European Journal of Soil Science,June2012,63,332–340doi:10.1111/j.1365-2389.2012.01442.x Soil phenolics in a continuously mono-cropped cucumber (Cucumis sativus L.)system and their effects on cucumber seedling growth and soil microbial communities
X.Z H O U a,G.Y U b&F.W U a
a Department of Horticulture,Northeast Agricultural University,Mucai59,Xiangfang,Harbin150030,P.R.China,and
b Department of Horticulture,Zhejiang University,Yuhangtang Road886,Hangzhou310058,P.R.China
Summary
Phenolic compounds have been implicated as autotoxins of cucumber under mono-cropping management
systems.Inhibition of cucumber growth may result from direct uptake of phenolic compounds or an indirect
effect resulting from changes in soil micro?ora.In the present study we monitored the dynamics of soil
phenolics in a continuously mono-cropped cucumber system and then assessed the effects of these compounds
on soil microbial communities.Six phenolic compounds were identi?ed in all soil samples in the continuously
mono-cropping system.Soil total phenolic content increased extensively in the?rst cropping,but maintained
a relatively stable level in the following croppings.Amendments of phenolics at the concentration detected in
the soil showed inhibitory effects on cucumber seedling growth and stimulatory effects on soil dehydrogenase
activity,soil microbial biomass carbon content and soil bacteria and fungi community sizes.Amendments of
phenolics caused shifts in soil microbial community structures and soil bacteria and fungi communities had
different responses.Our results suggested that direct phytotoxic effects of phenolics on cucumber probably did
not happen in continuous mono-cropping systems,but they might indirectly in?uence cucumber performance
by changing soil microbial communities.
Introduction
Autotoxicity is a type of intra-speci?c allelopathy,whereby a plant species inhibits the growth of its own kind through the release of toxic chemicals into the environment(Singh et al., 1999).Cucumber(Cucumis sativus L.)is a crop of much economic importance in many countries.Cucumber root exudates and plant debris have been shown to have autotoxicity potential(Yu &Matsui,1994;Yu et al.,2000).Autotoxins,including some derivatives of benzoic and cinnamic acids,have been identi?ed from the root exudates of cucumber(Yu&Matsui,1994).
A large body of literature supports the view that,at proper concentrations and conditions,some phenolic compounds are active allelochemicals,which could in?uence plant growth and development(Inderjit&Duke,2003).In order to restrict plant growth,allelochemicals must accumulate and persist at phytotoxic levels in the rhizosphere soil(Jilani et al.,2008).Previous studies have mainly focused on the identi?cation and quanti?cation of phenolic compounds in soil and plant tissues(Politycka et al.,
Correspondence:F.WU.E-mail:fzwu2006@https://www.doczj.com/doc/7817157141.html,
Received21June2011;revised version accepted13February20121984;Dalton et al.,1987;An et al.,2001).However,there are relatively few reports about the changes of soil phenolics in continuous mono-cropping systems,and much uncertainty remains regarding whether these compounds could really accumulate in the soil after long-term cropping(Ye et al.,2004).
Soil is a complex and dynamic environment in which microor-ganisms mostly govern the biological activity(Paul,2007).It is believed that soil micro-organisms could in?uence the persistence, availability and biological activities of allelochemicals in the soil (Inderjit,2005;Jilani et al.,2008).Root exudates or allelochem-icals are known as one of the most important factors affecting soil microbial characteristics(Bais et al.,2004),and play a key role in plant–microorganism interactions by in?uencing the struc-ture and function of soil microbial communities(Shi et al.,2011). Allelopathy can therefore be better understood in terms of soil microbial ecology(Inderjit,2005).Studies of plant root exudates or certain phenolic acids on soil microorganism populations have been carried out(Shafer&Blum,1991;Blum et al.,2000;Qu &Wang,2008).However,most of these previous studies inves-tigated the soil microorganism population with culture-dependent techniques,which are limited in that only a small fraction of the microorganisms are accessible for study.The analysis of DNA
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Autotoxicity of phenolics in a mono-cropping system333
obtained directly from environmental samples has been shown to be a powerful tool for investigating microbial communities (Pace,1997).Molecular?ngerprinting techniques,such as dena-turing gradient gel electrophoresis(DGGE)analysis of ribosomal DNA(rDNA)fragments,have stimulated a remarkable progress in microbial ecology research(Muyzer et al.,1993).Quantitative polymerase chain reaction(qPCR)provides the potential for accu-rate measurement of initial quantities of target DNA,and has been successfully applied in microbial-ecology research for the quan-ti?cation of bacteria and fungi(Rousk et al.,2010).Analysis of soil microbial communities with molecular techniques can fur-ther our understanding of how putative allelochemicals affect soil microbial communities.
