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
]. Such soil-borne disease is the result of disturbances in rhizosphere ecology reported in various crops, like peanut, grape, and apple [3
]. 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
]. 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 mycorrhizal apple to establish a higher soil pH value and nutrient levels. In grapes, mycorrhizal inoculations with Glomus etunicatum
, and G
considerably increased superoxide dismutase (SOD) activities, resulting in a lower oxidative damage [11
]. These results indicate a positive role of AMF in alleviating soil replant disease in plants; however, the underlying mechanisms are not clear.
Earlier studies reported that roots of peanut increased disease resistance signal substances such as salicylic acid (SA) and jasmonic acid (JA) in response to infection by Ralstonia solanacerum
, thus reducing the extent of damage caused by replant disease [12
]. SA and JA play a vital role in neutralizing the invasion of pathogens [13
]. These two signaling molecules are of two different types: SA is synthesized through a response pathway of living trophic microbes, while JA operates through dead trophic microbes [15
]. SA and JA could jointly facilitate a series of signal transductions to induce disease resistance in plants, eventually activating the expression of pathogenesis-related genes (PRs). In addition, SA inhibits the activity of cell wall-degrading enzymes secreted by pathogens and also activates the expression of disease-related genes, such as PR-3 and PR-2 encoding chitinase and glucanase, thereby further inhibiting pathogenic growth and reproduction [16
]. JA is also reported to induce gene expression in plants in response to pathogen infection [13
]. As reported by Zhang et al. [17
], inoculation with an arbuscular mycorrhizal fungus (Paraglomus occultum
) up-regulated the expression of the allene oxide synthase gene (a JA-related gene) in Xanthomonas axonopodis
-infected roots of trifoliate orange.
The present study aimed to investigate the effect of AMF inoculation on carbohydrate contents, antioxidant enzyme activities, and JA, SA, lignin, and total soluble phenol concentrations in roots of peach affected by replant disease, in addition to the changes in expression levels of essential enzyme genes involved in the synthetic pathway of SA and JA.
2. Materials and Methods
2.1. 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/m2/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).
2.2. 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 KMnO4
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 ddH2
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−ΔΔCt
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
2.3. 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.
Our study indicated a considerable reduction in root AMF colonization in peach with A
under R soil condition. This is in agreement with earlier studies of Zhang et al. [31
] 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
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
]. 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
, 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 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
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
, 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
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.