SOILS, SEC 1 • SOIL ORGANIC MATTER DYNAMICS AND NUTRIENT CYCLING • RESEARCH ARTICLE

Effects of vermicompost amendment as a basal fertilizer on soilproperties and cucumber yield and quality under continuouscropping conditions in a greenhouse

Hai-Tao Zhao1,2 & Tian-Peng Li1,2 & Yang Zhang1,2 & Jian Hu1,2& Yan-Chao Bai1,2 &

Yu-Hua Shan1,2& Feng Ke1,2

Received: 23 December 2016 /Accepted: 22 May 2017 /Published online: 9 June 2017# The Author(s) 2017. This article is an open access publication

AbstractPurpose We examined the effects of vermicompost applica-tion as a basal fertilizer on the properties of a sandy loam soilused for growing cucumbers under continuous cropping con-ditions when compared to inorganic or organic fertilizers.Materials and methods A commercial cucumber (Cucumissativus L.) variety was grown on sandy loam soil under foursoil amendment conditions: inorganic compound fertilizer(750 kg/ha,), replacement of 150 kg/ha of inorganic com-pound fertilizer with 3000 kg/ha of organic fertilizer orvermicompost, and untreated control. Experiments were con-ducted in a greenhouse for 4 years, and continuous plantingresulted in seven cucumber crops. The yield and quality ofcucumber fruits, basic physical and chemical properties ofsoil, soil nutrient characteristics, and the soil fungal commu-nity structure were measured and evaluated.Results and discussion Continuous cucumber cropping de-creased soil pH and increased electrical conductivity. However,application of vermicompost significantly improved several soilcharacteristics and induced a significant change in the rhizo-sphere soil fungal community compared to the other treatments.Notably, the vermicompost amendments resulted in an increasein the relative abundance of Ascomycota, Chytridiomycota,Sordariomycetes, Eurotiomycetes, and Saccharomycetes, and adecrease in Glomeromycota, Zygomycota, Dothideomycetes,

Agaricomycetes, and Incertae sedis. Compared to the organicfertilizer treatment, vermicompost amendment increased the rel-ative abundance of beneficial fungi and decreased those of path-ogenic fungi. Cucumber fruit yield decreased yearly under con-tinuous cropping conditions, but both inorganic and organic fer-tilizer amendments increased yields. Vermicompost amendmentmaintained higher fruit yield and quality under continuouscropping conditions.Conclusions Continuous cropping decreased cucumber yieldin a greenhouse, but basic fertilizer amendment reduced thisdecline. Moreover, basal fertilizer amendment decreased ben-eficial and pathogenic fungi, and the use of vermicompostamendment in the basic fertilizer had a positive effect on thehealth of the soil fungal community.

Keywords Continuous cropping . Cucumber . Greenhouse .

Mineral nutrition . Soil fungal community . Vermicompost

1 Introduction

Vermicompost is produced by earthworms’ digestion of or-ganic waste (e.g., food waste, horticultural waste, poultrydroppings, and food industry sludge) (Yadav and Garg 2011;Mendoza-Hernández et al. 2014; Huang et al. 2014; Lalanderet al. 2015). This material has received increased attention inrecent years because of its interesting physical, chemical, andbiological characteristics. Vermicompost is a sustainablesource of macro- and micro-nutrients (Atiyeh et al. 2000a,b), and the mineral nutrient elements in vermicompost areeasily absorbed by plants (Edwards and Burrows 1988;Orozco et al. 1996; Atiyeh et al. 2000a, b). Furthermore,vermicompost has a fine granular structure with a largesurface area, which allows it to absorb and retain nutrients(Zhao and Huang 1991). A large number of plant hormones

Responsible editor: Caixian Tang

* Jian Huhuj@yzu.edu.cn

1 College of Environmental Science and Engineering, YangzhouUniversity, Yangzhou, Jiangsu 225127, China

2 Jiangsu Collaborative Innovation Center for Solid Organic WasteResource Utilization, Nanjing, Jiangsu 210095, China

J Soils Sediments (2017) 17:2718–2730DOI 10.1007/s11368-017-1744-y

are found in vermicompost (e.g., IAA, GA3, kinetin) and theirpresence may be the result of jointing activity of earthwormsand microorganisms (Ravindran et al. 2016). Overall,vermicompost has the basic characteristics associated with amaterial that could be employed to improve soil quality. Theapplication of vermicompost has been found to be an effectivemethod for rejuvenation of soil fertility, enrichment of avail-able nutrient pools, and conservation of water (Makode 2015).Vermicompost amendments can increase the dry mass andyield of greenhouse crops (Norman and Clive 2010;Warman and Anglopez 2010; Little et al. 2011), and signifi-cantly suppress root pests via the modulation of soil propertiesand plant defenses, particularly for susceptible plants (Xiaoet al. 2016). The use of vermicompost as an organic fertilizeris considered a better alternative to inorganic fertilizers (Joshiet al. 2013).

Soil microorganisms play an important role in the recyclingand transformation of nutrients. The sustainability of terrestri-al agroecosystems is indicated by microbial diversity and ac-tivity (Su et al. 2015), and the application of both organic andinorganic fertilizers are beneficial for the stability of the orig-inal soil microorganism community structure (Ding et al.2016). Soil fungi are important soil microorganisms, andsome species promote the healthy growth of plants, whereasothers can cause harm to plants. Beneficial fungi includeChaetomium globosum (Aggarwal et al. 2014), unculturedGlomus (Hassan et al. 2013), uncultured Trichoderma(Sharma et al. 2012), and Mortierella alpina (Al-Shammariet al. 2013). Harmful fungi include Fusarium oxysporum(Fravel et al. 2003), Cladosporium sp. (Papazlatani et al.2016), Acremonium cucurbitacearum (Bruton et al. 2000),Fusarium solani (Oddino et al. 2008), Verticillium dahliae(Lopez-Escudero and Blanco-Lopez 2005), and Rhizopycnisvagum (Westphal et al. 2011). As soil fungi have a clear anddirect relationship with soil quality (Gilsotres et al. 2005; Sixet al. 2006), it is important to pay attention to the interactiveeffects of vermicompost on soil fungal communities, particu-larly in continuous cropping schemes in greenhouses.

