Effect of Hydrogen Peroxide on the Soil MicrobialCommunity Structure and Growth of Malus hupehensis Rehd. Seedlings under Replant Conditions (2024)

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ACS Omega. 2023 Feb 21; 8(7): 6411–6422.

Published online 2023 Feb 8. doi:10.1021/acsomega.2c06665

PMCID: PMC9947989

PMID: 36844530

Xin Xu,^† Yifan Zhou,^‡ Xiaoqi Wang,^† Weitao Jiang,^† Lei Qin,^† Jian Wang,^† Haijun Yu,^§ Xuesen Chen,^† Xiang Shen,^† Chengmiao Yin,*^† and Zhiquan Mao*^†

Author information Article notes Copyright and License information PMC Disclaimer

Associated Data

Supplementary Materials

Abstract

Apple replant disease(ARD) is common in apple production, whichseriously affects the growth and development of apples. In this study,hydrogen peroxide with a bactericidal effect was used to treat thereplanted soil, and the effects of different concentrations of hydrogenperoxide on replanted seedlings and soil microbiology were investigatedin order to seek a green, clean way to control ARD. Five treatmentswere set up in this study: replanted soil (CK1), replanted soil withmethyl bromide fumigation (CK2), replanted soil + 1.5% hydrogen peroxide(H1), replanted soil + 3.0% hydrogen peroxide (H2), and replantedsoil + 4.5% hydrogen peroxide (H3). The results showed that hydrogenperoxide treatment improved replanted seedling growth and also inactivateda certain number of Fusarium, while the Bacillus, Mortierella, and Guehomyces alsobecame more abundant in relative terms. The best results were obtainedwith replanted soil + 4.5% hydrogen peroxide (H3). Consequently, hydrogenperoxide applied to the soil can effectively prevent and control ARD.

1. Introduction

Apple replant disease (ARD) commonly refers to a situation wherethe old trees were removed, and they become weak or even die whenyoung trees are replanted in the same orchard soil.¹ Due to frequent tree species optimization, but limitedland resources, it is difficult to avoid replanting cultivation inthe original site during orchard renovation, which in turn leads tothe widespread occurrence of ARD, and significantly restricts thesustainable development of the apple industry.² Replanted young apple trees usually exhibit such phenomena as showdelayed root extension, slow plant metabolism, poor resistance tostress, and even death of the whole plant.³ ARD causes serious economic losses throughout the whole life cycleof an orchard.⁴ Thus, it is urgent to developalternative clean and green measures to control ARD.

ARD wascaused by a combination of reasons,^5,6 forexample, nematodes, oomycetes, and chemosensitive autotoxic substanceimbalance in the structure of soil microbiology,⁷⁻⁹ among whichsoil microbiological imbalance is known to be the main reason forARD.^10,11 When fruit trees are planted in orchardsfor successive years, beneficial bacteria decrease and the numberof soil pathogenic fungi increases, which eventually results in adecrease in the yield of fruit trees.¹² In South Africa, Washington, Italy, and other apple producing countries, Fusarium, Pythium, Phytophthora, and Cryptococcus were considered to be harmfulpathogens that cause ARD.^13,14 Liu¹⁵ found that the frequency of Fusarium washigh when isolating and identifying harmful fungi in the soil of replantedorchards. Fusarium showed high pathogenicity to the M. hupehensis Rehd. seedlings.¹⁶

In the United States, the use of brassicaceae seedmeal to mitigateARD has achieved better results.^4,17 However, in China,due to various reasons, soil fumigation is still a common way to controlARD.^18,19 However, although traditional soil chemicalfumigants have obvious effects, they also have disadvantages suchas easy residue, pollution of the natural environment, and potentialhazards to human health.^20,21 Therefore, it is veryimportant to seek a green, efficient, and reliable measure to solveARD. Hydrogen peroxide is a simple, safe, economical, and environmentallyfriendly oxidizing agent, and the by-product of hydrogen peroxideis water, which is often called the “Green Oxidant”.Therefore, the use of hydrogen peroxide has been concerned by researchersfor a long time.²² It is widely used inmedicine, agriculture, food safety, and clinical and environmentalapplications.^23,24 For example, in the medical field,hydrogen peroxide was often used as a bactericide or disinfectant.²⁵ In the food processing industry, hydrogen peroxidewas often used to kill microorganisms on food packaging bags, containers,and disinfectants such as drinking water, which could effectivelyinhibit the growth of microorganisms.²⁶ In the agricultural field, Kyeong-Hwan et al²⁷ used hydrogen peroxide vapor that interfered with the growthof disease-causing microorganisms. This was because hydrogen peroxidecould react with lipid double bonds in the microbial cell wall andenter the microbial interior, acting on proteins and lipids and polysaccharides,altering cell permeability, and leading to cell lysis and death.²⁸ In addition, traditional chemical fumigationwas time-consuming and requires film covering. In contrast, hydrogenperoxide to kill harmful microorganism in replanted soils may be aclean, green measure. However, there was little literature describingthe application of hydrogen peroxide in affecting the severity ofARD by killing soil microorganisms.

