Experimental materials and treatment The experiment was carried out in 2010 in a 20-year-old Red Fuji apple orchard located in the suburb of Tai’An city, Shandong province of China; and at the fruit tree root system laboratory of the College of Horticultural Sciences and Engineering of Shandong Agricultural University, also located in Tai’An city, respectively. M. hupehensis Rehd., which is a commonly used rootstock for apple trees, was used in this study. The seeds were stratified for 30 days at 4 ◦ C. After shoot tip emergence, the seeds were planted in nursery plates. The seedlings with six leaves were transplanted to a clay pot (with an outside diameter of 29 cm and an inner diameter of 25 cm) that contained 7 kg of replant soil collected from the abovementioned apple orchard. In the 20-year-old Red Fuji apple orchard, its original rootstock was M. robusta, and the soil type was cinnamon soil. The soil was randomly collected from multiple locations in the orchard (locations were 1 m away from the trunk) at a soil depth of 5–40 cm, and the soil samples were mixed well. The organic matter used in this study consisted of chicken feces, sheep manure, cow dung, and wheat straw (2 cm in length) mixed in a ratio of 6:1:1:2 (v/v). The organic matter was routinely composted under natural conditions. The organic matter was fermented by placing the well-mixed organic matter into a white plastic bucket (50 cm in diameter and 120 cm in height) and adding water until the contents were submerged under 30 cm.
53
After the content of the organic matter was mixed with water using a stick, the bucket was sealed with a plastic membrane that contained ventilation holes. The bucket was then buried until 2/3 of the bucket was embedded underground, stirred 5 times with a stick during the whole fermentation process and the fermentation process also proceeded under natural conditions. The compost and fermentation liquid were prepared on May 1, June 15, and July 20. The weight ratio of organic matter (prepared for solid compost and fermented fluid forms) to replant soil (supplemented with solid compost and fermented fluid forms) was 1:25. The ratio originated from growers’ planting experience. Considering the increasing temperature over time, we gradually shortened the organic matter preparation time. The organic matter (compost and fermented fluid forms) was applied to the pots on June 20, July 20, and August 20, respectively. The 4% solid compost of replant soil weight in each pot was supplemented to the surface of soil; subsequently, dug the surface of replant soil repeatedly with a small hoe until the solid compost was mixed even with the soil surface. As for fermented fluid forms, initially mixed the content well in the white plastic bucket with a stick, shared the fermented fluid forms and watered the pilot seedlings in succession; after the fermented fluid forms drying, treated them as the abovementioned compost. Four treatments were set up as follows: T1, newly cropping soil collected from the neighboring wheat field outside the orchard; T2, replant soil collected from the orchard supplemented with solid compost; T3, replant soil collected from the orchard supplemented with fermented fluid from the organic matter; and T4, replant soil collected from the orchard (control). The nutrient and water were managed as usual, and the N, P, and K contents in the soil were normalized among the treatment pots. Two seedlings were planted in each pot, and 30 pots (6 pots were spare to prevent undesirable condition among them, only 24 pots were occupied for each treatment in the experiment in fact) were prepared for each treatment. Each treatment contained 3 biological replications at the same point in time. For the antioxidant enzymes, every biological replication consisted of 4 seedlings, namely 4 seedlings (2 pots) are one set and as a sampling unit. Roots less than 5 cm in length were gathered and mixed well for antioxidant enzymes assay. For the soil enzyme and quantity of soil microbes, the pot soil (∼4 cm radius around each seedling and ∼6 cm in depth) in 2 pots was collected and mixed well as a biological replication. The samples were collected at 9:00 a.m. to 10:00 a.m. at a time. The first sampling was done on June 20 (June) before the application of organic matter on the same day. And the rest samplings were done after 20 days treatment; namely, on July 10 (July), August 10 (August) and September 10 (September), respectively. And the collected samples were prepared for further assay.
