Effects of short-term rotation and Trichoderma application on the soil environment and physiological characteristics of Malus hupehensis Rehd. seedlings under replant conditions

Acta Ecologica Sinica 37 (2017) 315–321

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Effects of short-term rotation and Trichoderma application on the soil environment and physiological characteristics of Malus hupehensis Rehd. seedlings under replant conditions Fengbing Pan, Li Xiang, Sen Wang, Jiajia Li, Xiang Shen, Xuesen Chen, Chengmiao Yin ⁎, Zhiquan Mao State Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agricultural University, Taian, Shandong 271018, China

a r t i c l e

i n f o

Article history: Received 12 July 2016 Received in revised form 30 November 2016 Accepted 8 January 2017

a b s t r a c t In this study, we explored the effects of short-term rotation and Trichoderma application on the plant biomass, root respiration rate, root protective enzymes of Malus hupehensis Rehd., and soil environment under replant conditions, to provide a basis for the prevention of the apple replant disease (ARD). In 2014, old apple orchard soils were treated by rotating peanut, Allium fistulosum L., fallow, and natural grass, and replanted apple orchard soil was used as the control. The soils of the different treatments were mixed well with Trichoderma at a concentration of 0.5% (w/w), and 6 kg of each treated soil was added to a pot (outer diameter, 27 cm; inner diameter, 23 cm; height, 18 cm). Uniform seedlings of Malus hupehensis Rehd. were transplanted to the pots on April 23, 2015. Two seedlings were planted in each pot, and 30 pots were prepared for each treatment. The samples were collected at the end of August, when there were visible differences in seedling growth among the treatments. The results showed that rotating Allium fistulosum L. mixed with Trichoderma could significantly improve the populations of bacteria and actinomycetes in the soil, which increased by 189.4% and 107.1%, respectively. The biomass of M. hupehensis significantly improved, and plant fresh weight, dry weight, height, and diameter increased by 186.3%, 205.9%, 58.8%, and 33.2%, respectively. In addition, the total fine root length, total fine root surface area, total fine root volume, and fine root tip number increased by 147.2%, 225.9%, 298.5%, and 331.3%, respectively. Compared with the control seedlings, the treated seedlings showed higher respiration rate of the root system, Superoxide Dismutase activity and Peroxidase activity by 131.0%, 46.3%, and 110%, respectively. Analysis of the terminal restriction fragment length polymorphism profiles showed that the treatment of short-term crop rotation and Trichoderma changed the soil fungal community structure; improved the Simpson index of the soil fungal community; and reduced the Shannon index, Pielou index, and Margalef index. The results suggest that shortterm rotation of Allium fistulosum L. mixed with Trichoderma could alleviate ARD. © 2017 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

1. Introduction Apple is one of the most important fruit trees in China. Apple replant disease (ARD) is widespread in China because repeated production in the same field is a common practice owing land scarcity and the replant of aged apple orchards. ARD has become a grave problem in the dominant production area of China, and it is urgent to solve the problem of production [1]. The problems are typically expressed as reduced plant growth and development, inhibited root system development, with a subsequent shortened production life and reduced yield [2–3]. The traditional methods to overcome ARD include crop rotation, intercropping, soil disinfection, organic amendment, resistance breeding and biological control [4–5]. However, the traditional methods cannot improve the

⁎ Corresponding author. E-mail address: [email protected] (C. Yin).

http://dx.doi.org/10.1016/j.chnaes.2017.09.003 1872-2032/© 2017 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

continuous cropping soil timely and effectively because of some limitations of the methods. Lv et al. [6] found that Allium fistulosum L. rotation can increase the content of soil organic matter, enhance the ratio of bacteria and fungi, reduce the content of phenolic acids in the soil, improve the root enzyme activity of M. hupehensis Rehd. seedlings, and increase the ground and underground biomass. Wheat cultivars substantially enhanced the growth of apple which was associated with a reduction in root infection by the dominant fungal pathogens resident to the respective orchard soils [7]. Studies have shown that tomato rotation and microbial fertilizers could improve the physiochemical properties of continuous cucumber cropping soil, enhance the activities of soil enzymes, and increase the quantity of bacteria and actinomycetes, but decrease the quantity of fungi. Meanwhile, the compound amendment of tomato rotation with microbial fertilizers had significantly positive effects on soil properties compared to each single amendment [8]. Wang et al. [9] found that rotation improved sorghum rhizosphere soil environment,