In the present experiment,we evaluated the feasibility of hydrogenperoxide to mitigate ARD under replanting conditions. We aimed to(1) determine the optimal concentration of hydrogen peroxide; (2)how different concentrations of hydrogen peroxide affect the soilmicrobial community structure; (3) response of replanted seedlingsto various concentrations of hydrogen peroxide, thus providing newinsights into the ARD mitigation.

2. Materialsand Methods

2.1. Experimental Materials

The replantedsoil was obtained from an old orchard in Manzhuang Town where appletrees have been planted for 34 years. The soil type was sandy loam.After the top soil was cleaned off, multiple points soil was randomlyselected within a depth of 20–40 cm and mixed well. Supplementary Table 1 describes soil basic physicochemicalproperties.

The experiment material was M. hupehensis Rehd. seedlings, and it is a very widely planted rootstock of applein China. M. hupehensis Rehd. seedswere stratificated for roughly 40 days at 4 °C. The seeds wereplanted in seedling cups contained with seedling substrates when whiteradicles were seen. When the seedling grew to 5–6 true leaves,the seedlings with similar growth, complete leaves, and no pests anddiseases were transplanted into tile pots with different soil treatments(pot diameter 24 cm, height 18 cm, and soil weight 7 kg).

Methylbromide fumigation products are provided by Jiangsu LianyungangDead Sea Bromide Co.

Hydrogen peroxide was purchased from JinanKunfeng Chemical Co.,Ltd., with a concentration of 27.5%.

2.2. ExperimentalDesign

The experimentwas performed from April to October 2021 at the Science and TechnologyInnovation Park of Shandong Agricultural University (36.16°N,117.1 6°E). A total of 5 treatments were examined: replantedsoil (CK1), replanted soil with methyl bromide fumigation (CK2), replantedsoil + 1.5% hydrogen peroxide (H1), replanted soil + 3.0% hydrogenperoxide (H2), and replanted soil + 4.5% hydrogen peroxide (H3).

Methyl bromide fumigation treatment and hydrogen peroxide treatmentwere performed 15 days before the planting of M. hupehensis Rehd. seedlings (performed in mid-April 2021). Methyl bromide fumigantwas mixed with replanting soil and put into the sealed trellis filmfor sealing, the soil layer is controlled about 20 cm, and 50 g ofmethyl bromide fumigation per square meter of soil is applied. Ifconverted to pot, 0.125 g/kg is applied to each pot, and the temperatureis controlled at 15 °C.¹⁹ Hydrogenperoxide was diluted in water in three different proportions (1.5,3.0, and 4.5%). The soil was irrigated until the upper-middle layerof soil reached a saturated state (518 mL). Equal amounts of sterilewater were added to CK1 and CK2 treatments as control. Five repetitionswere set up for each treatment.

On May 01, 2021, replanted seedlingswith consistent growth wereplanted in each treatment (two seedlings per pot). Each treatmentreceived uniform pruning, irrigation, and management. Plant and soilsamples were taken from the 5 treatments on August 15. Each treatmentgroup was sampled by randomly chosen three seedlings. Rhizospheresoil was collected from the pot by removing soil at the top and aroundthe pot, and it was mixed thoroughly. The rhizosphere soil sampleswere sealed in a resealable bag and divided into three parts aftersieving through 20 mesh; one part was air-dried under natural conditionsand performed for the measurement of soil enzyme activity; one partwas put into a 4 °C refrigerator for the determination of soilmicroorganisms; and the other was placed at −80 °C forIllumina MiSeq sequencing. When the plant samples were collected,the M. hupehensis Rehd. seedlings werewashed. Vigorous white roots were excised and stored in liquid nitrogenfor the determination of root enzymes and viability. Fresh and intactleaves were selected from the plant samples and stored at −20°C to determine the chlorophyll content.

2.3. Indicatorsfor Plant and Soil Measurements

The plant heights and grounddiameters were measured with conventionalmethods such as a pylon ruler and dial calipers. The soil on the seedlingswas washed off by water, excess water was wiped off, and the freshseedlings were weighed with an electronic balance. After the determination,it was quenched at 105 °C for 30 min and dried at 65 °C,and the dry mass was weighed. The determination of the chlorophyllcontent was carried out by extraction with ethanol²⁹ (Supplementary Material 1.1).The determination of photosynthetic parameters was carried out onAugust 14 (sunny day, no wind) from 9:00 am to 11:00 am (Supplementary Material 1.2). The TTC method wasused to determine the root respiration rate³⁰ (Supplementary Material 1.3). The determinationof root antioxidant enzyme activity refers to the method of Singhet al.³¹ (Supplementary Material 1.4).

2.4. DataAnalysis

The original sequencingwas filtered out using fastp software to eliminate the ones smallerthan 50 bp. According to their overlapped sequences, sequences over10 bp were assembled using flash software. Operational taxonomic unit(OTU) clustering analysis was usually conducted with 97% similarity.Based on OTU results, the alpha diversity index was calculated indifferent treatments by using mothur (https://mothur.org/wiki/calculators/.version 1.30.2) and plotted with origin 2018 software (Origin LabCorporation, USA). The rarefaction curves were plotted by R language,and the R language (version 3.3.1) vegan package was employed forCluster heat map analysis. Principal coordinate analysis (PCoA) wascalculated for each sample at the genus level. The significance ofthe difference between multiple groups was tested by the Kruskal–Wallis H test. Functional annotation and prediction of bacterialand fungal communities were performed using PICRUSt1 and FUNGuildprediction analysis, respectively. SPSS 26.0 (IBM SPSS Statistics,IBM Corporation, USA) was used for mean comparison and single-factoranalysis of variance. The Duncan-style new multiple range method wasused for significant difference comparison, and data were composedof mean ± standard error. Origin 2018 software was used to constructthe figures.

