A Negative Feedback Regulation of Replanted Soil Microorganisms on Plant Growth and Soil Properties of Peach

Replant disease is one of the main growth limiting factors, interfering with plant growth and yield of stone fruit trees such as peach trees. The ecological feedback mechanisms by replanted soil microbes regulating peach growth and soil structure are rarely known. In our study, rhizosphere soils collected from 18-year-old peach trees were used to plant new peach seedlings, and all soil microbes (R) and soil microbes with the size of < 100 μm (R<100) and < 40 μm (R<40) were applied into peach rhizosphere. After 90 days of microbial inoculation, compared with no microbe treatment (R0), the treatments such as R, R<40, and R<100 reduced plant growth performance (biomass, leaf number, plant height, and stem diameter) and root morphology (total length, projected area, surface area, diameter, and volume), with treatment R being the most inhibition of all other treatments. Similar response of treatment R was observed on changes in concentrations of chlorophyll a, chlorophyll b, and carotenoid. Interestingly, compared with R0 treatment, R<100 treatment produced a significant increase in glomalin-related soil protein (GRSP), percentage of water-stable aggregates in size of 0.25-0.5 mm, soil polyphenol oxidase activities and soil catalase activities. However, R treatment dramatically decreased the percentage of water-stable aggregates in size of 2-4 mm and soil peroxidase activities. Our results suggested that replanted soil microbes, especially with the size of < 100 μm, played a strong negative role on plant growth and rhizosphere biology of peach.


Introduction
Peach (Prunus persica L. Batsch) is globally a kind of deciduous fruit trees, which has a long history of cultivation.However, peach cultivation often faces a common problem, popularly known as soil replant disease, which results in a variety of symptoms like reduced growth, severe gummosis, leaf chlorosis, root browning, reduced yield, and even tree death (Bent et al., 2009;Tewoldemedhin et al., 2011).Changes soil microbiome are considered most important driving factor to such problem due to gradual shift from high-fertility bacterial to low-fertility fungal types (Sun et al., 2015).According to Benzri et al. (2005), soil bacteria such as Bacillus, decomposed cyanogenic glycosides under peach roots to produce some toxic substances (i.e., benzoic acid, cyanohydrogen acid, etc.).
Soil microorganisms including bacteria, fungi, actinomycetes, and symbiotic microbes such as rhizobia and mycorrhizae have an important role in process like nutrient acquisition (Sprent, 2001), nitrogen and carbon cycling (Kowalchuk and Stephen, 2001;Hogberg et al., 2001), and soil aggregate formation (Rillig and Mummey, 2006).In addition, soil microbes can operate through two major mechanisms, popularly known as positive or negative feedback regulation mechanism in the process of interaction between plants and soils (Lü and Wu, 2018).In positive feedback, roles of soil microbes such as plant growth promoting rhizobacteria (PGPR), phosphate-solubilizing bacteria (PSB), and arbuscular mycorrhizal fungi (AMF) are well established in promoting plant growth, improving soil structure, and strengthening the resistance against biotic and abiotic stress (Bhattacharyya and Jha, 2012;Vimal et al., 2017).Zhang et al. (2015) further demonstrated that inoculation with an arbuscular mycorrhizal fungus Funneliformis mosseae on a replanted peach significantly improved soil structure.In negative feedback, soil microbes accelerate the off-site movement of nutrients, hinder plant growth, and reduce crop productivity.In this regard, large number of pathogens, parasites, and some plant-eating animals compete with plants for the carbon and other nutrients (Bever et al., 1997;Burdon et al., 2006).mm was determined using the wet-sieving procedure (Kemper and Rosenau, 1986) with a soil aggregate analyzer (DM200-IV, Shanghai, China).Mean weight diameter (MWD) as an indictor of aggregate stability was calculated by the following formula (Kemper and Rosenau, 1986): , where Xi is mean diameter of the i sieve opening (mm), Wi is proportion of the i size fraction in the total sample mass, and n is number of size fractions.
Soil organic carbon (SOC) was determined by spectrophotometry using dichromate wet oxidation method (Rowell, 1994).Determination of soil easily extractable glomalin-related soil protein (EE-GRSP) and difficulty extractable glomalin-related soil protein (DE-GRSP) was carried out following the protocol as outlined by Wu et al. (2015).Total glomalin-related soil protein (T-GRSP) is the sum of EE-GRSP and DE-GRSP.
Determination of soil polyphenol oxidase (PPO), peroxidase (POD), and catalase (CAT) activities was assayed following the procedure as suggested by Yan (1988).