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enhanced soil microbial enzyme activity, alleviated the obstacle of continuous cropping and thus increased the sorghum yield. Chen [10] demonstrated that bacterial fertilizer can promote the growth of crops under conditions of continuous cropping. It has not been reported that the effect of rotation and bacterial fertilizer on ARD. In this study, we explored the effects of short-term rotation and Trichoderma application on the plant biomass, root respiration rate, root protective enzymes, and soil environment under replant conditions, to provide a basis for the prevention of the ARD. 2. Materials and methods 2.1. Experimental materials The experiment was conducted in April 2014 to October 2015 at the experiment base of National Research Center for Apple Engineering and Technology, College of Horticultural Sciences and Engineering, Shandong Agricultural University. Soil used for this experiment was obtained from a 25-year-old red Fuji apple orchard located in suburb of Taian city, Shandong province of China, which rootstock was Malus robusta Rehd. Soil type was cinnamon soil, pH 6.2, the content of organic matter was 9.35 g/kg, available phosphorus was 15.85 mg/kg, available potassium was 152.05 mg/kg, ammonium nitrogen was 13.09 mg/kg and nitrate nitrogen was 7.68 mg/kg. The experimental microbial fertilizer was Trichoderma spp., living bacteria count is 2 × 109 cfu/g. Application rate of Trichoderma was 0.5% weight of soil, then thoroughly mixed with the soil. M. hupehensis Rehd., which is a commonly used rootstock for apple trees, was used in this study. 2.2. Experimental design Old apple orchard soils were treated by rotating peanut, Allium fistulosum L., fallow, and natural grass in big pot (outer diameter, 38 cm; inner diameter, 28 cm; height, 26.5 cm), and replanted apple orchard soil was used as the control, on April 20, 2014. The soils of the different treatments were mixed well with Trichoderma at a concentration of 0.5% (w/w), and 6 kg of each treated soil was added to a small pot (outer diameter, 27 cm; inner diameter, 23 cm; height, 18 cm). M. hupehensis Rehd. seedlings were transplanted to the pots on April 23, 2015. Two seedlings were planted in each pot, and 30 pots were prepared for each treatment. The samples were collected at the end of August (124d), when there were visible differences in seedling growth among the treatments. The specific experimental design was shown in Table 1.

(Omega Bio-tek. Omc. USA) according to the manufacturer's protocols. Three soil samples from each treatment were extracted. The PCR amplifications were carried out with the fungi internal transcribed spacer (ITS) region primers ITS1F and ITS4. The PCR products were digested for 15 min with FastDigest Hha I (Thermo Scientific, Waltham, MA, USA) as described in the manufacturer's protocol. The digested samples were analyzed by Sangon Biotech Co. Ltd. using an ABI 3730LX DNA Analyzer [3]. The diversity index, cluster analysis and principal component analysis were analyzed on the sequencing results [12]. Determination of biomass: Plant height and rhizome roughness were measured using tape and vernier, respectively. Dry weight and fresh weight was measured using an electronic balance. Determination of root respiration rate: The root respiration rate was performed as previously described by Zhou [13]. 0.05 g of the seedling roots cut into uniform size (1.0 mm) and 1.5 mL reaction medium were added to the reaction cup of Hansatech Qxytherm System, determine for 3–5 min. Root respiration rate of the samples were calculated by the response curves [14]. Determination of the morphology of fine root: the total fine root length, total fine root surface area, total fine root volume, and fine root tip number were determined by NUS700 root scanner reference for Xiao et al. [15]. Determination of 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 [16]. The SOD activity was defined as the amount of enzyme causing half of the maximum inhibition of nitroblue tetrazolium (NBT) reduction. The POD activity assay was performed according to the method developed by Zhang et al. [17]. The POD activity was assayed spectrophotometrically by monitoring changes in absorbance at 470 nm. The CAT activity assay was also performed spectrophotometrically by monitoring changes in absorbance at 240 nm [18]. 2.4. Statistical analysis The experimental data was expressed as means and standard errors (SE) with three biological replications. Statistical calculations were performed with SPSS v.19.0 statistical software and Microsoft Excel 2010. Mean difference comparison among different treatments was done by ANOVA and Duncan's multiple range test at a 0.05 probability level. 3. Results 3.1. Effect of different treatments on soil microorganisms