3. Results

3.1. Influencesof Hydrogen Peroxide on the Growthof Replanted Seedlings

There were significant differencesin the growth status of replanted seedlings under different concentrationsof hydrogen peroxide treatment (Figure Figure11). H2 and H3 treatments obviously improved the growthof replanted seedlings; among them, H3 treatment had the best effect.The plant height, ground diameter, fresh weight, and dry weight ofthe H3 treatment increased 0.63, 1.27, 8.47, and 9.69 times, respectively,compared to CK1. Compared with CK2, H3 treatment increased 0.23, 0.06,0.82, and 0.87 times in the plant height, ground diameter, fresh weight,and dry weight, respectively (Figure Figure11).

Effect of Hydrogen Peroxide on the Soil MicrobialCommunity Structure and Growth of Malus hupehensis Rehd. Seedlings under Replant Conditions (5)

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Figure 1

Growth status of replanted seedlings. (a) Plant height;(b) grounddiameter; (c) fresh weight; (d) dry weight; CK1: replanted soil; CK2:replanted soil with methyl bromide fumigation; H1: replanted soil+ 1.5% hydrogen peroxide; H2: replanted soil + 3.0% hydrogen peroxide;H3: replanted soil + 4.5% hydrogen peroxide; letters reflect differentdegrees of treatment variation (P < 0.05).

3.2. Influences of HydrogenPeroxide on the ChlorophyllContent and Photosynthetic Parameters of Replanted Seedlings

Replanted soil + 1.5% hydrogen peroxide (H1), replanted soil + 3.0%hydrogen peroxide (H2), and replanted soil + 4.5% hydrogen peroxide(H3) all significantly increased the chlorophyll a and b contents(Table 1). Among them,the treatment with replanted soil + 4.5% hydrogen peroxide (H3) hadthe best effect, followed by CK2. Compared with CK1, chlorophyll a,b increased by 15.8 and 11.0% in the replanted soil + 1.5% hydrogenperoxide (H1) treatment; 23.6 and 24.5% in the replanted soil + 3.0%hydrogen peroxide (H2) treatment; and 40.0 and 79.3% in replantedsoil + 4.5% hydrogen peroxide (H3) treatment, respectively. The differencesin chlorophyll a, b between H1 and H2 were not significant.

Replanted soil + 1.5% hydrogen peroxide (H1), replanted soil + 3.0%hydrogen peroxide (H2), and replanted soil + 4.5% hydrogen peroxide(H3) treatments all significantly improved replanted seedling photosyntheticparameters (Figure Figure22). The application of replanted soil + 4.5% hydrogen peroxide (H3)was shown to be significantly different from all other treatments.Compared with CK1, intercellular carbon dioxide concentration, netphotosynthetic rate, stomatal conductance, and transpiration rateof replanted soil + 4.5% hydrogen peroxide (H3) treatment increasedby 14.7, 106.3, 81.8, and 56.7%, respectively. The difference in theintercellular carbon dioxide concentration between replanted soil+ 1.5% hydrogen peroxide (H1) and replanted soil + 3.0% hydrogen peroxide(H2) treatments was not significant.

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Figure 2

Effect of hydrogen peroxide treatmenton photosynthetic parametersof replanted seedlings. (a) Net photosynthetic rate; (b) intercellularcarbon dioxide concentration; (c) transpiration rate; (d) stomatalconductance; CK1: replanted soil; CK2: replanted soil with methylbromide fumigation; H1: replanted soil + 1.5% hydrogen peroxide; H2:replanted soil + 3.0% hydrogen peroxide; H3: replanted soil + 4.5%hydrogen peroxide; letters reflect different degrees of treatmentvariation (P < 0.05).

Table 1

Effects of Different Treatments onChlorophyll a and Chlorophyll b in the Leaves of Replanted Seedlings^a

treatment	chlorophyll a (mg·g^–1·FW)	chlorophyllb (mg·g^–1·FW)
CK1	18.45± 0.77d	5.46 ± 0.54d
CK2	23.44 ± 0.20b	8.23±0.45ab
H1	21.37 ± 0.68c	6.06 ± 0.35d
H2	22.81±0.56bc	6.80 ± 0.49cd
H3	25.83 ± 0.68a	9.79 ± 0.74a

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^aNote: letters reflect differentdegrees of treatment variation (P < 0.05).

3.3. Influences of HydrogenPeroxide on Root AntioxidantEnzyme Activities and the Root Respiration Rate of Replanted Seedlings

Replanted soil + 1.5% hydrogen peroxide (H1), replanted soil +3.0% hydrogen peroxide (H2), and replanted soil + 4.5% hydrogen peroxide(H3) treatments all significantly improved root antioxidant enzymeactivity (Figure Figure33).Compared with CK2, the superoxide dismutase (SOD) and catalase (CAT)activities of H3 increased by 21.55 and 4.93%, respectively. Significantdifferences were observed in the root antioxidant enzyme activitiesof replanted soil (CK1) and hydrogen peroxide treatment. The H1, H2,and H3 treatments also significantly increased the root respirationrates by 26.1, 51.3, and 96.4%, respectively, compared to replantedsoil (CK1).