Statistical analysis
The data (means ± SD, n = 5) were statistically analyzed by one-way variance (ANOVA) in SAS v8.1 (SAS Institute Inc., Cary, USA), and the Duncan's Multiple Range Tests were used to compare the significance between treatments at the 0.05 level.

Results and Discussion
Plant growth performance Plant growth performance is considered as a feedback indicator of rhizosphere properties (Ngullie et al., 2015).Compared with R0 treatment, R treatment registered a lower shoot weight, root weight, and total biomass weight (shoot plus root weight), leaf number, stem diameter, and plant height by 74%, 55%, 69%, 34%, 36%, and 36%, respectively (Table 1), suggesting that replant soil microbes had a negative feedback effect on plant growth parameters of peach.Such reduction in growth of peach plants is attributed to pathogens, parasites, and some plant-eating animals in the replant soil (Jonsson et al., 2001;Maherali and Klironomos, 2007;Van Der Heijden et al., 2008).While, compared with R0 treatment, R<100 treatment significantly reduced only plant height, shoot weight, and total weight by 11%, 30%, and 25%, respectively.The treatment R<40 likewise showed a higher magnitude of reduction in plants height and stem diameter by 20% and 16%, respectively, indicating that replant soil microbes with the size of <100 μm were more inhibitory than <40 μm (Fig. 1).As a result, replant soil microbes mainly including pathogens in nature inhibited plant growth (Griffiths et al., 2000;Loreau, 2001;Garbeva et al., 2004).Earlier studies by Huang et al. (2018) showed that the application of higher concentration of soil water extract from Angelica sinensis rhizosphere, produced a distinct reduction in growth response, in terms of plant height, root length, and fresh weight.While, studies by Lau and Lennon (2011) revealed that complex soil microbial community could promote leaf number, above-ground biomass, and flower number in Brassica rapa.
In this background, the present study was aimed to evaluate the inoculation response of replant soil microbes in plant growth and soil properties of peach (P.persica L. Batsch) under controlled potted conditions.

Experimental set-up
Seeds of peach provided by the Institute of Fruit and Tea, Hubei Academy of Agricultural Sciences, Wuhan, China, were germinated in autoclaved (121 °C, 0.11 MPa, 2 h) sands with an average day/night temperature of 27/20℃ and relative humidity of 80%.After one month, 6-leaf-old peach seedlings with uniform size were transplanted into 2.5-L plastic pots supplied with 2.5 kg autoclaved (121 °C, 0.11 MPa, 2 h) soils.The soil was collected from the rhizosphere of 18-year-old P. persica cv.Yuhualu grafted on P. persica L. Batsch in the Boksugol (30°25′15.1′′Nand 112°08′06.6′′E)near the west campus of Yangtze University in Jingzhou, Hubei, China.The characteristics of the soil are pH 6.1, available P 12.5 mg/kg, and organic carbon 11.2 mg/kg.
After five days of transplanting, peach seedlings with R<100 and R<40 were watered with 100 mL supernatants of < 100 μm and < 40 μm microbes into pots at every five days, whilst a 100 mL distilled water/pot was supplied into the Rand R0-treated pots.

Variable determinations
All the peach seedlings were harvested after 90 days of growth and divided into shoots and roots, whose dry biomass was determined after drying thoroughly.The soil adhered on root surface was collected for onward analysis.Each root system from all treatments was scanned with an EPSON Flat-Scanner (V700, Seiko Epson Corp, Japan) and analyzed with the WinRHIZO 2007d (Regent Instruments Inc., Quebec, Canada) for root morphological traits including root total length, area, volume, and diameter.
Leaf chlorophyll concentration was measured as proposed by Knudson et al. (1977) using the extraction with 80% acetone solution.
Distribution of soil water-stable aggregates (WSAs) at the size of 2.00-4.00,1.00-2.00,0.50-1.00,and 0.25-0.50xxx xxx These studies are strongly suggestive of the rhizosphere microbial diversity depending upon the nature and properties of crop-specific rhizosphere biology.