Cucumber is one of main greenhouse-cultivated cropsaround the globe. In this study, we compared the

performances of cucumber plants grown under continuouscropping on soils that had received amendments of organicfertilizer, inorganic compound fertilizer, or vermicompost asthe basal fertilizer. The following characteristics wereassessed: (1) cucumber fruit yield and quality under continu-ous cropping conditions and (2) the influence of the fertilizeramendments on soil properties under continuous cropping.We specifically focused on the influence of a vermicompostamendment on the soil fungal community, and explored therelationship of vermicompost properties and the soil fungalcommunity. Based on the widely reported positive effects ofvermicompost on crops, we hypothesized that replacement ofa portion of inorganic fertilizer with vermicompost in a green-house would have the following consequences: (1) improvedfruit yield and quality, (2) increased mineral nutrient suppliesin the soil, and (3) enhanced physicochemical and microbio-logical properties in soil under continuous croppingconditions.

2 Materials and methods

2.1 Experimental materials and design

The following soil amendments were used in this study: inor-ganic compound fertilizer (N-P2O5-K2O 15-15-15) (JiangsuMiddle East Group Co., Ltd., China,), chicken manure-basedorganic fertilizer (Nanjing Jiahe Organic Fertilizer Co., Ltd.,China), and vermicompost, a by-product of the digestion ofcow manure by earthworms (Eisenia fetida) (YangzhouAgricultural Environmental Safety Technical Service Center,Jiangsu, China). The experimental soil had a sandy loamtexture, and had been used in rice cultivation prior to theexperimental period. The properties of the organic fertilizer,vermicompost, and soil were determined according to Lu(2000) and are summarized in Table 1.

The experiments were conducted in greenhouses (8.0 × 50m)at the Yangzhou University Shatou test base (32.5°N, 119.31°E),located in the Yangtze River region where modern agriculture inChina was developed. In this experiment, cucumber (Cucumis

Table 1 The properties oforganic fertilizer, vermicompost,and soil

Indicators Soil Organic fertilizer Vermicompost

pH 7.49 6.62 7.21

Electrical conductivity (EC) (mS/cm) 0.11 6.56 1.73

Total nitrogen (TN) (g/kg) 1.65 16.95 9.83

Ammonium nitrogen (NH4+-N) (mg/kg) 10.74 25.41 52.47

Nitrate nitrogen (NO3−-N) (g/kg) 0.02 3.12 0.22

Organic matter (OM) (%) 0.26 47.59 27.23

Total phosphorus (TP) (g/kg) 0.47 6.28 44.76

Available phosphorus (Olsen-P) (g/kg) 0.04 1.67 4.68

Available potassium (AK) (g/kg) 0.14 28.20 3.37

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sativus L.) was planted in large (60 cm) and small (30 cm) inter-val patterns, and plants were spaced 30 cm apart. Cucumberswere planted twice per year (i.e., April to July and Septemberto December), and cucumber crops were continuously cultivatedfor 4 years (April 2012 to July 2015) in greenhouses that wereconstantly covered with plastic film. The soil remained fallowwhen cucumbers were not being cultivated. A randomized blockdesign with three replicates was used, and 12 plots (3.0 × 9.0 m)were established. To prevent contamination, a ground separator(5 cm in height) was placed between each plot. Inorganic com-pound fertilizer, organic fertilizer, and vermicompost were ap-plied as the basal fertilizer to the soil surface, and rotary tillageand ridging were then performed during the 15 days prior to thetransplantation of cucumber seedlings. Based on a model of3000 kg/ha of organic material equates to 150 kg/ha of inorganicfertilizer, the following basic fertilizer amendments were used:inorganic compound fertilizer (750 kg/ha) amendment (FT), in-organic compound fertilizer (600 kg/ha) and organic fertilizer(3000 kg/ha) amendment (FC), inorganic compound fertilizer(600 kg/ha) and vermicompost (3000 kg/ha) amendment (VP),and no basic fertilizer amendment (CK). Inorganic compoundfertilizer was also applied as a topdressing (750 kg/ha) to alltreatments when the cucumber plants entered the fruiting stage.The management measures employed in the greenhouse wereconsistent with those used in field production.

2.2 Cucumber quality and yield

During the early stage of cucumber seedling growth, six plantswere selected from each plot, and the yield per plant was deter-mined by measuring the weight of each plant during the 2012–2015 spring seasons (April to July). Samples were collected todetermine the quality of cucumber fruit before the topdressingapplication. The quality of cucumber fruits was measured inJune of 2015. The nitrate content of the fruit was extractedusing boiling water and determined by UV spectrophotometry(Qi 2003). The soluble solid content was measured using anAbbe refractometer (2WA-G, Shanghai Fifth OpticalInstrument Factory, China), soluble protein content was deter-mined using Coomassie brilliant blue G-250 staining, solublesugar content was measured using the anthrone colorimetricmethod, organic acid content was determined by the NaOHdirect titration method, and vitamin C content was measuredusing a titration method with 2,6-dichlorophenol (Qi 2003).The sugar-acid ratio of fruit was calculated as the ratio of solu-ble sugar to organic acid contents.

2.3 Basic soil and nutrient properties

Soil samples were collected at a depth of 0–15 cm before theannual topdressing application in June of each year. Each samplewas divided into two parts, one part was directly used for thedetermination of soil microbial nitrogen (MBN) and soluble

organic carbon (DOC) and the second partwas air-dried and usedfor the determination of other variables. The pH and electricalconductivity (EC) were measured from 2012 to 2015, but othervariables were only measured in 2015. The methods describedby Lu (2000) were used to estimate soil properties. A soil sus-pension was prepared at a 1:5 ratio of soil-to-water (m:v) usingdouble-distilledwater and used tomeasure pH (PXSJ-216F, Rex,China) and EC (DDSJ-308F, Rex, China). The amount of totalnitrogen (TN) in the soil was determined by distillation titration,the ammonia nitrogen (NH4

+-N) content was measured usingindophenol blue spectrophotometry, and the nitrate nitrogen(NO3

−-N) content was estimated using an ultraviolet spectropho-tometer. Soil samples were fumigated with chloroform andmixed with K2SO4 solution, and the resulting alkali distillationliquid was then used to determine MBN. The organic matter(OM) content was determined using a K2Cr2O7-H2SO4 solutionand external heating method. A clarification solution was pre-pared after extraction using a 1:5 ratio of soil-to-water (m:v) indouble-distilled water, and this was used to measure the DOCusing a total organic carbon analyzer (TOC-L, Shimadzu, Japan).Total phosphorus (TP)was obtained viaHClO4-H2SO4 digestionand then molybdenum blue colorimetry. Determination of theavailable phosphorus (Olsen-P) was performed using NaHCO3

solution extraction andmolybdenum blue colorimetry. The avail-able potassium (AK) was measured using ammonium acetatesolution extraction and a flame photometric method. Soil bulkdensity was determined using a cutting ring, and the soil watercontent was measured by drying the soil at 80 °C.