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Figure 3

Effects of root antioxidant enzymes and the root respiration rateof replanted seedlings under hydrogen peroxide treatment. (a) Superoxidedismutase activity; (b) catalase activity; (c) peroxidase activity;(d) root respiration rate; letters reflect different degrees of treatmentvariation (P < 0.05).

3.4. Influences of Hydrogen Peroxide on Soil EnzymeActivity

Replanted soil + 1.5% hydrogen peroxide (H1), replantedsoil + 3.0% hydrogen peroxide (H2), and replanted soil + 4.5% hydrogenperoxide (H3) treatments all significantly reduced the activity ofsoil enzymes (Figure Figure44). Among them, replanted soil with CK2 had the largest decrease,followed by replanted soil + 4.5% hydrogen peroxide (H3) treatment.Compared to CK1, sucrase, urease, and phosphatase activity were reducedby 3.80, 22.2, and 14.1%, in replanted soil + 1.5% hydrogen peroxide(H1); 17.5, 33.3, and 25.3%, in replanted soil + 3.0% hydrogen peroxide(H2); and 24.9, 44.4, and 36.5% in replanted soil + 4.5% hydrogenperoxide (H3), respectively.

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Figure 4

Effect of hydrogen peroxide treatment on soilenzyme activity.(a) Sucrase activity; (b) urease activity; (c) phosphatase activity.CK1: replanted soil; CK2: replanted soil with methyl bromide fumigation;H1: replanted soil + 1.5% hydrogen peroxide; H2: replanted soil +3.0% hydrogen peroxide; H3: replanted soil + 4.5% hydrogen peroxide;letters reflect different degrees of treatment variations (P < 0.05).

3.5. Analysisof Culturable Microorganisms andRT-qPCR Analysis of Fusarium oxysporum

The addition of different concentrations of hydrogen peroxidecan significantly improve the environment of replanted soil (Figure Figure55). In particular,replanted soil + 1.5% hydrogen peroxide (H1), replanted soil + 3.0%hydrogen peroxide (H2), and replanted soil + 4.5% hydrogen peroxide(H3) treatments significantly reduced the bacteria, fungi, and Fusarium oxysporum numbers in the soil. Replantedsoil + 4.5% hydrogen peroxide (H3) treatment had the largest decrease,followed by replanted soil + 3.0% hydrogen peroxide (H2) and finallyreplanted soil + 1.5% hydrogen peroxide (H1). Compared with the replantedsoil (CK1), soil bacteria and fungi were decreased by 34.6 and 58.9%in the replanted soil + 1.5% hydrogen peroxide (H1) treatment; 73.1and 70.5% in the replanted soil + 3.0% hydrogen peroxide (H2) treatment;and 91.0 and 87.6% in the replanted soil + 4.5% hydrogen peroxide(H3) treatment, respectively. In addition, compared with replantedsoil (CK1), Fusarium oxysporum of replantedsoil + 1.5% hydrogen peroxide (H1), replanted soil + 3.0% hydrogenperoxide (H2), and replanted soil + 4.5% hydrogen peroxide (H3) treatmentsdecreased by 44.5, 66.6, and 90.8%, respectively. The gene copy numberof F. oxysporum in the other treatmentswas lower than that in replanted soil (CK1).

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Figure 5

Changes in soil microbialpopulation under different treatments.(a) Number of soil bacteria; (b) number of soil fungi; (c) gene copynumber of F. oxysporum; CK1: replantedsoil; CK2: replanted soil with methyl bromide fumigation; H1: replantedsoil + 1.5% hydrogen peroxide; H2: replanted soil + 3.0% hydrogenperoxide; H3: replanted soil + 4.5% hydrogen peroxide; letters reflectdifferent degrees of treatment variations (P <0.05).

3.6. Analysisof Soil Microbial Community Compositionat the Genus Level

By sequencing, the dilution curve of fungiand bacteria tend to be flat, which indicated that the data volumeof microbial communities of soil environmental samples was close tosaturation, and it indicated that the current sequencing quantitiescould reflect the majority of microbial diversity information in thesamples (Figure S1). The abundance heatmapfor bacteria and fungi genera was constructed from the top 10 dominantspecies in the sample. Arthrobacter, Sphingomona, RB41, Bacillus, and Gaiella were the main bacterial species, except for unclassified bacteria(Figure Figure66a). Mortierella, Pseudallescheria, Fusarium, Lophiostoma, Guehomyces, Humicota, Trichoderma, and Kermia were the dominant fungal species except for the unclassifiedfungi (Figure Figure66b).The species of bacterial and fungal genera were basically the samebetween treatments, but their relative abundance differed significantly.The comparative abundance of Bacillus increased by14.03% for the H3 treatment compared to CK1; the comparative abundanceof Fusarium decreased by 40.29% for the H3 treatmentcompared to CK1. In addition, compared with CK1, CK2, H1, H2, andH3 treatments obviously increased the comparative abundance of Mortierella, and the largest increase was observed in thetreatment of replant soil + 4.5% hydrogen peroxide (H3), followedby CK2 treatment. Compared to CK1, replanted soil + 1.5% hydrogenperoxide (H1), replanted soil + 3.0% hydrogen peroxide (H2), and replantedsoil + 4.5% hydrogen peroxide (H3) treatments significantly increasedthe comparative abundance of Trichoderma, with CK2showing the largest increase.

