E ﬀ ects of Mycorrhizae on Physiological Responses and Relevant Gene Expression of Peach A ﬀ ected by Replant Disease

: A potted experiment was carried out to evaluate the e ﬀ ect of an arbuscular mycorrhizal fungus (AMF), Acaulospora scrobiculata , on peach seedlings grown in non-replant (NR) and replant (R) soils, to establish whether AMF inoculation alleviated soil replant disease through changes in physiological levels and relevant gene expression. After 15 weeks of mycorrhization, root mycorrhizal colonization was heavily inhibited by R treatment versus NR treatment. AMF plants under NR and R soil conditions displayed signiﬁcantly higher total plant biomass than non-AMF plants. AMF inoculation signiﬁcantly increased root sucrose and fructose concentrations and root catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonialyase activities under R conditions. Likewise, salicylic acid, jasmonic acid, chitinase, total soluble phenol, and lignin concentrations in roots were signiﬁcantly higher in AMF than in non-AMF seedlings grown in R soil. Over-expression of PpCHI , PpLOX1 , PpLOX5 , PpAOC3 , PpAOC4 , and PpOPR2 in roots was observed in AMF-inoculated seedlings, as compared to that of non-AMF-inoculated seedlings grown in R soils. Thus, mycorrhizal fungal inoculation conferred a greater tolerance to peach plants in R soil by stimulating antioxidant enzyme activities, disease-resistance substance levels, and the expression of relevant genes. total soluble phenol and lignin concentrations in mycorrhizal peach seedlings than in non-mycorrhizal


Introduction
Soil replant disease is a major problem in the production of peach (Prunus persica L. Batsch) trees, which causes the abnormal growth of trees and an inferior fruit yield and quality [1][2][3]. Such soil-borne disease is the result of disturbances in rhizosphere ecology reported in various crops, like peanut, grape, and apple [3,4]. It is documented that soil replant disease originates from soil physical-chemical imbalance, soil microflora imbalance, allelopathy, and autotoxicity [3].
Arbuscular mycorrhiza (AM) is a reciprocal symbiosis between arbuscular mycorrhizal fungi (AMF) and the roots of approximately 80% of land plants [5]. The mycorrhizal plants form extraradical hyphae developed on the root surface to acquire nutrients, coupled with an elevated photosynthetic efficiency [6]. Inoculation with AMF stimulates antioxidant enzyme activities to scavenge reactive oxygen species (ROS) induced by the pathogen invasion of the host plants [7]. AMF increased the structural rigidity of cell walls to produce a mechanical barrier and also induced phenolic substances, chitinase, and pathogenesis-related proteins to degrade or inhibit pathogenic infection [7,8].Čatská [9] reported the mitigating effect of mycorrhizal fungi on apple replant disease. Mehta and Bharat [10] further revealed the increase in the number of fungi, bacteria, and actinomycetes in replant soils of

Experimental Set-Up
The experiment was carried out using a completely randomized factorial design involving a total of four treatments using two factors with five replications. The first factor comprised mycorrhizal inoculations with Acaulospora scrobiculata (+AMF) and without A. scrobiculata (-AMF). The second factor consisted of the use of replanted (R) soil and non-replanted (NR) soil as growing medium in the pot.
The experiment was conducted during March-July 2017 in a greenhouse of the Yangtze University campus with an average day/night temperature of 27/20 • C, a photosynthetic photon flux density of 768 µmol/m 2 /s, and a relative humidity of 72%. The R soil was collected from the rhizosphere of 18-yr-old P. persica cv. Yuhualu in Boksugol (30 • 25 15.1 N and 112 • 08 06.6 E), near the west campus of Yangtze University, in Jingzhou, China. The NR soil was selected from the soil area, 500 m away from the R site, where no peach trees were planted. Both types of soils (R and NR soils) belonged to the Xanthi-Udic-Ferralsols (FAO system). The physiochemical soil characteristics were pH 6.3, available phosphorus 16.56 mg/kg, and available nitrogen 11.6 mg/kg.
The soil was sterilized in flowing steam at 0.11 MPa for 2 h before filling the experimental pots. The six-leaf-old seedlings of peach with uniform sizes grown in autoclaved sands were transplanted into 2.5 L plastic pots filled with 2.5 kg autoclaved soils. Approximately 120 g mycorrhizal inoculums containing 1500 spores and infected roots were applied into the rhizosphere of the potted peach seedlings to develop the mycorrhizal treatment. The non-AMF control was treated with an equal amount of autoclaved inoculum, along with a 2 mL filtrate (25 µm) of the inoculum for similar microflora except the mycorrhizal fungus. The mycorrhizal fungus used was Acaulospora scrobiculata Trappe (No.: BGC HK01), provided by the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences (Beijing, China), and propagated with white clover (Trifolium repens L.) as a host plant for 12 weeks at 22/18 • C (day/night temperature).