2.3. Index measurement Determination of the quantity of soil microbes: The quantity of soil microbes was determined using the agar plate dilution method. Beef peptone medium was used for the culture of bacteria, and PDA medium was used for the culture of fungi. Moreover, Gao 1 agar medium was used for the culture of actinomycetes [11]. Terminal restriction fragment length polymorphism analysis: The DNA was extracted from 0.5 g soil using an E.Z.N.A.™ Soil DNA Kit Table 1 Design of experiments. Treatment

Design of experiments

Fm Nm Pm Am S Cm Ci CK

Fallowing and microbial fertilizer Natural grasses and microbial fertilizer Peanut rotation and microbial fertilizer Allium fistulosum L. rotation and microbial fertilizer Sterilized soil Continuous cropping and microbial fertilizer Continuous cropping and inactivated microbial fertilizer Continuous cropping

The populations of bacteria in the sterilized soil recovered rapidly and was higher than other treatments significantly. Compared with Cm, Ci and CK, the populations of bacteria of sterilized soil increased by 208.8%, 278.5% and 427.6%, respectively. The results showed that short-term rotation mixed with Trichoderma could significantly improve the populations of bacteria in the soil. Compared with the control, the populations of bacteria of Fm, Nm, Pm and Am increased by 86.8%, 126.3%, 110.5% and 189.4%, respectively. Short-term rotation mixed with Trichoderma could significantly improve the populations of actinomycetes in the soil, compared with CK, the populations of actinomycetes of Fm, Nm, Pm and Am increased by 110.1%, 98.2%, 83.7% and 107.1%, respectively (Fig.1). 3.2. Effects of different treatments on fungal community The Shannon index, Pielou index and Margalef index of Nm and Am were lower other treatments significantly, Cm, Ci and CK were higher than other treatments. Simpson index of Nm was the highest, the other treatments were Am N Fm N S N CK N Cm N Pm N Ci (Table 2). PCA for T-RFLP patterns (Fig. 2) and Cluster analysis of T-RFLP patterns

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Fig. 1. Effect of different treatments on soil microorganisms.

(Fig. 3) showed that the short-term rotation and Trichoderma changed the fungal community in continuous cropping soil significantly. The treatments with Trichoderma had the similar fungal community, and were different from the treatments without Trichoderma. Duncan's new multiple range test, values followed by different letters in a column are significant at the 5% level. The same as follows. 3.3. Effect of different treatments on plant biomass of M. hupehensis Rehd. seedlings Short-term rotation and Trichoderma could significantly improve the biomass of M. hupehensis (Fig. 4). Compared with the control, the plant fresh weight of Fm, Nm, Pm, Am increased by 184.2%, 93.7%, 118.5%, and 186.3%, respectively. The dry weight of Fm, Nm, Pm, Am increased by 205.2%, 99.2%, 131.0%, and 205.7%, respectively. The height of Fm, Nm, Pm, Am increased by 70.8%, 38.8%, 44.0%, and 58.8%, respectively. The diameter of Fm, Nm, Pm, Am increased by 34.5%, 16.9%, 27.4%, and 33.5%, respectively. On the whole, the effect of rotating Allium fistulosum L. mixed with Trichoderma was the best. 3.4. Effect of different treatments on the morphology of fine root of M. hupehensis Rehd. seedlings The fine root of M. hupehensis Rehd. seedlings of rotating Allium fistulosum L. mixed with Trichoderma was better than other treatments. Compared with the control seedlings, the total fine root length, total fine root surface area, total fine root volume, and fine root tip number of rotating Allium fistulosum L. mixed with Trichoderma increased by 147.2%, 225.9%, 298.5%, and 331.3%, respectively (Table 3). 3.5. Effect of different treatments on root respiration rate and antioxidant enzymes activity of M. hupehensis Rehd. seedlings The root respiration rate of Fm, Nm, Pm, Am were significantly higher than the control, increased 80.1%, 96.3%, 51.6% and 131%, respectively. The treatment of rotating Allium fistulosum L. mixed with Trichoderma Table 2 Species diversity of soil fungi. Treatments