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Figure 6

Differences between species of bacterial genus(a) and fungal genus(b) for different treatments. CK1: replanted soil; CK2: replantedsoil with methyl bromide fumigation; H1: replanted soil + 1.5% hydrogenperoxide; H2: replanted soil + 3.0% hydrogen peroxide; H3: replantedsoil + 4.5% hydrogen peroxide.

3.7. Differences in the Microbial Community Compositionin Different Treatments

The PCoA plot showed that PC1 andPC2 explained 75.85% of the changes in bacterial communities and 61.98%of the changes in fungal communities, respectively (Figure Figure77). Microbial communities weresignificantly different between treatments, CK2 being the most distantfrom CK1, meaning that the differences were the greatest. The threedifferent hydrogen peroxide concentration treatments also had a certaindistance from the CK1, and the application of hydrogen peroxide inthe replanted soil might have an impact on the change of the microbialcommunity structure. The treatments were subjected to the Kruskal–Wallis H test based on the different microbial community structures,at the level of bacterial genera, Gaiella, Pedomicrobium, Romboutsia, and Turicibacter that were more abundant in the hydrogen peroxide-treatedsoils, with CK2 treatment being the most pronounced (Figure S2). At the fungal genus level, the comparative abundanceof Mortierella, Guehomyces in thesoil treated with hydrogen peroxide was significantly higher, whilethe comparative abundance of Fusarium and Lophiostoma was significantly lower (Figure S2).

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Figure 7

PCoA was calculated for each sample on the basis of theBray–Curtisdistance matrix at the bacterial genus (a) and fungal genus (b) level.CK1: replanted soil; CK2: replanted soil with methyl bromide fumigation;H1: replanted soil + 1.5% hydrogen peroxide; H2: replanted soil +3.0% hydrogen peroxide; H3: replanted soil + 4.5% hydrogen peroxide.

3.8. Analysis of Microbial AlphaDiversity underDifferent Concentrations of Hydrogen Peroxide Treatment

TheSimpson and Chao indices of bacteria and fungi in soil treatmentswith different concentrations of hydrogen peroxide were significantlydifferent. In general, the Simpson index often estimated microbialdiversity in samples, and the smaller the Simpson index, the higherthe community diversity. The Simpson indices of replanted soil + 1.5%hydrogen peroxide (H1), replanted soil + 3.0% hydrogen peroxide (H2),and replanted soil + 4.5% hydrogen peroxide (H3) treatments were allhigher than those of CK1, indicating that the microbial diversityof soil treated with hydrogen peroxide was lower (Figure Figure88a,c). The Chao index was usedto reflect the abundance of microorganisms, and the comparative abundanceof bacteria and fungi in the hydrogen peroxide-treated soil showeda decreasing trend, with significant differences in the Chao indexof bacterial genera between treatments. The Chao index of fungi generain CK2 and H3 treatments showed some differences (Figure Figure88b,d).

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Figure 8

Diversity of microorganismsunder different treatments. Simpsonindex for species of the genera bacteria (a) and fungi (c); Chao indexfor species of the genera bacteria (b) and fungi (d). CK1: replantedsoil; CK2: replanted soil with methyl bromide fumigation; H1: replantedsoil + 1.5% hydrogen peroxide; H2: replanted soil + 3.0% hydrogenperoxide; H3: replanted soil + 4.5% hydrogen peroxide; letters reflectdifferent degrees of treatment variations (P <0.05).

3.9. MicrobialCommunity Function Prediction

PICRUSt1 and FUNGuild predictionanalyses were made to analyzethe possible functions of bacterial and fungal communities in therhizosphere soil, respectively. PICRUSt1 results indicated that atotal of 24 taxon functions were obtained in the bacterial communities.Except for the unclassified functions and prediction-only functions,the top three functions were amino acid transport and metabolism,energy production and conversion, and signal transduction mechanisms,and their relative abundance in the bacterial community was 4.68,4.01, and 3.72%, respectively (Figure Figure99a). Among the fungal communities, undefined saprotroph,endophyte-litter saprotroph-soil saprotroph-undefined saprotroph,animal pathogen-endophyte-lichen parasite-plant pathogen-soil saprotroph-woodsaprotroph were the three function taxa with higher abundance (Figure Figure99b). Among them, theonly functional group that corresponds to the genus Mortierella belongs to is endophyte-litter saprotroph-soil saprotroph-undefinedsaprotroph, and it had the highest abundance in the H3 treatment.The only fungal genera corresponding to animal pathogen-endophyte-lichenparasite-plant pathogen-soil saprotroph-wood saprotroph was Fusarium, and the abundance of Fusarium was significantly lower in the replanted soil + 3.0% hydrogen peroxide(H2) and replanted soil + 4.5% hydrogen peroxide (H3) treatments thanin CK1(Table S2).