Determinations of Variables
Seedlings were harvested at 105 days after the imposition of treatments, and the total fresh biomass was determined. The roots were scanned with an EPSON Flat-Scanner (V700, Seiko Epson Corp., Suwa City, Japan) and analyzed with the WinRHIZO 2007b (Regent Instruments Incorporated, Quebec, QC, Canada) for total root length, projected area, surface area, and volume. The roots were stained with 0.05% trypan blue using the protocol described by Phillips and Hayman [18], and mycorrhizal colonization was expressed as the percentage of mycorrhizal colonized root length versus the total observed root length.
The concentration of fructose, glucose, and sucrose in the roots was determined colorimetrically according to the procedure outlined by Wu et al. [19]. Root catalase (CAT), SOD, peroxidase (POD), and polyphenol oxidase (PPO) activities were determined according to the method described by Aebi [20] using 0.1 mol/L KMnO 4 as the standard, the nitrogen blue tetrazolium method [21], the protocol described by Lurie et al. [22] with methyl catechol as the standard, and the protocol described by Aquino-Bolanos and Mercado-Silva [23] with pyrocatechol as the standard, respectively. Phenylalanine ammonialyase (PAL) activity in the roots was analyzed according to the colorimetric method at 290 nm [24].
The extraction of SA and JA from the roots was performed according to the method suggested by Segarrad et al. [25]. The concentration of SA and JA was determined using high-performance liquid chromatography-tandem mass spectrometry. Root chitinase [26], lignin, and total soluble phenol [27] concentrations were determined as per the suggested procedures.
The total RNA of roots was extracted in 0.1 g fresh root samples using the EASY spin plus plant RNA mini kit (RN38, Aidlab, Beijing, China), and reverse transcription was carried out with TRUEscript 1st Strand cDNA Synthesis Kit with gDNA Eraser (PC5402, Aidlab, Beijing, China). Sequences of SA and JA synthetic genes were observed based on the Genomics Database for Rosaceae (https://www.rosaceae.org/node/1). The specific primers (Table 1) of relevant genes for qRT-PCR analysis (10 µL SYBR GREEN PCR Master Mix, 6.4 µL ddH 2 O, 2 µL cDNA, and 0.8 µL each primer for forward and reverse) were designed using the Primer Premier 5.0 software (Palo Alto, CA, USA), according to cDNA sequences of Prunus persica genome. The qRT-PCR was conducted on the Bio-rad CFX connect-time system under the conditions characterized by 95 • C for 30 s, 40 cycles with 95 • C for 5 s, 60 • C for 10 s, and 72 • C for 30 s. The relative expression of genes was determined by the 2 −∆∆Ct method, as suggested by Kenneth and Schmittgen [28]. Translation elongation factor 2 (TEF2) was used to validate an RNA-seq analysis and identified as the best single peach reference gene to normalize gene expression based on earlier reports [29,30].

Statistical Analysis
The data were subjected to the two-factor analysis of variance (ANOVA) using SAS software (version 8.1; SAS Institute, Inc., Cary, NC, USA). Duncan's multiple range tests at the 0.05 level were used to compare the significance levels between treatments.