2.2. Antioxidant enzymes assay in root The activities of antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)) in root were determined. The SOD activity assay was performed as previously described (Zhang et al., 2009). The SOD activity unit (U) was defined as the amount of enzyme causing half of the maximum inhibition of nitroblue tetrazolium (NBT) reduction, and the enzyme activity was expressed as U g−1 FW. The POD activity assay was performed according to the method developed by Omran (1980). The POD activity was assayed spectrophotometrically by monitoring changes in absorbance at 470 nm. The POD activity unit (U) was defined as the amount of enzyme causing a change of 0.01 in the absorbance at 470 nm per minute, and the enzyme activity was expressed as U g−1 FW. The CAT activity assay was also performed spectrophotometrically by monitoring changes in absorbance at 240 nm (Singh et al., 2010). The CAT activity unit (U) was defined
54
Z. Zhang et al. / Scientia Horticulturae 146 (2012) 52–58
as the amount of enzyme causing a change of 0.1 in the absorbance at 240 nm/min, and the activity was expressed as U g−1 FW.
volumetric flask and diluted to 100 mL, respectively. The absorption was assayed at 570 nm when the color was kept relatively stable and drew the standard curve.
2.3. Soil enzyme assays 2.4. Determination of the quantity of soil microbes The soil enzyme assays were performed as previously described by Guan (1986). In detail, the activity of soil urease was assayed by using indophenol blue method with minor changes. 5 g air-dried soil was placed in a 50 mL volumetric flask, added 1 mL toluene, filled the top of the bottle with a stopper tightly and jiggled the volumetric flask for 15 min. Subsequently 5 mL 10% urea solution and 10 mL citrate buffer (pH 6.7) were added to the volumetric flask. The mixture was incubated at 37 ◦ C for 24 h after mixing well carefully. Then distilled water at 38 ◦ C was applied to dilute the mixture to 50 mL (the toluene should float above the scale), mixed them well, and the suspension was filtered. 1 mL filtrate was placed in a 50 mL volumetric flask, diluted with distilled water to 10 mL, followed by adding 4 mL sodium phenolate solution, and immediately joined 3 mL sodium hypochlorite solution. After the mixture was shaken well and placed for 20 min, the mixture was diluted to 50 mL. Its absorption was measured at 578 nm. The abovementioned steps without soil were used for a control. The D-value of absorption (sample minus control) was used to calculate the activity of soil urease. The activity of soil urease was expressed as mg released NH3 -N by 1 g soil at 37 ◦ C per 24 h. The ammonia nitrogen quantity was obtained according to the standard curve. Standard curve: added 0, 1, 3, 5, 7, 9, 11, 13 mL ammonium sulfate solution (0.01 mg mL−1 ) to a 50 mL volumetric flask, respectively. The above flasks were diluted to 20 mL with distilled water. Thereafter 4 mL sodium phenolate solution and 3 mL sodium hypochlorite solution were added. Mixed them well coupling with the adding process. The mixture colored after 20 min, set the volume. The absorption was determined at 578 nm within 1 h. For invertase activity with minor alterations, 5 g of air-dried soil was mixed with 15 mL 8% sucrose and 5 mL phosphate buffer at pH 5.5 and 0.1 mL toluene. The mixture was incubated for 24 h at 37 ◦ C. The suspension was filtered. 1 mL filtrate was transferred to a 50 mL volumetric flask. Added 3 mL 3,5-dinitrosali-cylic acid and heated it in a boiling water bath for 5 min, then the flask was moved to running water and cooled it for 3 min. Afterwards, diluted the mixture with distilled water to 50 mL. The glucose from sucrose hydrolysis catalyzed by invertase reacted with 3,5-dinitrosali-cylic acid. The product 3-aminonitrosalicylic acid was determined the absorption at 508 nm. The activity of invertase was calculated by mg released glucose by 1 g soil per 24 h. A control without soil was used for each sample. Standard curve: added 0, 1, 2, 3, 4, 5, 6, 7 mL glucose solution (0.5 mg mL−1 ) to a 50 mL volumetric flask, respectively. Operated them as the abovementioned method of assaying invertase activity, the standard curve was drawn on the basis of colorimetric absorbance for the vertical axis and glucose concentration as the abscissa. For neutral phosphatase with some modifications, 5 g of airdried soil was incubated at 37 ◦ C for 24 h with 1 mL toluene, 5 mL disodium phenyl phosphate (6.75 g C6 H5 PO4 Na2 ·2H2 O dissolved in 1 L distilled water) and 5 mL citrate buffer (pH 7.0). Afterwards, the mixture was diluted with distilled water at 38 ◦ C to 50 mL and filtered. 1 mL filtrate mixed well with 5 mL (borate buffer pH 9.0), 3 mL 2.5% potassium ferricyanide and 3 mL 0.5% 4-aminoantipyrine. The solution was diluted to 100 mL after changing into pink. The absorption was assayed at 570 nm when the color was kept relatively stable (about 20–30 min). A control without soil was used for each sample. The activity of neutral phosphatase was described as mg released phenol by 1 g soil per 24 h. The phenol content was obtained according to the standard curve. Standard curve: 1, 3, 5, 7, 9, 11 mL phenol solution (0.05 mg mL−1 ) was injected to a 100 mL