2.4 Soil microbial properties

Rhizosphere soil samples were collected in June 2015 to deter-mine the diversity of soil fungal communities; the samples wereobtained as described by Ling et al. (2014). All samples wereprocessed individually, and genomic DNAs present in each com-posite sample were extracted using a FastDNA® SPIN Kit forsoil (MP Biomedicals, USA) according to the manufacturer’sinstructions. Three replicate DNA extractions were conductedfor each soil sample. The composition of the fungal communityin each soil sample was determined using Illumina Miseq se-quencing, as described by Magoč and Salzberg (2011). The pro-tocol for sequencing the samples was as follows: (1) PCR wasused to amplify the internal transcribed spacer (ITS) ITS1 regionusing ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) andITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) primers, (2)Illumina sequencing libraries were prepared using PCR productsas templates, and (3) Illumina Miseq system was used to se-quence the ITS1 region.

Filtering and connection of the original double-terminalsequences were then conducted. Firstly, the FASTQ sequences(raw data obtained by IlluminaMiseq sequencing) of two endswere filtered based on the weight of the fragment using asliding window approach with a 5-bp window size and a 1-

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bp step. The sliding window began at the first base position,and the average weight of base was calculated based on theQ20 score (i.e., base accuracy ≥99%). The sequence was trun-cated when bases lower than Q20 were detected, and the finalsequence length was restricted to >150 bp (sequences withambiguous base calls (N) were excluded). Secondly, se-quences were connected with FLASH software (Lievenset al. 2007) using an overlap of >10 bp between reads 1 and2, and base mismatches were not allowed. Lastly, the effectivesequence of each sample (with fully matched indices) wasextracted based on the sequence information used to distin-guish the samples.

2.4.1 Quality sequence filtering

Quality sequences were filtered using Qiime software (version1.9.0) (Caporaso et al. 2010; Scarlett et al. 2013), and theuchime method found in the mothur software package (ver-sion 1.31.2; http://www.mothur.org/) was used to removechimeric sequences (Schloss et al. 2009; Edgar et al. 2011).Erroneous sequences were removed, including sequenceswith 5′ prime mismatched bases greater than 1, sequencescontaining ambiguous base calls (N), sequences containingmore than 8 contiguous bases, sequences less than 150 bp inlength, and chimeric sequences.

2.4.2 Clustering and annotation of operational taxonomicunits

The uclust method found in the Qiime software package wasused to cluster the high-quality sequences, and a 97% se-quence similarity criterion was used to assess sequence clus-ters (Edgar et al. 2011). The longest sequence of each classwas selected as the representative sequence. A BLAST search(Altschul et al. 1990) based on the Qiime software packagewas used to compare the sequences to those in sequence da-tabases, and taxonomic information on each operational taxo-nomic unit (OTU) sequence was obtained (Altschul et al.1990). The FastTree program (Price et al. 2009) was used toconstruct a phylogeny based on the representative OTU se-quences. In order to ensure the accuracy of the results, OTUswith an abundance value <0.001% of the total number ofsequences were removed (Bokulich et al. 2012). The ACEindex was used to measure community richness (Pitta et al.2010), and the Shannon index was used to evaluate microbialdiversity in soil samples (Shannon and Weaver 1949).

2.5 Statistical analysis

The data (n = 3) were subjected to one-way analysis of variance(ANOVA) and multivariate analysis of variance (MANOVA)using the SPSS software package for Windows (SPSS, version18.0, Chicago, USA). When statistical significance (p < 0.05)

was detected, the mean values subjected to Duncan’s multiplerange tests. Pearson’s two-tailed tests were performed to establishcorrelations between soil physical and chemical properties andcucumber development (n = 3). A principal component analysis(PCA) was used to analyze the characteristics of the soil fungalcommunity. The relationship between soil environmental factorsand soil fungal community composition was analyzed using aredundancy analysis (RDA).

3 Results

3.1 Cucumber yield and quality

Fruit yields in the greenhouse fell with continuous cropping(Table 2). This effect presented in all treatments over the exper-imental period, 2012 to 2015. The highest yield (1.53 kg/plant)was obtained in 2012 from plants in the vermicompost plusinorganic compound fertilizer treatment, and yields in the othertreatments were followed in the following sequence: organicfertilizer plus inorganic compound fertilizer, inorganic compoundfertilizer amendment, and control (no amendment). After 4 yearsof continuous cropping, the yield in theVP treatment declined by19.4%, yields in the FC, FT, andCK treatments declined by 41.0,30.9, and 22.2%, respectively. Basal fertilizer amendment clearlyincreased the yield of cucumber fruit regardless of whether or-ganic or inorganic fertilizers were used; the VP treatment ap-peared to be best at maintaining fruit yield under continuouscropping in a greenhouse than the FC and FT treatments.

Application of a basal fertilizer significantly increased cucum-ber fruit quality (Table 3). Compared to the CK treatment, VPtreatment significantly increased soluble solids, soluble sugar,vitamin C, and soluble protein contents as well as the sugar-acid ratio of cucumber fruits after 4 years of continuous cropping(in 2015). Some quality indices of FT were significantly higherthan those of the CK treatment, including vitamin C content,nitrate content, and the sugar-acid ratio. The level of nitrates inthe fruit from the FT treatment was 3.5-fold higher than in theCK treatment. Nitrate contents of fruit from the FC treatmentwere significantly higher than those in the CK treatment, butwere significantly lower than those of the FT treatment.Overall, the VP treatment was the best at maintaining the qualityof the fruit after 4 years of continuous cropping.