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Figure 9

Predicted function ofbacterial (a) community and fungal (b) community.CK1: replanted soil; CK2: replanted soil with methyl bromide fumigation;H1: replanted soil + 1.5% hydrogen peroxide; H2: replanted soil +3.0% hydrogen peroxide; H3: replanted soil + 4.5% hydrogen peroxide.

4. Discussion

ARD canlead to stunted tree growth, root rot, reduced fruit production,and then result in tree mortality, ultimately shortening the lifespan of replanted apple orchards.^37,38 The main causesof ARD include increased numbers of harmful fungi and changes in themicrobial community structure in the soil under replanting conditions.^19,39 This study showed that different concentrations of hydrogen peroxidecould promote the replanted seedling growth from the physiologicalindicators, and both the plant biomass of replanted soil + 3.0% hydrogenperoxide (H2) and replanted soil + 4.5% hydrogen peroxide (H3) treatmentswere obviously higher than that of CK1. This status was likely dueto hydrogen peroxide killing pathogenic fungi in the soil, improvingthe soil environment for replanting and promoting plant growth.⁴⁰ Plant growth cannot be achieved without photosynthesis,and chlorophyll is the main photosensitive pigment for photosynthesisto absorb light energy, which has an important impact on plant growth.⁴¹ Therefore, the chlorophyll content affects photosyntheticparameters, which in turn affects the growth of M.hupehensis Rehd. seedlings.⁴² Ozaki et al.⁴³ found that the photosyntheticrate of melon leaves was also enhanced when treated with hydrogenperoxide. Compared with CK1, the chlorophyll content and photosyntheticparameters were obviously varied among different hydrogen peroxidetreatments, with replanted soil + 4.5% hydrogen peroxide (H3) treatmentbeing the most effective. Hydrogen peroxide impacts the structureof soil microbial communities and changes the microbial-mediated soilnutrient conversion process to facilitate nutrient uptake by crops.Therefore, the growth of the plants was enhanced and their chlorophyllcontent and photosynthetic parameters were also enhanced.⁴⁴

Most studies have shown that in general,free radical productionand elimination in plants often remain in relative balance. When confrontedwith adversity conditions, the production rate of free radicals wasfar greater than the elimination rate, and the plant was damaged.⁴⁵ Under the condition of long-term continuouscropping, the deteriorating soil environment could create a threatof adversity in apple seedlings. The relative balance of free radicalswas broken, and seedling growth was threatened. Superoxide dismutase,peroxidase, and catalase were the three important factors in the protectionof plants from excessive free radicals. The three work synergisticallyto protect the inner membrane structure of the plant body and reducethe damage to the membrane structure by free radicals.⁴⁶ In this study, it was found that hydrogen peroxidetreatment enhanced the level of antioxidant enzymes in plants. Themain reason is that hydrogen peroxide has a strong oxidizing and sterilizingeffect, which purifies the soil environment and reduces microbialdamage to the root system, thus promoting the growth of plants.⁴⁷ The second may have a small amount of hydrogenperoxide surviving in the soil that can directly or indirectly activateantioxidant enzymes during stress, thus increasing the induced resistanceof the plant.⁴⁸ Studies have demonstratedthat hydrogen peroxide provides a more vigorous root system in wheat.⁴⁹ Hydrogen peroxide can also promote rooting androot growth of ground cover chrysanthemum plugs.⁵⁰

A good rhizosphere microbial community structurecould maintainthe balance of soil microhabitats and ensure normal growth of plants.⁵¹ Soil microorganisms were important for plantgrowth as an important indicator for assessing soil ecosystems.⁵² Generally speaking, beneficial soil microorganismshave a positive effect on plant development, while harmful microorganismscan hinder plant development, and even result in death. For example, Fusarium wilt in many important crops worldwide iscaused by Fusarium.³² RT-PCRanalysis illustrated that that CK2, H1, H2, H3 treatments obviouslydecreased the gene copy number of F. oxysporium. The methyl bromide fumigant has great potential to kill pests andpathogens in the soil and also reduces the population of Fusarium.(32) Hydrogen peroxide possesses microbicidaland sporicidal activity.⁵³

Researchshows that Mortierella has a symbioticor reciprocal relationship with plants.⁵⁴Mortierella converts insoluble phosphorus and potassiumin the soil into available phosphorus and potassium for plant uptakeand utilization, thus improving plant resistance.⁵⁵Mortierella was shown to have a negativerelationship with the occurrence of ARD and could play a vital rolein suppressing ARD by competing for nutrients or resisting nutrients.⁵⁶Fusarium was severely positivelyinterrelated with ARD in China,^57,58 and it exhibits strongpathogenicity.⁵⁶Bacillus not only inhibited the reproduction of pathogenic fungi, improvedmicrobial community structure, and enhanced plant disease resistancebut also secreted catabolic phytase to increase the amount of freephosphorus in the soil, promoted phosphorus uptake by plants, andimproved the crop yield.⁵⁹ Making fungifertilizer from Trichoderma asperellum strain both promoted replanted seeding growth and development, optimizedthe soil microhabitats, and effectively alleviated ARD.⁶⁰ In aquatic and terrestrial habitats, Lophiostom commonly occurs as sapwood on branches, stems,or bark in woody and herbaceous plants.⁶¹ Intaraudom et al.⁶² found that the secondarymetabolites of Lophiostoma bipolare BCC25910 had no obvious inhibitory effect on disease-causing bacteria.In this experiment, the soil microbial community after hydrogen peroxidetreatment was studied using amplicon sequencing technology. We foundthat soil treatment with hydrogen peroxide significantly improvedthe comparative abundance of Bacillus, Pedomicrobium, Mortierella, Guehomyces, and Trichoderma and significantly reduced the comparative abundanceof Fusarium and Lophiostoma, withthe most obvious effect of replanted soil + 4.5% hydrogen peroxide(H3) treatment. Hydrogen peroxide treatment significantly alteredthe soil microhabitats and distinctly reduced the number of Fusarium. Li⁶³ found that theaccumulation of hydrogen peroxide has the effect of directly poisoningand killing pathogenic bacteria.