The quantity of soil microbes was determined using the agar plate dilution method as previously described by Cheng and Xue (2000). Beef peptone medium was used for the culture of bacteria, and Martin’s medium was used for the culture of fungi. Moreover, Gao 1 agar medium was used for the culture of actinomycetes. 2.5. Statistical analysis The mean separation and analysis of variance were calculated using the Microsoft Excel 2003 and DPS V7.05 software. The experimental treatment consisted of three biological replicates. The data represent mean values ± standard errors (n = 3); different lower case letters a, b, c and d indicate statistical significance at the 0.05 level (Duncan’s new multiple range test, P < 0.05). 3. Results 3.1. Effects of organic matter on the biomass production of M. hupehensis seedlings under replant conditions in September 2010 The biomass of M. hupehensis seedlings was determined on September 10, 2010. Here, we only enumerated the biomass in September. The similar varying trend was observed in July 10 and August 10, but data are not shown to avoid repetition. The height, basal diameter, aboveground fresh biomass weight, and fresh root weight of the seedlings were the highest in treatment T1 (newly cropping soil collected from the neighboring wheat field outside the orchard) followed by those in treatment T3 (replant soil collected from the orchard supplemented with fermented fluid from the organic matter), T2 (replant soil collected from the orchard supplemented with solid compost), and T4 (replant soil collected from the orchard; control), which had the lowest values (Table 1). The height, basal diameter, aboveground fresh biomass weight, and fresh root weight of the seedlings grown in T3 were 0.82, 0.96, 0.79, and 0.83-fold greater than those grown in T1, respectively. However, they were 1.31, 1.13, 1.68, and 1.22-fold greater than those grown in T2, respectively; furthermore, 1.9, 1.56, 2.14, and 1.8-fold greater than those in grown in T4 (control group), respectively. 3.2. Influence of organic matter on the activities of SOD, POD, and CAT in the root system of M. hupehensis seedlings grown under replant conditions The application of organic matter to the replant soil enhanced the SOD activity in the root system of M. hupehensis seedlings with the time prolonged, and the enzyme activity significantly varied among the treatments (Fig. 1A). In July, the order of SOD activity in the root system of M. hupehensis seedlings among the treatments was T1 > T2 > T3 > T4, with only limited increases in all the treatments. In August, the SOD activity in the root system of M. hupehensis seedlings grown in T3 was the highest followed by the activity of the seedlings grown in T1 and T2, and all of these increases were significant, T4 was the lowest. In September, the SOD activities in the root system of M. hupehensis seedlings grown in T2 and T3 reached the highest level (approximately 222.5 U g−1 FW, and they were no significant difference), which was much higher than the activities observed in T1 and T4. With organic matter addition to the replant soil, the POD activity in the root system of M. hupehensis seedlings was also enhanced with the extension of time, and it significantly varied among the
Z. Zhang et al. / Scientia Horticulturae 146 (2012) 52–58
55
Table 1 Effects of organic matter on the biomass production of M. hupehensis seedlings under replant conditions in September 2010. Treatment
Plant height (cm)
Basal diameter (cm)
Aboveground fresh biomass weight (g)
Fresh root weight (g)
T1 T2 T3 T4
59.00 ± 0.29 a 37.00 ± 0.76 c 48.50 ± 0.29 b 25.50 ± 1.04 d
0.6030 ± 0.0055 a 0.5100 ± 0.0055 c 0.5780 ± 0.0044 b 0.3700 ± 0.0026 d
13.08 ± 0.05 a 6.16 ± 0.05 c 10.34 ± 0.02 b 4.84 ± 0.03 d
11.67 ± 0.06 a 8.00 ± 0.08 c 9.74 ± 0.03 b 5.41 ± 0.02 d
Values are means of three replicates ± standard error. Different lower case letters within the same column indicate significant difference at 5% level (Duncan’s new multiple range test).