3.2 Characteristics of soil properties

3.2.1 Characteristics of soil basic physical and chemicalproperties

Both continuous cropping of cucumbers and basal fertilizeramendments under greenhouse conditions resulted in a gradualdecrease in soil pH and a gradual increase in soil electrical con-ductivity (EC) (Table 2). After 4 years of continuous cropping, the

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use of basal fertilizer amendments had decreased soil bulk densityand increased soil water content (Table 3). Soil pH in the CKtreatment was significantly higher than that of VP-treated soil inthe spring seasons of 2014 and 2015, and was also significantlyhigher than those of the FTand FC treatments in 2015. The pH ofVP-treated soil (6.39) was significantly lower than that of theother treatments in 2015. The EC of FC-treated soil (0.41 mS/cm) in spring 2012was significantly higher than those of the othertreatments, while the ECof soil in theCK treatment (0.82mS/cm)in spring 2013 was significantly lower than those of the othertreatments. Moreover, the EC of FT-treated soil (2.76 mS/cm)was significantly higher than those of the other treatments in2015. The soil bulk density values of VP-treated soil were signif-icantly lower than those of the CK treatment, while the soil watercontent was significantly greater than that of the CK treatment in2015. The soil water content of FC-treated soil was significantlylower than that in the VP treatment in 2015. The various analysesindicated that theVP treatment led to the largest decline in soil pHand soil bulk density and the largest increase in soil water contentafter 4 years of continuous cropping. The FT treatment caused thelargest increase in soil EC.

3.2.2 Characteristics of carbon, nitrogen, phosphorus,and potassium in soil

In the three treatments in which basal fertilizer was applied, soilnitrogen characteristics were significantly different at the end ofthe experiment compared to the control (Table 3). TN contentwas highest in the FT treatment, NH4

+-N content was highest inthe CK treatment, and microbial nitrogen (MBN) content washighest in the VP treatment. Nitrate nitrogen (NO3

−-N) content inthe CK treatment was significantly lower than those of the FC,FT, and VP treatments, and the differences in TN, NH4

+-N,NO3

−-N, and MBN between FT and FC treatments were not

significant in 2015. The TN (1.95 g/kg) and NH4+-N (4.48 mg/

kg) contents in the FT treatment were significantly larger thanthose in the VP treatment, but the MBN (125.25 mg/kg) contentin the VP treatment was significantly larger than that in the FTtreatment. In comparison to the CK treatment, the FT and FCtreatments increased in TN and NO3

−-N contents, and causedreductions in soil NH4

+-N content. Furthermore, the VP treat-ment increased soil NO3

−-N andMBN and reduced soil NH4+-N

content.Basal fertilizer amendments also significantly influenced

soil carbon, phosphorus, and potassium contents (Table 3).Soil organic matter (OM), soluble organic carbon (DOC), totalphosphorus (TP), available phosphorus (Olsen-P), and avail-able potassium (AK) contents were significantly lower in2015 in the CK treatment than in treatments given basal fer-tilizer amendments. In the FC treatment, soil OM was signif-icantly higher than in the VP and FT treatments, but there wereno significant differences in soil DOC contents among the FC,VP, and FT treatments. Soil TP and AP contents in the VPtreatment were significantly higher than those of the FT andFC treatments, while the soil AK content of the FT treatmentwas significantly higher than those of the VP and FC treat-ments. These results indicate that the application of organicfertilizer promoted the accumulation of soil OM and that basalfertilizer amendments had no significant effects on soil DOCcontents. The VP amendment increased TP and AP accumu-lation, and the FT amendment increased soil AK.

3.3 Relationship between cucumber yield and qualityand soil properties

Cucumber fruit yield and quality were significantly affectedby soil properties. Thus, the increases in soil water, TP, AP,and MBN contents were positively correlated with cucumber

Table 2 Characteristics of the cucumber fruit yield and the soil pH and EC in different base fertilizer treatments from spring of 2012 to 2015

Indicators Treatments Spring of 2012 Spring of 2013 Spring of 2014 Spring of 2015

Fruit yield (g/plant) No base fertilizer 704 ± 274 d 6744 ± 12 d 548 ± 18 d 509 ± 21 c

Inorganic compound fertilizer only 1032 ± 40 c 915 ± 39 c 714 ± 5 c 598 ± 7 c

Organic fertilizer amendment 1392 ± 48 b 1021 ± 24 b 821 ± 16 b 786 ± 33 b

Vermicompost amendment 1533 ± 52 a 1345 ± 44 a 1236 ± 26 a 1317 ± 48 a

pH No base fertilizer 7.49 ± 0.23 a 7.63 ± 0.06 a 6.88 ± 0.11 a 6.81 ± 0.03 a

Inorganic compound fertilizer only 7.69 ± 0.10 a 7.24 ± 0.28 a 6.83 ± 0.03 ab 6.61 ± 0.03 b

Organic fertilizer amendment 7.62 ± 0.04 a 7.51 ± 0.08 a 6.83 ± 0.03 ab 6.67 ± 0.04 b

Vermicompost amendment 7.48 ± 0.03 a 7.45 ± 0.03 a 6.57 ± 0.07 b 6.39 ± 0.04 c

Electrical conductivity(EC) (mS/cm)

No base fertilizer 0.28 ± 0.01 b 0.82 ± 0.06 b 0.95 ± 0.03 c 1.71 ± 0.12 c

Inorganic compound fertilizer only 0.31 ± 0.01 b 1.32 ± 0.06 a 1.33 ± 0.04 b 2.76 ± 0.07 a

Organic fertilizer amendment 0.41 ± 0.03 a 1.18 ± 0.04 a 1.37 ± 0.10 ab 2.24 ± 0.05 b

Vermicompost amendment 0.34 ± 0.02 b 1.34 ± 0.05 a 1.68 ± 0.09 a 2.36 ± 0.13 b

Mean values ± standard error (n = 3) are shown; means shared with a common letter in the same column indicate no significant differences betweentreatments (p < 0.05) based on Duncan’s multiple range test

2722 J Soils Sediments (2017) 17:2718–2730

yield (Table 4). However, soil TN, NH4+-N, pH, and bulk

density had a significant negative correlation with cucumberyield. The NO3

−-N, MBN, DOC, TP, Olsen-P, and soil watercontents had a positive correlation with cucumber fruit quality,but the soil TN, NH4

+-N, pH, and bulk density had a signifi-cant negative opposite correlation. Soil OM content had asignificant positive correlation with the nitrate content of thefruit, and soil EC had a positive correlation with the sugar-acidratio of the fruit. The soil AK content was significantly andpositively correlated with vitamin C content, sugar-acid ratio,and nitrate content of fruit, but was significantly and negative-ly correlated with organic acid content.