AMF Colonization, Total Plant Biomass, and Root Morphology
The non-AMF-inoculated seedlings did not show mycorrhizal colonization in the roots, while the A. scrobiculata-inoculated seedlings represented 29.8 to 52.0% of mycorrhizal colonization in the roots ( Table 2). The R treatment heavily inhibited root mycorrhizal colonization. The AMF-inoculated peach seedlings displayed a relatively higher growth performance than the non-AMF seedlings in NR and R soils ( Table 2). Compared with non-AMF seedlings, mycorrhizal seedlings recorded a higher total plant biomass, total root length, root surface area, root projected area, and root volume by 32%, 16%, 22%, 26%, and 26%, respectively, in NR soil, and also registered a higher total plant biomass by 24% in R soil. Table 2. Effects of Acaulospora scrobiculata on mycorrhizal colonization, total plant biomass, and root morphology of peach (Prunus persica) seedlings grown in replant (R) and non-replant (NR) soil.

Treatments
Data (means ± SD, n = 5) followed by different letters among treatments indicate significant differences between treatments at p < 0.05.

Changes in Root Carbohydrate Concentrations
Root sucrose, fructose, and glucose levels were considerably higher under NR soil than under R soil, irrespective of inoculation with or without AMF (Figure 1). Compared with non-AMF seedlings, the seedlings colonized by A. scrobiculata recorded 16% and 11% significantly higher root sucrose and fructose concentrations and 14% lower root glucose concentrations under NR soil, and also had 25%, Agronomy 2020, 10, 186 5 of 10 59%, and 52% significantly higher root sucrose, fructose, and glucose concentrations, respectively, under R soil.

Changes in Root Antioxidant Enzyme Activities
Soil R treatment produced a significant increase in root CAT, POD, and PPO activity but a decrease in root SOD activity, as compared with soil NR treatment, irrespective of whether it was AMF inoculated (Figure 2). AMF inoculation increased root CAT, POD, and PPO activity in NR and R soils, relative to non-AMF treatment (Figure 2a,c,d). Compared to non-AMF seedlings, mycorrhizal seedlings showed higher root CAT, POD, and PPO activities: 129%, 32%, and 57% higher under NR soil and 403%, 84%, and 46% higher under R soil. Mycorrhizal treatment did not alter root SOD activity under NR and R soils (Figure 2b).

Changes in Root Antioxidant Enzyme Activities
Soil R treatment produced a significant increase in root CAT, POD, and PPO activity but a decrease in root SOD activity, as compared with soil NR treatment, irrespective of whether it was AMF inoculated (Figure 2). AMF inoculation increased root CAT, POD, and PPO activity in NR and R soils, relative to non-AMF treatment (Figure 2a,c,d). Compared to non-AMF seedlings, mycorrhizal seedlings showed higher root CAT, POD, and PPO activities: 129%, 32%, and 57% higher under NR soil and 403%, 84%, and 46% higher under R soil. Mycorrhizal treatment did not alter root SOD activity under NR and R soils (Figure 2b).

Root Physiological Responses
Soil R treatment significantly inhibited root PAL activity, chitinase activity, total soluble phenol levels, and lignin concentrations in non-mycorrhizal seedlings, but not in mycorrhizal seedlings (Table 3). Root SA level, JA level, PAL activity, and chitinase activity were higher in AMF seedlings than in non-AMF seedlings: 20%, 61%, 16%, and 10% higher under NR condition and 23%, 30%, 279%, and 53% higher under R condition. Also, AMF inoculation significantly reduced the root total soluble phenol content and lignin levels by 10% and 25% under NR soil, while increasing them by 10% and 45% under R soil, compared with the non-AMF control. Table 3. Effects of Acaulospora scrobiculata on salicylic acid (SA), jasmonic acid (JA), phenylalnine ammonialyase (PAL), chitinase, total soluble phenol, and lignin in roots of peach seedlings grown in replant (R) and non-replant (NR) soil. Data (means ± SD, n = 5) followed by different letters among treatments indicate significant differences between treatments at p < 0.05.