treatments (Fig. 1B). In July, the POD activity in the root system of M. hupehensis seedlings grown in T2 was the highest, even surpassing that in T1, whereas T3 was lower than T1. Afterwards, the activity of T3 increased rapidly, in August, the POD activity in the root system of M. hupehensis seedlings grown in T3 reached its peak value and was 1.06, 1.15, and 1.54-fold greater than that of seedlings grown
in T1, T2, and T4, respectively. In September, the POD activity in the root system of M. hupehensis seedlings grown in T1 was the highest and was 1.2, 1.32, and 2.1-fold greater than that of seedlings grown in T2, T3, and T4, respectively. Compared to that in August, the POD activity of T3 in September decreased; in contrast, the POD activity of T2 in September rose. From July to September, there were no statistical difference in between T1 and T3, as well between T2 and T4. T1 and T3 had also higher CAT activity than T2 and T4. CAT activity in the root system was the highest in August (Fig. 1C). 3.3. Influence of organic matter on the activities of soil urease, soil invertase, and soil phosphatase Compared to those in June, the activities of soil urease except T4 in July ascended; those in August and September were greater than those in June. In July, the activity of soil urease was the highest in T2 followed by T1 and T3, with a significant difference among the treatments. In August, the addition of fermented fluid to the replant soil increased the soil urease activity in T3 to its highest level, with levels 1.21, 1.69, and 1.76-fold greater than the levels in T1, T2, and T4, respectively. In September, the highest activity of soil urease was found in T1, which was slightly higher than the activity found in T3, no obvious difference between them; but the both were significantly higher than the activity found in T2 and T4 (Fig. 2A). The activity of soil invertase in different treatments advanced with the progress of time. The addition of fermented fluid to the replant soil (T3) led to a slightly higher soil invertase activity than that of T4 in July and August, 1.13-fold (significant difference, Fig. 2B) and 1.03-fold (no-significant difference, Fig. 2B), respectively; but to a markedly higher soil invertase activity than that of T4 in September, up to 1.99-fold (significant difference, Fig. 2B). The activity of soil invertase in T1 was higher than that of T3, with levels 1.15, 1.26, and 1.61-fold greater than the levels in T3 in June, July, and August, respectively. In September, the activity of soil invertase was the highest in T3 followed by T1, and the activities in the both of these treatments were significantly higher than the activities in T2 and T4 (Fig. 2B). The activity of soil phosphatase varied significantly among the treatments from July to September, with the order of T1 > T3 > T2 > T4, and the enzyme activity in successive month gradually increased over time compared to the former month. For all treatments, the activity of soil phosphatase reached a maximum level in September, with levels 1.22 (T1), 1.0 (T2), 1.32 (T3), and 1.21-fold (T4) greater than the levels in August (Fig. 2C). 3.4. Influence of organic matter on the quantity of bacteria, fungi, and actinomycetes in the soil
Fig. 1. Effects of organic matter on the activities of (A), superoxide dismutase (SOD); (B), peroxidase (POD); and (C), catalase (CAT) in the root system of M. hupehensis seedlings grown under four treatments condition on June, July, August and September.