3.4 Characteristics of soil fungi

3.4.1 Structure of the soil fungal community

The number of reads sequenced from soil samples ranged from97,589 (FT treatment) to 86,476 (VP treatment), and the pro-portion of high-quality reads ranged from 75.7% (FT treatment)to 74.9% (VP treatment) (Table 5). The lengths of the high-quality sequences were in the range of 240–270 bp. In total,687 OTUs were identified from rhizosphere soil from all treat-ments; 359 common OTUs were identified (data not shown).

Application of basal fertilizer significantly affected thestructure of the soil fungal community. The VP treatment in-creased soil fungal diversity (Shannon index) and richness(ACE index) compared to the other treatments (Table 5). Aprincipal component analysis (PCA) indicated that the differ-ences in the fungal community structure between FT and CKtreatments were small. However, fungal community structuresin the FC and VP treatments differed from that in the CKtreatment, and these differences were especially prominentafter the VP treatment (Fig. 1).

The soil fungal community was dominated by theAscomycota (55.9%), and other phyla were present at much low-er levels, e.g., Basidiomycota (5.7%), Zygomycota (4.2%),Chytridiomycota (0.7%), and Glomeromycota (0.3%) (Fig. 2a).Compared with the CK treatment, the relative abundance ofAscomycota and Chytridiomycota increased significantly in theVP treatment, while the Glomeromycota and Zygomycota weresignificantly decreased. In the FC treatment, the relative abun-dance of Ascomycota, Glomeromycota, and Zygomycota de-creased significantly. In the FT treatment, the relative abundanceof Chytridiomycota significantly increased but that ofGlomeromycota and Zygomycota decreased. The relative abun-dance of Basidiomycota decreased significantly in the VP, FC,and FT treatments.

Table 3 Cucumber fruit qualities, and soil nitrogen, carbon, phosphorus, potassium, water content, and bulk density in the four different base fertilizertreatments in spring of 2015

Indices CK FT FC VP

Cucumber fruit Soluble solid (Bx) 3.01 ± 0.02 b 3.08 ± 0.02 b 3.05 ± 0.02 b 3.28 ± 0.05 a

Soluble sugar (mg/g) 31.8 ± 2.1 b 33.20 ± 0.7 ab 34.1 ± 1.4 ab 36.8 ± 0.5 a

Organic acid (g/kg) 1.4 ± 0.0 a 1.3 ± 0.0 a 1.4 ± 0.0 a 1.4 ± 0.1 a

Vitamin C (mg/kg) 66 ± 4 b 90 ± 4 a 68 ± 2 b 102 ± 5 a

Soluble protein (mg/g) 1.96 ± 0.04 b 1.92 ± 0.04 b 1.93 ± 0.04 b 2.34 ± 0.00 a

Sugar-acid ratio 0.22 ± 0.01 b 0.26 ± 0.01 a 0.24 ± 0.00 ab 0.26 ± 0.02 a

Nitrate concentration (mg/kg) 79.8 ± 8.2 c 280 ± 5.0 a 211 ± 11 b 77.6 ± 7.1 c

Soil Total nitrogen (TN) (g/kg) 1.76 ± 0.04 bc 1.95 ± 0.01 a 1.88 ± 0.07 ab 1.69 ± 0.07 c

Ammonium nitrogen (NH4+-N) (mg/kg) 18.3 ± 0.9 a 14.5 ± 0.4 b 14.2 ± 0.8 b 11.0 ± 0.4 c

Nitrate nitrogen (NO3−-N) (g/kg) 1.02 ± 0.04 c 1.50 ± 0.08 a 1.53 ± 0.13 a 1.53 ± 0.02 a

Microbial nitrogen (MBN) (mg/kg) 63.2 ± 3.8 b 68.1 ± 3.9 b 78.1 ± 3.7 b 125.2 ± 5.9 a

Organic matter (OM) (g/kg) 18.9 ± 0.1 c 20.9 ± 0.1 b 22.2 ± 0.4 a 20.7 ± 0.7 b

Soluble organic carbon (DOC) (mg/kg) 1.80 ± 0.05 b 2.17 ± 0.10 a 1.96 ± 0.03 ab 2.25 ± 0.08 a

Total phosphorus (TP) (g/kg) 0.50 ± 0.02 d 0.69 ± 0.02 b 0.62 ± 0.01 c 1.14 ± 0.02 a

Available phosphorus (Olsen-P) (mg/kg) 120 ± 2 c 169 ± 4 b 169 ± 5 b 202 ± 19 a

Available potassium (AK) (mg/kg) 130 ± 7 c 509 ± 29 a 337 ± 17 b 352 ± 14 b

Water content (%) 20.0 ± 0.3 c 21.3 ± 0.4 bc 22.1 ± 0.5 b 24.2 ± 0.5 a

Bulk density (g/cm3) 1.09 ± 0.02 a 1.07 ± 0.02 ab 1.04 ± 0.01 ab 1.00 ± 0.04 b

CK control, no fertilizer addition; FT inorganic fertilizer; FC organic fertilizer/inorganic fertilizer mixture; VP vermicompost/inorganic fertilizer mixture(see Sect. 2 for the description of the mixtures)

Mean values ± standard error (n = 3) are shown; means shared with a common letter in the same column indicate no significant differences betweentreatments (p < 0.05) based on Duncan’s multiple range test

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At the class level, Sordariomycetes (40.3%) predominatedover Dothideomycetes (4.2%), Eurotiomycetes (3.2%),Agaricomycetes (3.0%), and Tremellomycetes (2.7%) in rhi-zosphere soil (Fig. 2b). The relative abundance ofDothideomyceteswas significantly higher in the CK treatmentthan in fertilizer-amended soils. The relative abundance ofEurotiomycetes and Sordariomycetes in the VP treatmentand of Agaricomycetes and Tremellomycetes in the CK treat-ment was higher than in the other treatments.