Root morphology
Changes in root morphology affect the above-ground plant growth.An inhibitive effect of exogenously applied replant soil microbes (Fig. 1; Table 2) was observed over Ro.Compared to R0 treatment, R treatment significantly reduced the total root length, projected area, and root volume by 22%, 17%, and 35%, respectively (Table 2).The treatment R<100 though, decreased these growth parameters to a lesser extent compared to R treatment by 20%, 15%, and 25%, respectively.However, R<40 treatment failed to induce any change in these root traits.It seems that replant soil microbes, especially with Φ < 100 µm, but not Φ < 40 µm, exhibited strong negative effects on root morphology of replanted peach.The soil microbe size of Φ < 100 µm directly hindered the root development possibly by producing toxic exudates to restrict the root growth (Caffaro et al., 2011;Sugiyama and Yazaki, 2012).On the other hand, R<40 treatment did not induce changes in growth root parameters such as root length, root area, root diameter, and root volume compared with R0 treatment, suggesting that Φ < 40 µm size soil microbes are less growth inhibitive on root growth.In previous studies, soil beneficial microbes, for example, AMF (Φ 10 µm) recorded much higher magnitude of growth response than Φ < 40 µm (Allen, 2009).These microbes stimulated the root growth by reducing the relative abundance of toxic substances and producing auxins and polyamines, much to the added advantage in accelerating the root growth (Van Der Heijden et al., 2008;Zou et al., 2015;Lü and Wu, 2017).

Chlorophyll concentrations
Response on plant biomass is by and large regulated through changes in chlorophyll concentration.Our results displayed that compared to R0 treatment, both R<40 and R<100 did not produced no significance difference in concentration of leaf chlorophyll a, chlorophyll b, and carotenoid concentrations (Fig. 2).While, R treatment drastically reduced the leaf chlorophyll a, chlorophyll b, and carotenoid concentration.
These results are in agreement with Huang et al. (2018) in Angelica membranaceu and Cannabis sativa.Lau and Lennon (2011) on the other hand, reported that complex microbial community significantly improved chlorophyll concentrations of Brassica rapa through increase photosynthetic efficiency, compared to simple microbial community.Our observations suggested that all soil microbes derived from fresh replant soil produce a negative feedback regulation on chlorophyll synthesis of peach plants, possibly related to reduce availability of nutrients like Mg, N, and Fe.
Glomalin-related soil protein (GRSP), WSAs and SOC GRSP produced by AMF is closely related to soil organic matter and plays an important role in the formation of soil aggregates, soil carbon budget and resistance to stresses in plants (Barea et al., 2013).GRSP is considered to be the focal point of structural stability of soil (Wu et al., 2016).The concentration of soil EE-GRSP, DE-GRSP and T-GRSP were observed to increase by 9%, 8%, and 8% with R<40 treatment, by 91%, 32%, and 47% with R<100 treatment, and by 55%, 5%, and 19% with R treatment, compared to R0 treatment (Fig. 3).These observations further suggested that peach rhizosphere soils with R<100 treatment produced relatively higher soil GRSP levels, due to stimulation in PGPR via spore germination and mycelium elongation of xxx AMF, leading to better production efficiency of GRSP (Driver et al., 2005;Bedini et al., 2009;Wu et al., 2016), however, in agreement with replanted peach inoculated with Funneliformis mosseae (Zhang et al., 2015).Soil microorganisms such as nematodes and collembolan are reported to have a negative effect on GRSP production by AM hyphae to reduce further proliferation and spread of hyphae network (Bedini et al., 2009).
Changes in soil structure are directly guided by SOC.Soil WSAs are closely associated with water infiltration rate, tilth and aeration in soils (Kemper and Rosenau, 1986).WSAs are an important index of soil structural stability, which is regulated by a variety of aggregate stabilizing agents like roots, SOC, soil enzymes, and soil microbial community (Rillig, 2004;Rillig and Mummey, 2006;Wu et al., 2012;Peng et al., 2013).SOC plays an adhesion role in the initial formation of soil aggregates (Bronick and Lal, 2005).Soil microbes can potentially affect soil WSAs at different levels through hyphae network and their secreted organic polymers (Rillig and Mummey, 2006;Griffiths et al., 2008;Wang et al., 2014).In our study, compared to R0 treatment, R treatment significantly reduced WSA2.00-4.00mm by 65% and increased WSA0.50-1.00 mm and WSA0.25-0.50mm by 38% and 54% (Table 3).R<100 treatment significantly reduced WSA2.00-4.00 mm and WSA0.50-1.00mm by 48% and 31%, and increased WSA0.25-0.50mm by 62%, over R0 treatment.Soil MWD and SOC respectively, declined by 41% and 19% with R and 38% and 10% with R<100, compared with R0 treatment (Table 3).Although a distinct increase in soil GRSP level was observed in inoculated seedlings, a lower soil WSA distribution, SOC, and MWD in inoculated seedlings showed that microbial release of GRSP is not the deciding factor in soil structural improvement, because root surface, root exudates, SOC, and soil enzymes all modulate soil structure formation (Wu et al., 2015).90±1.49b0.33±0.03c0.70±0.15cNote: Data (means ± SD, n = 5) followed by different letters among treatments indicate significant differences at 5% level.Abbreviations: R0 = autoclaved replanted soil without any microbes; R<40 = autoclaved replanted soil treated by Φ < 40 µm soil microbes; R<100 = autoclaved replanted soil treated by Φ < 100 µm soil microbes; R = non-autoclaved replanted soil.Soil antioxidant enzyme activities Soil enzymes play an important role in energy transformation and nutrient cycling in soil-based ecosystems (Bowles et al., 2014).Soil enzymatic activities are closely related to soil microbial flora, quantity and biodiversity, heavily promoting soil metabolic processes (Taylor et al., 2002;Finkenbein et al., 2013).Soil PPO can oxidize the aromatic compounds in soil and generate organic matters and pigments, thus completing the soil aromatic compounds cycle (Toscano et al., 2003).PPO on the other hand, holds an equally important role in transforming heterocyclic compounds (i.e., PAHs) in soils, coupled with positive effects of GRSP on the availability of PAHs (Sun et al., 2012;Gao et al., 2017).Hence, soil microbes, especially with the size of Φ <100 μm significantly increased soil PPO activities, in association with an increase of GRSP (Fig. 4a).Soil POD mainly comes from soil microorganisms, which plays an important role in the formation of humus (Wang et al., 2010).Our results indicated that three soil microbial treatments significantly reduced soil POD activities (Fig. 4b).This is in agreement with Zhang et al. (2015) earlier reported similar response on soil POD and soil PPO activities of replanted peach.Toxic substances released by roots or soil microbes required POD to perform degradation, thereby, resulted a decrease in soil POD activity (Kong, 2007;Wang et al., 2010).Soil CAT is derived from the decomposition of plants and exerts effects on removing the toxic effect of soil hydrogen peroxide (Liu et al., 2008).CAT is closely related to soil microbial abundance and plant roots biomass, which breaks down hydrogen peroxide in the soil to reduce its toxic effects on plants (Liu et al., 2008).Such a considerable increase in soil CAT activities by soil microbial treatments (Fig. 4c) as seen through our observations would partly alleviate toxic effects of hydrogen peroxide on plants.
Furthermore, R<100 treatment represented strong inhibitory effects on biomass production, root development Data (means ± SD, n = 5) followed by different letters among treatments indicate significant differences at the 5% level xxx and soil physicochemical traits, relative to R<40 treatment.It might be due to the fact that R<100 treatment contained a mass of pathogenic organisms with the size of 40-100 μm, such as Rhizoctonia solani (Qin et al., 2014), Ralstonia solanacerum (Chen et al., 2011), Fusarium wilt (Akköprü andDemir, 2005), Pythium, Phytophthora (Spies et al., 2011), etc.Therefore, such pathogens of R<100 treatment might trigger the inhibitive effect.