Shannon index

Simpson index

Pielou index

Margalef index

Fm Nm Pm Am S Cm Ci CK

2.11 ± 0.03abc 1.85 ± 0.24c 2.22 ± 0.01ab 1.98 ± 0.03c 2.21 ± 0.04ab 2.26 ± 0.06ab 2.34 ± 0.01a 2.15 ± 0.08abc

0.16 ± 0.01bc 0.23 ± 0.05a 0.14 ± 0.00c 0.20 ± 0.00ab 0.15 ± 0.00bc 0.13 ± 0.01c 0.13 ± 0.00c 0.16 ± 0.01bc

0.80 ± 0.02a 0.7 ± 0.06b 0.83 ± 0.00a 0.74 ± 0.01b 0.80 ± 0.01a 0.82 ± 0.00a 0.82 ± 0.00a 0.79 ± 0.03a

2.91 ± 0.10bc 2.74 ± 0.38c 3.18 ± 0.16abc 2.83 ± 0.21bc 3.43 ± 0.22ab 3.62 ± 0.11a 3.73 ± 0.07a 3.34 ± 0.17abc

had the best effect. The trends of all treatment were S N Am N Nm N Fm N Pm N Cm N Ci N CK (Fig. 5). Short-term rotation and Trichoderma could significantly improve the activity of SOD and POD of M. hupehensis Rehd. seedlings. Compared to the control, SOD of Fm, Nm, Pm, Am increased by 43.7%, 48.0%, 39.5% and 46.2%, respectively. POD of Fm, Nm, Pm, Am increased by 43.7%, 48.0%, 39.5% and 46.2%, respectively. The activity of CAT of Fm and Am were higher than other treatments significantly, increased by 233.5% and 158.9% compared to the control. 4. Discussion ARD is common in all of the major apple-growing regions of the world [19]. ARD is widespread in China because repeated production in the same field is a common practice owing land scarcity and the replant of aged apple orchards, and it has become an important factor restricting the sustainable development of China's apple industry [20]. Therefore, how to alleviate the ARD effectively has become an important issue in apple production in China [21–22]. In this study, shortterm rotation and Trichoderma were used to relieve the negative constraints of ARD. The results showed that the comprehensive measures to prevent ARD were more effective than the single control measures. The possible reason was that the causes of ARD are diverse and complex [23], and short-term rotation and Trichoderma would improve the soil environment from different aspects [24–25], inhibit the occurrence of ARD [26]. Soil microbes have a close relationship with the stability and function of the soil system. In aged apple orchards, the structure of bacterial and fungal communities in the rhizosphere soil has changed over time. And the accumulation of harmful microorganisms increased the incidence of soil-borne diseases [27–28]. In the present study, the main pathogenic fungi associated with ARD were Fusarium, Cylindrocarpon, Phytophthora, Rhizoctonia and Pythium [29]. The results of this study showed that the treatment of short-term rotation and Trichoderma significantly increased the population of bacteria and actinomycetes in the continuous cropping soil, which was beneficial to improve the microbial environment of continuous cropping soil. The changing pattern of soil microbial community might be useful to monitor desertification and soil degradation [30].Studies have shown that T-RFLP was used to study the microbial community of fungi, which could be analyzed by Shannon index, Pielou index, Margalef and Simpson index [31]. Qin et al. [32] used T-RFLP find that the fall of the diversity of bacteria community gave way to the rise of fungi community under continuous cropping, causing imbalance in microbial community. The result of TRFLP of this study showed that the Simpson index of soil fungal community increased significantly when Trichoderma was applied, which meant Trichoderma successfully colonized and became the dominant microflora in the soil. The possible reason was that the mass multiplication of Trichoderma compressed the living environment of pernicious fungi, and inhibited the growth of harmful fungi.

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Fig. 2. PCA for T-RFLP patterns of different treatments.