Changes in Relative Expression Levels of Genes
AMF inoculation up-regulated the root Pp4CL3 gene expression level under NR and R treatment conditions, respectively, compared with that observed upon non-AMF inoculation ( Figure 3). The relative expression of PpPAL1 in roots was increased upon mycorrhizal

Root Physiological Responses
Soil R treatment significantly inhibited root PAL activity, chitinase activity, total soluble phenol levels, and lignin concentrations in non-mycorrhizal seedlings, but not in mycorrhizal seedlings (Table 3). Root SA level, JA level, PAL activity, and chitinase activity were higher in AMF seedlings than in non-AMF seedlings: 20%, 61%, 16%, and 10% higher under NR condition and 23%, 30%, 279%, and 53% higher under R condition. Also, AMF inoculation significantly reduced the root total soluble phenol content and lignin levels by 10% and 25% under NR soil, while increasing them by 10% and 45% under R soil, compared with the non-AMF control. Table 3. Effects of Acaulospora scrobiculata on salicylic acid (SA), jasmonic acid (JA), phenylalnine ammonialyase (PAL), chitinase, total soluble phenol, and lignin in roots of peach seedlings grown in replant (R) and non-replant (NR) soil. Data (means ± SD, n = 5) followed by different letters among treatments indicate significant differences between treatments at p < 0.05.

Changes in Relative Expression Levels of Genes
AMF inoculation up-regulated the root Pp4CL3 gene expression level under NR and R treatment conditions, respectively, compared with that observed upon non-AMF inoculation (Figure 3). The relative expression of PpPAL1 in roots was increased upon mycorrhizal inoculation under NR soil, while it was reduced under R soil with AMF treatment. Compared with the non-AMF treatment, Figure 3. Effects of Acaulospora scrobiculata on relative expressions of PpPAL1, Pp4CL3, PpCHI, PpAOC3, PpAOC4, PpLOX1, PpLOX5, and PpOPR2 genes in roots of peach (Prunus persica) seedlings grown in replant (R) and non-replant (NR) soil. Data (means ± SD, n = 3) are significantly different (p < 0.05) if followed by different letters above the bars.

Discussion
Our study indicated a considerable reduction in root AMF colonization in peach with A. scrobiculata under R soil condition. This is in agreement with earlier studies of Zhang et al. [31,32] on peach inoculated with another arbuscular mycorrhizal fungus, Funneliformis mosseae. The negative response of root colonization to soil R treatment is due to toxic substances accumulated in the rhizosphere that further restrict spore germination and the hyphal growth of AMF [33]. In this study, inoculation with A. scrobiculata showed a favorable improvement in the total plant biomass, irrespective of soil NR or R conditions. A similar result was reported in apple, grapevine, strawberry, and ginkgo [11,34,35]. The growth improvement of plants by mycorrhizal fungi is likely attributed to the nutrient acquisition by mycorrhizal extraradical hyphae.
Carbohydrates are the power source for energy assurance to mycorrhizal development, signal transduction, and metabolic activities in plants [6]. In this study, mycorrhizal peach seedlings had significantly higher root fructose and sucrose concentrations and lower root glucose concentrations under NR condition and higher root fructose, glucose, and sucrose concentrations under R condition. It is documented that AMF primarily utilized glucose from the sucrose cleavage of roots to maintain symbiotic requirements [19]. Mycorrhizal peach grown in R soil maintained relatively higher fructose, glucose, and sucrose contents than nonmycorrhizal peach in R soil, thereby maintaining the requirement of mycorrhizal activities.
The present study showed that root CAT, POD, PPO, and PAL activities were increased in response to mycorrhization with A. scrobiculata, regardless of soil NR and R conditions. Li et al. [36] also observed higher POD and PAL activities in the root of replanted watermelon after inoculation with Glomus versiforme. Greater antioxidant enzyme activities of mycorrhizal plants aided in alleviating oxidative damage, thereby, enhancing the tolerance capacity of AM plants