3.4.2 Beneficial and harmful fungi

Next, we examined the relative abundance of harmful fungiin soils from the different treatments. The relative abundanceof F. solani and R. vagum in the CK treatment was significant-ly higher than in the other treatments; Cladosporium sp. weresignificantly more abundant in the FT treatment. The relativeabundance of F. oxysporum in the CK and FT treatments was

significantly greater than that in the FC and VP treatments(Fig. 3a). In the FT treatment, a decrease in the relative abun-dance of F. solaniwas observed. In the FC and VP treatments,a reduction in the relative abundance of F. solani, F.oxysporum, Cladosporium sp., and R. vagum was found.Overall, the application of basal fertilizers to the soil decreasedthe relative abundance of harmful fungi. The organic fertil-izers had a significantly greater effect on the reduction ofharmful fungi than the inorganic fertilizer; the beneficial ef-fects of VP were especially pronounced. Application of basalfertilizers also significantly changed the relative abundance ofbeneficial soil fungi. The relative abundance ofM. alpina andC. globosum were significantly higher in the CK treatmentthan in fertilizer-treated soil. The relative abundance of uncul-tured Trichoderma was significantly greater in the FT treat-ment than that in other treatments (Fig. 3b). In general, fertil-ization treatments decreased the abundance of beneficial fungiin the soil. In the FC treatment, the abundance of M. alpina

Table 4 Correlation analysis of soil physical and chemical properties with development of cucumber

Indexes Soluble solid Soluble sugar Organic acid Vitamin C Soluble protein Sugar-acid ratio Nitrate of fruit Fruit yield per plan

TN −0.392 −0.117 −0.325 −0.153 −0.643** 0.227 0.869** −0.551*NH4

+-N −0.705** −0.421 0.077 −0.676** −0.519* −0.548* −0.042 −0.775**NO3

−-N 0.551* 0.590* −0.094 0.542 0.347 0.647* 0.423 0.502

MBN 0.922** 0.764** 0.522 0.738** 0.935** 0.458 −0.364 0.975**

OM 0.067 0.269 −0.031 −0.002 −0.203 0.331 0.706* 0.187

DOC 0.708* 0.721** −0.093 0.932** 0.606* 0.809** 0.203 0.695*

TP 0.961** 0.684** 0.006 0.839** 0.887** 0.652** −0.311 0.890**

Olsen-P 0.690** 0.624** −0.067 0.660* 0.448 0.735** 0.243 0.643*

AK 0.295 0.244 −0.566* 0.571* −0.018 0.742** 0.754** 0.190

pH −0.797** −0.465 0.236 −0.779** −0.659* −0.668* 0.027 −0.805**EC 0.316 0.092 −0.557 0.561 0.006 0.740** 0.673 0.191

Water content 0.892** 0.752** 0.046 0.588* 0.757** 0.663** −0.146 0.818**

Bulk density −0.505 −0.128 0.223 −0.359 −0.560* −0.310 0.290 −0.617*

TN total nitrogen, NH4+ -N ammonium nitrogen, NO3

− -N nitrate nitrogen,MBN microbial nitrogen, OM organic matter, DOC soluble organic carbon,TP total phosphorus, Olsen-P available phosphorus, AK available potassium, EC electrical conductivity

*Significant correlation at the 0.05 level (bilateral), **significantly related to the 0.01 level (bilateral)

Table 5 Summary of pyrosequencing data and diversity estimates for the rhizosphere soil of different base fertilizer treatments in spring of 2015

Treatments Number of reads Number of high quality reads Number of OTUs ACE index Shannon index (H) Good coverage ratio (%)

CK 97,320 ± 7,410 a 73,538 ± 5,478 a 437 ± 3 b 483.7 ± 6.5 b 5.57 ± 0.03 b 99.9 ± 0.0 a

FC 88,946 ± 2,466 a 67,074 ± 2,143 a 427 ± 1 b 479.5 ± 3.0 b 4.30 ± 0.07 c 99.9 ± 0.0 ab

FT 97,589 ± 1,631 a 73,879 ± 1,246 a 428 ± 12 b 479.3 ± 8.5 b 5.57 ± 0.09 b 99.9 ± 0.0 a

VP 86,476 ± 3,637 a 64,753 ± 2,788 a 470 ± 3 a 528.7 ± 3.1 a 5.95 ± 0.06 a 99.9 ± 0.0 b

CK control, no fertilizer addition; FT inorganic fertilizer; FC organic fertilizer/inorganic fertilizer mixture; VP vermicompost/inorganic fertilizer mixture(see Sect. 2 for the description of the mixtures)

Mean values ± standard error (n = 3) are shown; means shared with a common letter in the same column indicate no significant differences betweentreatments (p < 0.05) based on Duncan’s multiple range test

2724 J Soils Sediments (2017) 17:2718–2730

was significantly decreased compared to the VP treatment,although the abundance of uncultured Trichodermawas great-er in the FC treatment.

3.5 Relationship of soil fungal community and soilproperties

Redundancy analysis (RDA) indicated that soil environmentalfactors altered the composition of soil fungal communities(Fig. 4). The first two RDA axes explained 88.77% of the cumu-lative variance of the fungi-environment relationship, suggestinga strong relationship between the composition of the fungal com-munity and environmental variables. Since the scaling is focusedon inter-sample distances, we can independently interpret eacharrow as pointing in the direction in which the sample scoreswould shift with an increase in the value of that environmentalvariable. The length of the arrow allows us to compare the size ofsuch an effect across the environmental variables, and the anglebetween the arrows and axes of the environmental variables canbe used to approximate their correlations. The first RDA axisexplained 68.54% of the cumulative variance; therefore, envi-ronmental variables such as soil water, MBN, TP, pH, andNH4

+-N contents play important roles in fungal communitycomposition (p < 0.05), based on their relatively greater lengthsand smaller angles relative to the first axis.

In the ordination diagrams, pH and NH4+-N generally

showed the strongest positive correlations with the first axis,while soil water, MBN, and TP showed the strongest negativecorrelations. The results agreed with those of the correlationanalysis between yield and environmental variables (Table 4),indicating the presence of intrinsic connections among soilproperties, fungal community composition, and cucumberdevelopment.

If we project the sample point perpendicular to the arrow ofa species, we obtain an approximate ordering of the values ofthis species across the projected samples. In the ordinationdiagrams, it can be seen that ordering the positions of thevalues for harmful fungi (e.g., F. solani, F. oxysporum,Acremonium sp., Humicola nigrescens, and Cladosporiumsp.) place the VP treatment at the lowest position, suggestingthat vermicompost amendment decreased the abundance ofharmful fungi. Compared to the control, the abundance ofbeneficial fungi in the vermicompost treatment also exhibiteda downward trend, but their abundance was higher than that ofthe other fertilizer application treatments. A sharp decrease inharmful fungi and the presence of relatively abundant benefi-cial fungi in the soil shows that vermicompost amendment hada positive effect on the development of healthy soil microbiota.