In the present study,cucumber was continuously mono-cropped in pots under glasshouse conditions for nine croppings. Dynamics of soil phenolic contents in these different croppings were determined by high performance liquid chromatography (HPLC).Afterwards,the effects of a mixture of these identi?ed phenolics on cucumber seedling growth and soil microbial properties were assessed.Soil bacterial and fungal community structures and abundances were analysed by PCR-DGGE and qPCR methods,respectively.The main aims of the present study were to(i)monitor the dynamics of soil phenolic contents in the continuously mono-cropped cucumber system,(ii)determine how amendments of phenolics would affect soil microbial communities and(iii)assess direct phytotoxic effects of phenolics on cucumber in our mono-cropping system.
Materials and methods
Glasshouse experiment
A glasshouse experiment was conducted in plastic pots(30-cm diameter,25-cm height)in a glasshouse located in the experimental station of Northeast Agricultural University,Harbin, China(45?41 N,126?37 E),from April2005to July2009.Two crops of cucumber(C.sativus L.cv.Jinlv3)(April to July,July to October)were grown in every year.There was one cucumber plant per pot.Fresh soil was taken each year(April25)from the upper soil layer(0–15cm)of a?eld under grass for more than 15years and used to set up a series of pots(8kg per pot)for new plantings.Soil in other previously cropped pots was retained with no further soil added.Cucumber was sown on April25 and July25in each of the5years.In the?nal year,the plants grown on soils that had one,three,?ve,seven and nine previous crops of cucumber were harvested(Figure S1).There were60 pots per cropping.All pots were placed in the glasshouse using a completely randomized design.Fertilizers were added according to local recommendations with decomposed swine manure(15% organic matter,0.5%N,0.5%P,0.4%K)used as basal fertilizer at the rate of0.75kg per pot.Urea fertilizer(46%N)was used as a top-dressing at the rate of25g per pot31days after cucumber sowing.The pots were irrigated twice weekly with ground water. The soil water content was not controlled rigorously,but frequent irrigation made sure that plants did not experience drought stress and there was no standing water in the pots throughout the growing season.Weeds were removed manually.The soil from the open ?eld was a black soil(Mollisol)(Buol et al.,2003)with sandy loam texture,containing the following:organic matter,3.67%; available N(nitrate and ammonium),89.02mg kg?1;Olsen P, 63.36mg kg?1;available K,119.15mg kg?1;EC(1:2.5,w:v), 0.33mS cm?1;pH(1:2.5,w:v),7.78.
Plant biomass
Cucumber plants were sampled at40days after sowing.Plant dry mass was measured after oven drying at70?C to constant mass. Soil phenolics extraction and determination
Soil samples were collected from the pots at40days after cucumber sowing,and sieved(2mm mesh)to remove root tissues. Soil phenolics were extracted with methods previously described (Dalton et al.,1987).Brie?y,25g soil was added to150ml 2m NaOH and agitated for24hours on a reciprocal shaker at 25?C.The suspension was centrifuged at6000g for15minutes, and the supernatant was?ltered through?lter papers.The pH of the?ltrate was adjusted to2.5with5m HCl,and extracted?ve times with ethyl acetate.The resulting extracts were pooled and evaporated to dryness at40?C.The residue was dissolved in5ml 80%methanol and kept in the dark at4?C.
Total phenolics were measured by the Folin-Ciocalteau method (Box,1983).A calibration curve was constructed with different concentrations of ferulic acid.Units of total phenolics in the soil were expressed asμg of ferulic acid equivalents per gram of soil dry-mass.
The methanol solution of soil extracts was?ltered through 0.22μm?lter membranes for HPLC analysis.Soil phenolic contents were determined with a Waters HPLC system(Waters, Milford,MA).The mobile phase was a mixture of80% methanol and20%water.The?ow rate was kept constant at0.8ml minute?1.Detection was performed at280nm.The injection volume was15μl and the column temperature was maintained at25?C.Identi?cation and quanti?cation of phenolic compounds were con?rmed by comparing retention times and areas with pure standards.The content of each identi?ed phenolic compound in soil was expressed asμg per gram of soil dry mass. Radicle elongation experiment
Soil extracts and a mixture of phenolic compounds were used in this phytotoxic bioassay.First,5ml10-fold diluted methanol solution of soil extracts was added to a Petri dish(9-cm diameter), which contained two layers of?lter papers,and put in darkness to evaporate the methanol,and then5ml distilled water was added. Second,5ml phenolic compounds mixture at concentrations detected in the soil(50.8,26.0,7.6,11.6,86.9and15.0μg ml?1 for p-hydroxybenzoic acid,syringic acid,vanillic acid,vanillin, p-coumaric acid and ferulic acid,respectively)were added to a
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Petri dish,which contained two layers of?lter papers.Solution pH was adjusted to7.0with0.1m NaOH.Afterwards,10germinated cucumber(C.sativus L.cv.Jinlv3)seeds with radicles of1mm length were separately placed in these Petri dishes.Cucumber seeds treated with only distilled water added to the dish were used as the control.There were15Petri dishes per treatment, which were wrapped with Para?lm and incubated in the dark at 25?C.Radicle length was measured10days after treatments had been added.