Conclusions
Replant soil microbes inhibited plant growth performance, root growth, soil aggregation, SOC, and soil PPO activities, especially the soil microbes locked within the size of Φ < 100 μm showed the heaviest negative response.With peach as continuous rotation, soil microbes with Φ < 100 μm need to be eliminated to mitigate the issues related to soil replant disease.However, it remains to be seen, after how many rotations (cropping seasons) such replant issue becomes more distinctive in a given crop-soil-climate setup, alongside the associated biochemistry involved.

Fig. 2 .
Fig. 2. Effects of different sized replanted soil microbes on chlorophyll a, chlorophyll b and carotenoid concentrations of peach (Prunus persica) seedlings.Data (means ± SD, n = 5) followed by different letters among treatments indicate significant differences at the 5% level Fig. 3. Effects of different sized replanted soil microbes on easily extractable glomalin-related soil protein (EE-GRSP), difficulty extractable glomalin-related soil protein (DE-GRSP), and total glomalin-related soil protein (T-GRSP) concentrations of peach (Prunus persica) seedlings.Data (means ± SD, n = 5) followed by different letters among treatments indicate significant differences at the 5% level

Table 1 .
Effects of different sized soil microbes on plant growth performance of replanted peach (Prunus persica) seedlings

Table 2 .
Effects of different sized soil microbes on root morphological traits of replanted peach (Prunus persica) seedlings

Table 3 .
Effects of different sized soil microbes on distribution of water-stable aggregate (WSA), mean weight diameter (MWD), and soil organic carbon (SOC) concentrations of replanted peach (Prunus persica) seedlings