4 Discussion

4.1 Yield and quality responses to soil vermicompostamendment

Fertilization increases crop yield, and the beneficial effects ofVP amendments are more obvious than other treatments(Yang et al. 2015). Similar results were obtained in our studyin that the fertilizer amendment increased the yield of cucum-ber fruit even though the yield decreased yearly under contin-uous cropping conditions. Providing timely and adequate sup-plies of mineral nutrients for cucumber growth is part of themechanism that improves yield. The use of the VP amend-ment aided cucumber fruit yield under continuous croppingconditions. This outcome might result from the excellentphysical characteristics and abundant nutrients of VP (Zhaoet al. 2010a, b; Ravindran et al. 2016) and the ability of VP toeffectively regulate soil properties (Oo et al. 2015).

Nitrate content is used to evaluate the safety of vegetables,and a negative correlation exists between nitrate content andthe nutritional quality of vegetables. Moreover, nitrate, aminoacids, proteins, sugars, organic acids, mineral, and vitaminsare closely interrelated (Blom-Zandstra 1989; Huang et al.2011). Our previous study suggested that the overall qualityof cucumbers was improved by applying VP and VP-inorganic mixed fertilizer under greenhouse conditions(Zhao et al. 2010a, b). The difference between the nitratecontents of cucumber fruits was not significant between VPamendment and lack of fertilizer amendment, despite thefact that the VP application provided a large number ofmineral nutrients. Soil properties, especially biologicalproperties, are altered by BP treatment, which also im-proves the development of the cucumber plants. Thesemay be the main mechanisms that underlie the improve-ment of cucumber fruit quality. Vitamin C is known to havemarked nucleophilic properties and the ability to capture

-0.2 -0.1 0.0 0.1 0.2 0.3 0.4

-0.2

-0.1

0.0

0.1

0.2 FC

CK

VP

FT

PCA-PC1 vs PC2PC

2 (2

0.01

%)

PC1 (76.36%)

Fig. 1 Principal component analysis (PCA) for the rhizosphere soil ofdifferent base fertilizer treatments in spring of 2015. CK control, no fer-tilizer addition; FT inorganic fertilizer; FC organic fertilizer/inorganicfertilizer mixture; VP vermicompost/inorganic fertilizer mixture (seeSect. 2 for the description of the mixtures)

J Soils Sediments (2017) 17:2718–2730 2725

and deactivate free radicals or reactive oxygen species pro-duced by metals (Jiraungkoorskul and Sahaphong 2007).The application of inorganic compound fertilizers and VP

has been shown to significantly increase vitamin C in fruit,but organic fertilizer amendments do not have this effect.Vitamin C content in fruit is related to the supply of mineral

0

20

40

60

80

100Unidentified

ZygomycotaGlomeromycotaChytridiomycotaBasidiomycotaAscomycota

Phylum

Rela

tiv

e a

bu

nd

an

ce (

%)

TOTAL CK FT FC VP

0

20

40

60

80

100Class Unidentified

Incertae_sedisSaccharomycetesTremellomycetesAgaricomycetesEurotiomycetesDothideomycetesSordariomycetesOthers

Treatments

a

b

Fig. 2 The distribution of soilfungal species (phylum and class)in different base fertilizertreatments in spring of 2015. CKcontrol, no fertilizer addition; FTinorganic fertilizer; FC organicfertilizer/inorganic fertilizermixture; VP vermicompost/inorganic fertilizer mixture (seeSect. 2 for the description of themixtures)

Mortierella alpina Chaetomium globosum uncultured Trichoderma

CK

FT

FC

VP

a

Beneficia fungi

b

bc

d

a

b

c c

a

bb

c

Fusarium solani Fusarium oxysporum Cladosporium sp Rhizopycnis vagum

0

2

4

6

8

10

12

14

16

Pathogenic fungi

Rel

ativ

e ab

un

dan

ce (

%)

Fungi

a

b

cc

a a

b

ba

b

c ca

bc bc

a b

Fig. 3 The relative abundance of soil pathogenic fungi and soil beneficialfungi for the rhizosphere soil of different base fertilizer treatments inspring of 2015. CK control, no fertilizer addition; FT inorganicfertilizer; FC organic fertilizer/inorganic fertilizer mixture; VPvermicompost/inorganic fertilizer mixture (see Sect. 2 for the

description of the mixtures). Mean values ± standard error (n = 3) areshown; means shared with a common letter in the same column indicateno significant differences between treatments (p < 0.05) based onDuncan’s multiple range test

2726 J Soils Sediments (2017) 17:2718–2730

nutrients, and the uptake of mineral nutrients by plants de-pends on soil characteristics, such as cation exchange ca-pacity, pH, microorganisms, and metal ions (Fernandes andHenriques 1991). Therefore, the vitamin C content of cu-cumber fruits might depend on the combined effects of soilmineral nutrients, soil properties, plant responses, and fer-tilizer properties.

4.2 Effects of vermicompost application on soil properties

In the present study, we found that the continuous cropping ofcucumbers in a greenhouse setting resulted in decreased soilpH and increased EC, and these outcomes are consistent withprevious studies. The changes result from the addition of alarge number of base cations to the soil at the time of fertili-zation (Orozco et al. 1996; Atiyeh et al. 2000a, 2000b) and tothe fact that crops selectively absorb ions, such as hydrogenions. After seven cucumber crops, the application of VPamendment resulted in decreased soil pH that differed fromthose after FT and FC amendments. However, there were nosignificant differences detected between the EC after FC andVP treatments. The cause of this effect might be the ability of

VP to promote crop system development more effectivelythan other fertilizers and the capacity to absorb more mineralions while simultaneously producing hydrogen ions (Edwardsand Burrows 1988; Orozco et al. 1996). The unique charac-teristics of VP and the beneficial interaction between VP andsoil (Ngo et al. 2012; Oo et al. 2015) suggest that VP amend-ments could significantly increase soil water-holding capacityand lower bulk density, which is conducive to the healthygrowth of crops.