The application of organic matter to the replant soil significantly changed the quantity of bacteria in the soil, which was consistent with the change in the activity of soil phosphatase (Fig. 2C). Namely, from July to September, the number of soil bacteria was in the order of T1 > T3 > T2 > T4, with increasing over time. In September, the
56
Z. Zhang et al. / Scientia Horticulturae 146 (2012) 52–58
Fig. 2. Effects of organic matter on the activities of (A), soil urease; (B), soil invertase; and (C), soil phosphatase during the process of different treatments on June, July, August and September.
bacterial quantity in all the treatments peaked, and the number of bacteria in T1 was 1.96, 1.52, and 3.26-fold greater than the number of bacteria in T2, T3, and T4, respectively (Fig. 3A). The addition of organic matter to the replant soil also increased the quantity of fungi in the soil. In July, the number of fungi in T2 and T3 was 1.48 and 1.7-fold greater than that in T4, respectively; also higher than that in T1, but T1 was higher than T4. In August, the number of fungi in T1 was slightly higher than that of T3, with the fungal number being 1.31, 1.05, and 1.95-fold greater than the fungal number in T2, T3, and T4, respectively. In addition, the number of fungi in T3 was remarkably higher than that of T2. In September, the number of fungi in T1 was still the highest, with the fungal number being 1.12, 1.19, and 1.26-fold greater than the fungal number in T2, T3, and T4, respectively; while T2 surpassed T3 (Fig. 3B). From June to September, the number of actinomycetes in all treatments increased, and the content of actinomycetes in T1 was significantly higher than that of T2, T3, and T4. The addition of either fermented fluid (T3) or compost (T2) to the replant soil significantly enhanced the content of actinomycetes in the soil as compared to
Fig. 3. Effects of organic matter on the quantities of (A), bacteria; (B), fungi; and (C), actinomycetes in the soils with different treatments on June, July, August and September.
T4 (Fig. 3 C). In July, the content of actinomycetes in T3 was slightly higher than that of T2, with a ratio of 1.05; on the contrary, in both August and September, the actinomycetes content in T2 was higher than that in T3, with ratios of 1.14 and 1.06, respectively. However, there were no significant differences between T2 and T3 in September (Fig. 3 C). 4. Discussion In this study, we showed that the application of fermented fluid to replant soil increased the biomass production of M. hupehensis seedlings. It has also been previously reported that the application of fermented fluid from organic matter increases crop yield (Khalil et al., 2000). Thus, the fermented fluid played a role in alleviating ARD. This study demonstrated that the addition of fermented fluid or compost to replant soil increased the activities of SOD, POD, and CAT in the roots of M. hupehensis seedlings compared to the activities in the roots of seedlings grown in replant soil without
Z. Zhang et al. / Scientia Horticulturae 146 (2012) 52–58
supplements. Antioxidant enzymes, such as SOD, POD, and CAT, are important for the removal of excessive radicals in plant tissues (Chaparzadeh et al., 2004; Uzildaya et al., 2012). Numerous studies have demonstrated that the application of organic matter improves the systematic resistance in plants (Vallad et al., 2003; Wu et al., 2008). The compost protects plants from oxidative damage, and this protection is performed via significant increase in the activities of the antioxidant enzymes (Tartoura and Youssef, 2010). Their research results coincide with the conclusion that compost components, i.e. organic matters, humic substances, biotic agents, are effectively contributed in the physiological metabolism to counteract oxidative stress induced by Cd2+ contaminated soil (Tartoura and Youssef, 2010). The present result showed that the fermented fluid from organic matter induced higher activities of SOD, POD, and CAT in the roots of M. hupehensis seedlings than the solid