The diversity and richnessof species were commonly expressed byAlpha diversity. In this study, the simpson index of replanted soil+ 1.5% hydrogen peroxide (H1), replanted soil + 3.0% hydrogen peroxide(H2), and replanted soil + 4.5% hydrogen peroxide (H3) treatmentswas higher than that of CK1, indicating that the microbial diversityof replanted soil + 1.5% hydrogen peroxide (H1), replanted soil +3.0% hydrogen peroxide (H2), and replanted soil + 4.5% hydrogen peroxide(H3) treatments was lower than that of CK1. Li⁶⁴ found a decreased microbial community diversity after soilfumigant was applied, and this is the same result as that in our study.Hydrogen peroxide is a broad-spectrum disinfectant that inhibits harmfulmicroorganisms while also having an effect on beneficial microorganisms,causing a “vacuum” in the soil.⁶⁵ Soil enzymes are the results of soil microbial metabolism and decompositionof plant and animal residues,⁶⁶ and itis involved in a series of biochemical reactions in the soil.⁶⁷ In this study, hydrogen peroxide treatment reducedthe soil enzyme activity, and the higher the application concentration,the more obvious the reduction in soil enzyme activity. At the sametime, the soil microbial population also decreased to different degrees.This is probably due to the application of hydrogen peroxide thatkilled some of the microorganisms associated with soil enzyme activityin the soil, which in turn led to a decrease in soil enzyme activity.This is in agreement with the findings of Klose et al.⁶⁸ who used bromomethane fumigation of soil tocause a decrease in soil microbial population and soil enzyme activity.

In the present study, 24 bacterial taxa functions were obtainedby PICRUSt1 for bacteria community prediction analysis, among whichamino acid transport and metabolism functions were more abundant,which was consistent with the results of Yang et al.⁶⁹ FUNGuild prediction analysis showed that animal pathogen-endophyte-lichenparasite-plant pathogen-soil saprotroph-wood saprotroph correspondedto the fungi genus was only Fusarium, and the higherfunctional abundance of Fusarium corresponded toits higher abundance in the functional group composition. The highfunctional abundance of Fusarium was in line withthe higher abundance of its functional taxa. Correspondingly, the Fusarium abundance in replanted soil (CK1) was higher. FUNGuildsuggests that Fusarium was the key causal agent ofARD. The abundance of Mortierella in soil after hydrogenperoxide treatment was obviously high, compared to replanted soil,and the most obvious treatment was with replanted soil + 4.5% hydrogenperoxide (H3) treatment. Free radicals produced by hydrogen peroxideare known to damage DNA of spores in one of the species.⁷⁰ This indicates that hydrogen peroxide reducedthe abundance of Fusarium and increased the numberof beneficial microorganisms, which effectively improved the soilmicrohabitat and alleviated ARD.

5. Conclusions

In this experiment, we found that the hydrogen peroxide treatmentpromoted the growth and development of replanted seedlings and improvedthe chlorophyll content, photosynthetic parameters, and root antioxidantenzyme activities of seedlings. Soil microhabitat also changed todifferent degrees, especially, increased the comparative abundanceof Bacillus, Mortierella, decreasedthe comparative abundance of Fusarium. In summary,the effect of replanted soil + 4.5% hydrogen peroxide (H3) treatmentwas the most significant, which effectively alleviated ARD.

Acknowledgments

The research was supported by the ChinaAgricultureResearch System of MOF and MARA (Grant No. CARS-27), the NationalNatural Science Foundation of China (Grant No. 32072510), the QingchuangScience and Technology Support Project of Shandong Colleges and Universities(No. 2019KJF020), the Natural Science Foundation of Shandong Province(Grant No. ZR2020MC131), Shandong Agricultural Major Applied TechnologyInnovation Project (Grant No. SD2019ZZ008), the National Key Researchand Development Program of China (Grant No. 2020YFD1000201), TaishanScholars Funded Project (No. ts20190923), and the Fruit InnovationTeam in Shandong Province, China (Grant No. SDAIT-06-07).

Supporting Information Available

The Supporting Information isavailable free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c06665.

Description of methodsfor the determination of indicatorssuch as root antioxidant enzyme activity and soil culturable microorganisms;information about soil physicochemical properties; dilution curves;the Kruskal–Wallis test used to statistically compare the relativeabundance of bacterial and fungal genera on different treatments;and distribution of OTUs of functional taxa (PDF)

Author Contributions

X.X. and Y.F.Z.contributed equally to this work as first author. All authors readand approved the manuscript.