Discussion
Our study indicated a considerable reduction in root AMF colonization in peach with A. scrobiculata under R soil condition. This is in agreement with earlier studies of Zhang et al. [31,32] on peach inoculated with another arbuscular mycorrhizal fungus, Funneliformis mosseae. The negative response of root colonization to soil R treatment is due to toxic substances accumulated in the rhizosphere that further restrict spore germination and the hyphal growth of AMF [33]. In this study, inoculation with A. scrobiculata showed a favorable improvement in the total plant biomass, irrespective of soil NR or R conditions. A similar result was reported in apple, grapevine, strawberry, and ginkgo [11,34,35]. The growth improvement of plants by mycorrhizal fungi is likely attributed to the nutrient acquisition by mycorrhizal extraradical hyphae.
Carbohydrates are the power source for energy assurance to mycorrhizal development, signal transduction, and metabolic activities in plants [6]. In this study, mycorrhizal peach seedlings had significantly higher root fructose and sucrose concentrations and lower root glucose concentrations under NR condition and higher root fructose, glucose, and sucrose concentrations under R condition. It is documented that AMF primarily utilized glucose from the sucrose cleavage of roots to maintain symbiotic requirements [19]. Mycorrhizal peach grown in R soil maintained relatively higher fructose, glucose, and sucrose contents than non-mycorrhizal peach in R soil, thereby maintaining the requirement of mycorrhizal activities.
The present study showed that root CAT, POD, PPO, and PAL activities were increased in response to mycorrhization with A. scrobiculata, regardless of soil NR and R conditions. Li et al. [36] also observed higher POD and PAL activities in the root of replanted watermelon after inoculation with Glomus versiforme. Greater antioxidant enzyme activities of mycorrhizal plants aided in alleviating oxidative damage, thereby, enhancing the tolerance capacity of AM plants to biotic stresses like soil replant disease. On the other hand, PAL is a key enzyme for accomplishing the reaction of phenylpropanoids, where the intermediate products (phenolic substances) and end products (lignin, flavonoids, etc.) are important components of defense resistance against pathogens. Our study further indicated higher total soluble phenol and lignin concentrations in mycorrhizal peach seedlings than in non-mycorrhizal Agronomy 2020, 10, 186 8 of 10 peach seedlings under R condition, but not under NR condition. The study of Chen et al. [37] on secondary metabolites produced by F. mosseae-inoculated cucumber plants showed that AMF effectively induced an accumulation of phenolics, flavonoids, and lignin. These observations further suggested that AMF inoculation might stimulate the reaction of phenylpropanoids to enhance the tolerance against soil R disease in peach.
Chitinase hydrolyses chitin, a component of the cell wall of many pathogens, plays a defensive role against pathogen infection [38]. In the present work, regardless of NR and R condition, inoculation with A. scrobiculata significantly increased chitinase activity in roots of AMF-inoculated seedlings when compared to that in non-AMF-inoculated seedlings. In addition, AMF inoculation under R condition up-regulated the expression levels of PpCHI gene encoding chitinase, further suggesting that mycorrhizal symbiosis collapsed the cell wall of pathogen-infected roots under R condition.
The present study also indicated that AMF inoculation significantly increased root SA and JA levels in peach grown in NR and R soils, compared to the non-AMF treatment. Nevertheless, inoculation with AMF down-regulated the expression levels of root PpPAL1 and up-regulated the expression levels of Pp4CL3 under R condition. These observations suggested that AMF-modulated Pp4CL3 gene expression in SA synthetic pathway was more efficiently than AMF-modulated PpPAL1 expression. In the JA synthetic pathway, root PpAOC3, PpAOC4, PpLOX1, PpLOX5, and PpOPR2 were over-expressed in roots of mycorrhizal peach seedlings when compared to those found in roots of non-mycorrhizal seedlings under R condition, implying that AMF inoculation effectively stimulated the JA pathway under R condition. Methyl ester jasmonic acid, a kind of JA, stimulated the accumulation of disease-resistant substances in plants, according to López-Ráez et al. [39].

Conclusions
AMF-inoculated peach seedlings displayed higher total plant biomass, root CAT, POD, and PPO activities, and root sucrose and fructose concentrations under both NR and R soil conditions. Mycorrhization strongly increased PAL and chitinase activities and SA, JA, and total soluble phenol and lignin levels in roots of peach seedlings grown in R soil. In this process, JA played a dominant role in offering the required resistance of mycorrhizal plants against replant disease through the over-expression of PpCHI, PpLOX1, PpLOX5, PpAOC3, PpAOC4, and PpOPR2 genes in roots triggered by mycorrhization.