compost, and perhaps the fermented fluid was more helpful to activate a stronger antioxidant capability in the plant, thus mitigating the damages from environmental stress to the plant. Soil enzymes are derived from plant residues, animal carcasses, and soil microbes and they are key indicators for topsoil health ˇ (Snajdr and Baldrian, 2006). In the present study, the addition of organic matter in the form of fermented fluid or compost to replant soil increased the activities of soil urease, soil invertase, and soil phosphatase, which may have resulted from increased soil humus content, enhanced viability of soil microbes, and accelerated carbon and nutritional cycling in the soil (Casucci et al., 2003; Tian ˇ et al., 2010; Stursová and Baldrian, 2011). The application of maize residues to soil without earthworms significantly enhances the five soil enzyme activities (soil protease, urease, invertase, alkaline phosphatase and dehydrogenase) compared with the control treatment during rice and wheat cultivation (Tao et al., 2009). The abovementioned result demonstrates organic matter can promote the soil enzymes. Applications of compost and compost extracts are identified as promising, practical tools for managing ARD, especially under the marginal production conditions of South African apple producing regions (van Schoor et al., 2009). Furthermore, van Schoor et al. (2009) also verifies that compost and compost extracts still significantly increase seedling growth parameters for several of the ARD soils tested; suggesting that they can ameliorate the effects of ARD, in addition to supplying nutrients. Thus, the compost contributed to alleviating ARD. Moreover, we found the fermented fluid led to higher soil enzyme activities than the compost, which might be due to the difference in nutrient availability ˇ in the two supplements, as supported by the study of Snajdr and Baldrian (2006); and they conclude that nutrient availability affects the activities of soil enzymes. Soil microbes have a close relationship with the stability and function of the soil system (Singh et al., 2007; TerhoevenUrselmans et al., 2009). In aged apple orchards, the structure of bacterial and fungal communities in the rhizosphere soil has changed over time (Rumberger et al., 2007). The addition of organic matter to the replant soil dramatically changed the structure of soil microbe communities, including increased quantities of bacteria, fungi, and actinomycetes; this change may have been due to richer energetic contents supplied by the organic matter to the soil microbes in the replant soil (Patra et al., 1995). Laurent et al. (2008) consider that there is a positive correlation between biomass production of replanted apple seedlings and the amount of soil bacteria and soil fungi. Consistent with the present study, Tewoldemedhin et al. (2011a) also advocate that actinomycetes may not be responsible for the occurrence of ARD. The alleviation of ARD observed in the present study may have resulted from the increase of beneficial fungi. Soil fertility, abundance, and diversity of fungi have been reported to relieve ARD (Laurent et al., 2008). In the present study, the addition of organic matter compost or fermented fluid to replant soil improved M. hupehensis seedling biomass
57
production and could create a more comfortable and friendly soil environment. The study of organic matter application in the form of fermented fluid to replant soil provided a new approach for the prevention of ARD. The effects of two types of organic matter supplements (fermented fluid and compost) on ARD control were compared in this study. However, the mechanism behind the difference, especially the mechanism responsible for the better ARD control with fermented fluid, remains to be addressed in future studies. Acknowledgments The research was supported by the earmarked fund for National Modern Agro-industry Technology Research System (CARS–28) and the Key Innovation Project for Agricultural Application Technology of Shandong Province. References Bernard, E., Larkin, R.P., Tavantzis, S., Erich, M.S., Alyokhin, A., Sewell, G., Lannan, A., Gross, S.D., 2012. Compost rapeseed rotation, and biocontrol agents significantly impact soil microbial communities in organic and conventional potato production systems. Appl. Soil Ecol. 52, 29–41. Brown, M.W., Tworkoski, T., 2004. Pest management benefits of compost mulch in apple orchards. Agr. Ecosyst. Environ. 