Notes

The authors declare nocompeting financial interest.

Supplementary Material

ao2c06665_si_001.pdf^{(2.7M, pdf)}

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Effect of Hydrogen Peroxide on the Soil Microbial Community Structure and Growth of Malus hupehensis Rehd. Seedlings under Replant Conditions? ›

The results showed that hydrogen peroxide treatment improved replanted seedling growth and also inactivated a certain number of Fusarium, while the Bacillus, Mortierella, and Guehomyces also became more abundant in relative terms.

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How long does hydrogen peroxide stay active in soil? ›

Under aerobic soil metabolism conditions, hydrogen peroxide degrades with a half-life of ca. 1.4 hours in diluted test solutions. This half-life value was extrapolated to ca. 7 hours for concentrated test solutions.

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What does hydrogen peroxide do to the soil? ›

H202 kills the bacteria and fungi in the soil responsible for root rot, and restores oxygen to help remaining roots recover faster. Directions: 1:1 ratio of Oxygen Plus 3% and water. Bottom water or top water so potting mix is fully saturated. Avoid leaves (pouring at this strength on leaves can burn them).

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Is hydrogen peroxide good for seedlings? ›

All plants can generally tolerate hydrogen peroxide, however it must be diluted. If it is left at full strength, it can bleach or damage leaves.

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How does hydrogen peroxide affect microbes? ›

Hydrogen peroxide is responsible for certain bactericidal effects observed in biological systems, such as growth inhibition of one bacterial species by another and killing of invading microorganisms by activated phagocytic cells.

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How long does it take for hydrogen peroxide to decompose? ›

Hydrogen peroxide is relatively unstable and decomposes quickly. In a sealed container, hydrogen peroxide lasts approximately 3 years. However, as soon as you open the container, it starts to break down. You might be surprised to learn that it's only effective for 1 to 6 months once the container is opened.

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How long is hydrogen peroxide active? ›

Hydrogen peroxide.

You need to replace hydrogen peroxide six months after opening it, but it will last for three years unopened. To test whether it is still effective, you can pour it in to the sink and see if it fizzes and bubbles. If it does, it's still good. Expired hydrogen peroxide is ineffective but not harmful.

What percentage of hydrogen peroxide kills bacteria? ›

Hydrogen peroxide does kill germs, including most viruses and bacteria. A concentration of 3% hydrogen peroxide is an effective disinfectant typically found in stores. Hydrogen peroxide can damage some surfaces, and is a more dangerous chemical than some disinfectants, so be cautious when handling it.

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Which form of hydrogen peroxide is most effective at killing microorganisms? ›

Hydrogen peroxide in liquid and gaseous forms has been shown to provide excellent antimicrobial activities against a broad spectrum of organisms.

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Why is hydrogen peroxide bad for organisms? ›

Hydrogen peroxide produces highly reactive hydroxyl radicals via Fenton's reaction. Reduced iron Fe(II), which is found only inside cells, donates an electron to H₂O₂ and splits the molecule to form these radicals, making H₂O₂ a potent biocide.

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Does hydrogen peroxide evaporate over time? ›

The rate at which hydrogen peroxide evaporates depends on various factors such as concentration , temperature , and surface area . Generally , it takes about 10 - 30 minutes for hydrogen peroxide to evaporate completely at room temperature . However , in warmer environments , it can evaporate much faster .

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How long can you soak plants in hydrogen peroxide? ›

Does hydrogen peroxide break down in sunlight? ›

Hydrogen peroxide is also decomposed through the effect of UV-light as well as by certain enzymes (catalase).

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Effect of Hydrogen Peroxide on the Soil Microbial Community Structure and Growth of Malus hupehensis Rehd. Seedlings under Replant Conditions (2024)

Associated Data

Abstract

1. Introduction

2. Materialsand Methods

2.1. Experimental Materials

2.2. ExperimentalDesign

2.3. Indicatorsfor Plant and Soil Measurements

2.4. DataAnalysis

3. Results

3.1. Influencesof Hydrogen Peroxide on the Growthof Replanted Seedlings

3.2. Influences of HydrogenPeroxide on the ChlorophyllContent and Photosynthetic Parameters of Replanted Seedlings

Table 1

3.3. Influences of HydrogenPeroxide on Root AntioxidantEnzyme Activities and the Root Respiration Rate of Replanted Seedlings

3.4. Influences of Hydrogen Peroxide on Soil EnzymeActivity

3.5. Analysisof Culturable Microorganisms andRT-qPCR Analysis of Fusarium oxysporum

3.6. Analysisof Soil Microbial Community Compositionat the Genus Level

3.7. Differences in the Microbial Community Compositionin Different Treatments

3.8. Analysis of Microbial AlphaDiversity underDifferent Concentrations of Hydrogen Peroxide Treatment

3.9. MicrobialCommunity Function Prediction

4. Discussion

5. Conclusions

Acknowledgments

Supporting Information Available

Author Contributions

Notes

Supplementary Material

References

FAQs

Effect of Hydrogen Peroxide on the Soil Microbial Community Structure and Growth of Malus hupehensis Rehd. Seedlings under Replant Conditions? ›

What percentage of hydrogen peroxide kills bacteria? ›