The nitrogen characteristics of soil directly reflect its fertil-ity, and influence the absorption and utilization of mineralelements by plants. The addition of VP has been shown tosignificantly increase soil mineral nutrition (e.g., NH4

+-N,NO3

−-N, and phosphorus), decrease soil organic matter con-tent, and influence the rates of nutrient cycling (Arancon et al.2006; Yang et al. 2015). Our results here showed that appli-cation of fertilizer resulted in the accumulation of NO3

−-N inthe soil. The microbial nitrogen (MBN) content of VP wasalso significantly higher than those of the other treatments.

We observed a decrease in the carbon and nitrogen contentsin soils amended with fertilizer, and the VP amendment didnot alter organic carbon content. However, compost amend-ment has been shown to increase soil organic carbon contentcompared to initial soil properties (Ngo et al. 2012). Our dataindicated that the soil organic matter (OM) and soluble organ-ic carbon (DOC) contents increased significantly followingapplication of fertilizer over a 4-year period; these results areconsistent with previous studies that showed increased carbonstorage by exogenous organic amendments in addition to im-provements in several soil functions related to the presence oforganic matter (Lashermes et al. 2009; Ngo et al. 2012). In thepresent study, the FTand VP treatments were more conduciveto soil carbon conversion and the enhancement of soil micro-bial activity, because the soil microbes could use DOC moreefficiently. Soil available-N and AP were increased by VPtreatment, and the integration of bioorganic nutrient supple-ments significantly maintained soil health and productivity ona long-term basis for sustainable crop production (Yadav et al.2016). Our data demonstrated that an integrated nutrient man-agement scheme for chemical fertilizers and organic manuresmight offer a feasible and environmentally friendly option forsoil conservation (Singh et al. 2016).

4.3 Effect of vermicompost application on soil funguscharacteristics

Environmental variables play an important role in soil micro-bial flora divergence (Rodrigues et al. 2014); thus, soil pH andcontents of NH4

+-N, NO3−-N, Ca, and total carbon influence

the characteristics of the soil microbiota (Ligi et al. 2014). Ithas been shown that crop growth can increase microbial nu-trient mobilization capacity and alter the distribution of thesoil microbial population (Marschner et al. 2011). Our results

Fig. 4 Redundancy analysis (RDA) of the fungi community for the rhi-zosphere soil of different base fertilizer treatments in spring of 2015. CKcontrol, no fertilizer addition; FT inorganic fertilizer; FC organicfertilizer/inorganic fertilizer mixture; VP vermicompost/inorganic fertiliz-er mixture (see Sect. 2 for the description of the mixtures); S1 Fusariumsolani; S2 Fusarium oxysporum; S3 Acremonium sp.; S4 Humicolanigrescens; S5 Cladosporium sp.; S6 Rhizopycnis vagum; S7 Fusariumoxysporum; S8 Mortierella alpina; S9 Chaetomium globosum; S10 un-cultured Trichoderma; S11 Fusarium nematophilum; S12 Alternariadestruens; S13 Ascomycota sp.; S14 Emericellopsis sp.; S15 Hypoxylonticinense; S16 Mrakia robertii; S17 uncultured Sordariales; S18Aspergillus ochraceus; S19 uncultured Hydnum; S20 Trichosporonasahii; S21 Aspergillus flavus; TN total nitrogen; AN ammonium nitro-gen; NI nitrate nitrogen; MBN microbial nitrogen; OM organic matter;DOC soluble organic carbon; TP total phosphorus; AP available phos-phorus (Olsen-P); AK available potassium; SW soil water content; BD soilbulk density

J Soils Sediments (2017) 17:2718–2730 2727

indicated that the soil fungal community after VP amendmentexhibited greater differentiation than those associated with theFC and FT amendments. In soil amended with the VP treat-ment, MBN and TP were significantly and positively corre-lated with the soil fungal communities; this result is consistentwith previous reports (Liu et al. 2014; Ligi et al. 2014).

Applied organic and chemical fertilizers can greatly alter thesoil microorganism population (Cai et al. 2003), and an increasedmicrobial population and their related activities in VP amendedsoil are key factors that influence plant resistance or tolerance tocrop pathogens and nematode attacks (Arancon et al. 2006). Inthis study, the effect of FC treatment on the reduction of harmfulfungi was significantly better than that of the FT treatment, andthe beneficial effects of VP were more prominent.

Application of a suitable amount of VP might inhibit soilpests and soil-borne diseases (Edwards and Norman 2004),and might significantly decrease the number of plant parasiticnematodes and infection rates in plants (Arancon et al. 2002).Additionally, VP treatment might suppress diseases such asphytophthora, fusarium, and plasmodiophora in tomatoes(Chaoui et al. 2002). Clearly, the excellent biological proper-ties of VP, which contains biologically active substances andlarge amount of functional microorganisms, are another majormechanism responsible for its beneficial effects (Tomati et al.1987; Grappelli et al. 1987).

5 Conclusions

Continuous cropping over 4 years resulted in a decreased yield ofcucumber fruit in a greenhouse, and the application of a basalfertilizer mitigated this decline in output. Moreover, basal fertil-izer amendments decreased the levels of beneficial and patho-genic fungi in the soil, although VP amendment had a positiveeffect on the composition of a healthy soil fungal community.The use of VP amendment as the basal fertilizer significantlyimproved the basic soil physicochemical properties, mineral nu-trient, and biological properties, and it also increased cucumberyield, and improved fruit quality. Future research will focus onthe effects of VP amendment on soil bacterial community struc-ture in a continuous cropping system.

Acknowledgements This researchwas supported by theNationalNaturalScience Foundation ofChina (ProgramNo. 31000939), theNational Scienceand Technology Support Project of China (Program No. 2013BAD21B03),the Jiangsu Province Agricultural Science and Technology IndependentInnovation Fund (Program Nos. CX(15)1019), and the Science andTechnology project of Changshu city (ProgramNo.CS201408).

Open Access This article is distributed under the terms of the CreativeCommons At t r ibut ion 4 .0 In te rna t ional License (h t tp : / /creativecommons.org/licenses/by/4.0/), which permits unrestricted use,distribution, and reproduction in any medium, provided you give appro-priate credit to the original author(s) and the source, provide a link to theCreative Commons license, and indicate if changes were made.

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