103, 465–472. Casucci, C., Okeke, B.C., Frankenberger, W.T.J., 2003. Effects of mercury on microbial biomass and enzyme activities in soil. Biol. Trace Elem. Res. 94, 179–191. Chang, C.Y., Chao, C.C., Chao, W.L., 2008. Community structure and functional diversity of indigenous fluorescent Pseudomonas of long-term swine compost applied maize rhizosphere. Soil Biol. Biochem. 40, 495–504. Chaparzadeh, N., D’Amico, M.L., Khavari-Nejad, R.A., Izzo, R., Navari-Izzo, F., 2004. Antioxidative responses of Calendula officinalis under salinity conditions. Plant Physiol. Biochem. 42, 695–701. Cheng, L.J., Xue, Q.H., 2000. Microbiology Experiment Technology. World Publishing Corporation, Xi’an, China, pp. 80–83 (in Chinese). Crecchio, C., Stotzky, G., 2001. Biodegradation and insecticidal activity of the toxin from Bacillus thuringiensis subsp kurstaki bound on complexes of montmorillonite-humic acids-A1 hydroxypolymers. Soil Biol. Biochem. 33, 573–581. Ghorbani, R., Koocheki, A., Jahan, M., Asadi, G.A., 2008. Impact of organic amendments and compost extracts on tomato production and storability in agroecological systems. Agron. Sustain. Dev. 28, 307–311. Guan, S.Y., 1986. Soil Enzyme and its Research Method. Agricultural Press, Beijing, pp. 274–340 (in Chinese). Hagn, A., Engel, M., Kleikamp, B., Munch, J.C., Schloter, M., Bruns, C., 2008. Microbial community shifts in Pythium ultimum-inoculated suppressive substrates. Biol. Fertil. Soils 44, 481–490. Khalil, M.E.A., Badran, N.M., El-Emam, M.A.A., 2000. Effect of different organic manures on growth and nutritional status of corn. Egypt J. Soil Sci. 40, 245–263. Laurent, A.S., Merwin, I.A., Thies, J.E., 2008. Long-term orchard groundcover management systems affect soil microbial communities and apple replant disease severity. Plant Soil 304, 209–225. Leinfelder, M.M., Merwin, I.A., 2006. Rootstock selection, preplant soil treatments, and tree planting positions as factors in managing apple replant disease. HortScience 41, 394–401. Loria, E.R., Sawyer, J.E., Barker, D.W., Lundvall, J.P., Lorimor, J.C., 2007. Use of anaerobically digested swine manure as a nitrogen source in corn production. Agron. J. 99, 1119–1129. Manici, L.M., Ciavatta, C., Kelderer, M., Erschbaumer, G., 2003. Replant problems in South Tyrol: role of fungal pathogens and microbial population in conventional and organic apple orchards. Plant Soil 256, 315–324. Omran, R.G., 1980. Peroxide levels and the activities of catalase peroxidase, and indoleacetic acid oxidase during and after chilling cucumber seedlings. Plant Physiol. 65, 407–408. Patra, D.D., Chand, S., Anwar, M., 1995. Seasonal changes in microbial biomass in soils cropped with palmarosa (Cymbopogon martinii L.) and Japanese mint (Mentha arvensis L.) in subtropical India. Biol. Fertil. Soils 19, 193–196. Rumberger, A., Merwin, I.A., Thies, J.E., 2007. Microbial community development in the rhizosphere of apple trees at a replant site. Soil Biol. Biochem. 39, 1645–1654. Rumberger, A., Yao, S., Merwin, I.A., Nelson, E.B., Thies, J.E., 2004. Rootstock genotype and orchard replant position rather than soil fumigation or compost amendment determine tree growth and rhizosphere bacterial community composition in an apple replant soil. Plant Soil 264, 247–260. Singh, B.K., Sharma, S.R., Singh, B., 2010. Antioxidant enzymes in cabbage: variability and inheritance of superoxide dismutase, peroxidase and catalase. Sci. Hortic. 124, 9–13. Singh, K.P., Suman, A., Singh, P.N., Srivastava, T.K., 2007. Improving quality of sugarcane-growing soils by organic amendments under subtropical climatic conditions of India. Biol. Fertil. Soils 44, 367–376.