It was an inhibition of root growth, and then the inhibition of the uptake of water and nutrients when the plants in adversity conditions [33]. Allelochemicals could inhibit the growth of the succeeding crops in continuous cropping [34], root system was directly affected [35–36]. Vaughan et al. find that allelochemicals could inhibited an increase in the length of the main root Pisum sativum, the phenolic acids also inhibited cell division [37]. According to our results, the treatment of short-term rotation and

Trichoderma could promote the growth of root system of M. hupehensis Rehd. seedlings. And the total fine root length, total fine root surface area, total fine root volume, and fine root tip number significantly improved compared with control, which had a positive effect on nutrient and water uptake in continuous cropping condition [38–39]. Under normal growth conditions, the production and detoxification of free radicals in tissue cells existed a dynamic equilibrium. Abiotic and

Fig. 3. Cluster analysis of T-RFLP patterns of different treatments.

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Fig. 4. Effect of different treatments on plant biomass of M. hupehensis Rehd. seedlings.

biotic stresses could break the equilibrium and accumulate of reactive oxygen species (ROS), including O2\\, H2O2 and OH. Oxidative stress occurs when there is a serious imbalance between the production of ROS and antioxidant defense. Excessive production of ROS may lead to oxidative stress, DNA and protein damage, membrane permeability, lipid peroxidation, loss of cell function and, ultimately, cell death [40]. To scavenge ROS and to avoid oxidative stress, plants have antioxidant defense enzymes such as SOD, CAT, and POD [41–42]. In this study, short-term rotation mixed with Trichoderma improved the activity of SOD, POD, and CAT of the root, which could prevent excessive accumulation of ROS damaged to plant roots through the effective removal of ROS and other harmful substances. While short-term rotation mixed with Trichoderma increased plant root activity and root metabolism, promoted the plant growth and stress resistance. Rotating Allium fistulosum L. mixed with Trichoderma had the best effect. Root respiration, as an important process of plant metabolism, not only provided the raw material and energy for plant life activities, but also was a sensitive indicator of measuring the root function and indicating the root material and energy change, so it was important to root absorption, root regeneration, and plant growth [43]. In this experiment, rotating different crops mixed with Trichoderma could improve the

root respiration of Malus hupehensis seedling of different degree, in which the effect of rotating Allium fistulosum L. mixed with Trichoderma was the most obvious. Most of the energy produced by root respiration was used for the absorption of nutrients, and the decrease of respiration made the absorption of nutrient ions restrained and slowed down the growth of plants. Enhanced the root respiration rate could improve the root activity of Malus hupehensis seedling, promote root absorption of nutrients and plant growth, improve the resistance of plants [44], which had a very positive role to alleviate ARD.

5. Conclusion The results of this study showed that short-term rotation mixed with Trichoderma could improve the soil environment, increase the number of bacteria and actinomycetes, increase the activity of antioxidant enzymes and root respiration, and promote the root growth, thus effectively alleviate ARD. The effect of the rotation onion mixed and bacterial manure is the most obvious. Therefore, mixing short-term rotation Allium fistulosum L. mixed with Trichoderma could be used as a very effective measure to relieve the ARD in a short period of time.

Table 3 Effect of different treatments on the morphology of fine root of M. hupehensis Rehd seedlings. Treatments

Total fine root length/cm

Total fine root surface area/cm2

Total fine root Volume/cm3

Fine root tip number

Fm Nm Pm Am S Cm Ci CK

2292.7 ± 77.5c 1974.3 ± 106.0 cd 2157.9 ± 138.2 cd 3033.5 ± 477.0b 3928.2 ± 159.5a 1644.8 ± 98.8de 1251.3 ± 74.0e 1227.1 ± 27.6e

821.1 ± 21.5c 518.2 ± 19.4def 624.0 ± 85.5d 1054.5 ± 46.2b 1377.0 ± 133.2a 578.9 ± 47.8de 404.3 ± 30.4ef 323.6 ± 29.4f

25.7 ± 1.2b 13.5 ± 1.6c 16.4 ± 3.1c 25.9 ± 2.1b 34.1 ± 3.1a 13.6 ± 1.9 cd 7.6 ± 1.0de 6.5 ± 0.8e

9114.3 ± 1792.5bc 6038.7 ± 1846.1 cd 6443.3 ± 1058.3 cd 12,511.7 ± 2481.2ab 16,175.7 ± 2118.5a 4309.7 ± 175.8 cd 3816.3 ± 795.2d 2901.0 ± 336.2d

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Fig. 5. Effect of different treatments on root respiration rate and antioxidant enzymes activity of M. hupehensis Rehd. seedlings.

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