58
Z. Zhang et al. / Scientia Horticulturae 146 (2012) 52–58
ˇ Snajdr, J., Baldrian, P., 2006. Production of lignocellulose-degrading enzymes and changes in soil bacterial communities during the growth of Pleurotus ostreatus in soil with different carbon content. Folia. Microbiol. 51, 579–590. ˇ Stursová, M., Baldrian, P., 2011. Effects of soil properties and management on the activity of soil organic matter transforming enzymes and the quantification of soil-bound and free activity. Plant Soil 338, 99–110. Tao, J., Griffiths, B., Zhang, S., Chen, X., Liu, M., Hu, F., Li, H., 2009. Effects of earthworms on soil enzyme activity in an organic residue amended rice–wheat rotation agro-ecosystem. Appl. Soil Ecol. 42, 221–226. Tartoura, K.A.H., Youssef, S.A.G., 2010. Effect of compost on the antioxidant defense systems of cucumber (Cucumis sativus L.) against cadmium toxicity. Ann. Agric. Sci. (Cairo) 55, 191–203. Terhoeven-Urselmans, T., Scheller, E., Raubuch, M., Ludwig, B., Joergensen, R.G., 2009. CO2 evolution and N mineralization after biogas slurry application in the field and its yield effects on spring barley. Appl. Soil Ecol. 42, 297–302. Tewoldemedhin, Y.T., Mazzola, M., Labuschagne, I., McLeod, A., 2011a. A multiphasic approach reveals that apple replant disease is caused by multiple biological agents with some agents acting synergistically. Soil Biol. Biochem. 43, 1917–1927. Tewoldemedhin, Y.T., Mazzola, M., Botha, W.J., Spies, C.F.J., McLeod, A., 2011b. Characterization of fungi (Fusarium and Rhizoctonia) and oomycetes (Phytophthora and Pythium) associated with apple orchards in South Africa. Eur. J. Plant Pathol. 130, 215–229. Tewoldemedhin, Y.T., Mazzola, M., Mostert, L., McLeod, A., 2011c. Cylindrocarpon species associated with apple tree roots in South Africa and their quantification using real-time PCR. Eur. J. Plant Pathol. 129, 637–651. Tian, L., Dell, E., Shi, W., 2010. Chemical composition of dissolved organic matter in agroecosystems: correlations with soil enzyme activity and carbon and nitrogen mineralization. Appl. Soil Ecol. 46, 426–435.
Uzilday, B., Turkan, I., Sekmen, A.H., Ozgur, R., Karakaya, H.C., 2012. Comparison of ROS formation and antioxidant enzymes in Cleome gynandra (C4 ) and Cleome spinosa (C3 ) under drought stress. Plant Sci. 182, 59–70. Vallad, G.E., Cooperband, L., Goodman, R.M., 2003. Plant foliar disease suppression mediated by composted forms of paper mill residuals exhibits molecular features of induced resistance. Physiol. Mol. Plant Pathol. 63, 65–77. van Schoor, L., Denman, S., Cook, N.C., 2009. Characterisation of apple replant disease under South African conditions and potential biological management strategies. Sci. Hortic. 119, 153–162. Wu, H.S., Yang, X.N., Fan, J.Q., Miao, W.G., Ling, N., Xu, Y.C., Huang, Q.W., Shen, Q.R., 2008. Suppression of Fusarium wilt of watermelon by a bio-organic fertilizer containing combinations of antagonistic microorganisms. BioControl 54, 287–300. Yao, S.R., Merwin, I.A., Abawi, G.A., Thies, J.E., 2006. Soil fumigation and compost amendment alter soil microbial community composition but do not improve tree growth or yield in an apple replant site. Soil Biol. Biochem. 38, 578–599. Yogev, A., Raviv, M., Hadar, Y., Cohen, R., Katan, J., 2006. Plant waste-based composts suppressive to diseases caused by pathogenic Fusarium oxysporum. Eur. J. Plant Pathol. 116, 267–278. Zaman, M., Matsushima, M., Chang, S.X., Inubushi, K., Nguyen, L., Goto, S., Kaneko, F., Yoneyama, T., 2004. Nitrogen mineralization, N2 O production and soil microbiological properties as affected by long-term applications of sewage sludge composts. Biol. Fertil. Soils 40, 101–109. Zhang, Y.K., Han, X.J., Chen, X.L., Jin, H., Cui, X.M., 2009. Exogenous nitric oxide on antioxidative system and ATPase activities from tomato seedlings under copper stress. Sci. Hortic. 123, 217–223.