Seedling growth experiment
Cucumber seedlings with two cotyledons were transplanted into cups containing150g soil,which was the starting soil in the glasshouse experiment.Cucumber seedlings at the one-leaf stage were treated with a phenolic compounds mixture(33.6,19.7, 6.0,9.1,77.4and12.3μg g?1soil for p-hydroxybenzoic acid, syringic acid,vanillic acid,vanillin,p-coumaric acid and ferulic acid,respectively)every other day(once every48hours)(Shafer &Blum,1991).The concentration of each phenolic acid was the average concentration of the?ve croppings subtracted from the concentration found in the soil in the?eld.Solution pH was adjusted to7.0with0.1m NaOH.Seedlings in soil but treated with distilled water were used as the control.All seedlings were maintained in a glasshouse(32?C day/22?C night,relative humidity of60–80%,16hour light/8hour dark).There were nine plants per treatment.Ten days later,plant height,leaf area and plant dry mass were measured,and soils in the cups were collected,sieved(2mm mesh)and stored at?70?C.
Soil dehydrogenase activity and microbial biomass carbon (MBC)estimation
Dehydrogenase activity was determined by using the reduction of2,3,5-triphenyltetrazolium chloride(TTC)method(Tabatabai, 1994).Fresh soil samples(5g)were incubated at37?C for 24hours in the dark,and the concentration of triphenylformazan (TPF)in the extracts was spectrophotometrically measured at 485nm with methanol as the blank.Dehydrogenase activity was expressed asμg TPF g?1soil24hour?1on an oven-dried soil basis.
Soil MBC content was determined on a15-g oven-dry equivalent?eld-moist soil sample(<5mm)by the chloroform-fumigation-extraction method(Vance et al.,1987).Fumigated and non-fumigated soil samples were extracted with0.5m K2SO4for 30minutes(1:5,w:v)and?ltered.Extractable organic carbon in soil extracts was titrated with FeSO4after dichromate digestion. An extractability factor of0.38was used to calculate MBC(Vance et al.,1987).
DNA extraction and PCR-DGGE
Soil bacterial and fungal community structures were analysed with the PCR-DGGE method.Total soil DNA was extracted with an E.Z.N.A.Soil DNA Kit(Omega Bio-Tek,Norcross,GA,USA). PCR ampli?cation of partial bacterial16S rRNA gene sequence was performed with the primer set of GC-338f/518r(Muyzer et al.,1993)according to methods previously described(Cahyani et al.,2003).A nested PCR protocol was used to amplify fun-gal internal transcribed spacer(ITS)regions of the rRNA gene (Gardes&Bruns,1993)with primer sets of ITS1F/ITS4(White et al.,1990;Gardes&Bruns,1993)and GC-ITS1F/ITS2(Gardes &Bruns,1993)for the?rst and second round of PCR ampli?-cations,respectively.DGGE was performed using an8%(w:v) acrylamide gel with30–70%and20–60%denaturant gradient for bacteria and fungi,respectively,and run in a1×TAE(Tris-acetate-EDTA)buffer for14hours under conditions of60?C and 80V with a DCode universal mutation detection system(Bio-Rad Lab,Hercules,CA,USA).After electrophoresis,the gel was stained in1:3300(v:v)GelRed(Biotium,Hayward,CA,USA) nucleic acid staining solution for20minutes.DGGE pro?les were photographed with an AlphaImager HP imaging system(Alpha Innotech Crop.,San Leandro,CA,USA)under UV light. qPCR for bacterial16S rRNA genes and fungal ITS regions
SYBR Green qPCR assays were used to estimate the sizes of bacterial and fungal communities with primer sets of338f/518r (Muyzer et al.,1993)and ITS1F/ITS4(White et al.,1990;Gardes &Bruns,1993),respectively.qPCR assays were conducted with an IQ5real-time PCR system(Bio-Rad Lab)in a20-μl volume containing10μl2×Real SYBR Mixture(Cowin Biotech,Beijing, China),0.2m m of each primer,8μg of bovine serum albumin and2.5ng of puri?ed soil DNA extracts.The PCR conditions were predenaturing at94?C for5minutes,denaturing at94?C for 45s,annealing at56?C for45s for bacterial16S rDNA gene (or57.5?C for45s for fungal ITS region),extension at72?C for90s(30cycles in total)and a?nal elongation at72?C for 10minutes.Standard curves were created with10-fold dilution series of plasmids containing the16S rRNA gene(or ITS region) from soil samples.Sterile water was used as a negative control to replace the template.All ampli?cations were in triplicate. The speci?city of the products was con?rmed by melting curve analysis and agarose gel electrophoresis.The threshold cycle(Ct) values obtained for each sample were compared with the standard curve to determine the initial copy number of the target gene. Statistical analysis
Data were analysed by analysis of variance(SAS Institute, 2003)and mean comparison between treatments was based on the least signi?cant difference(LSD)test.Banding patterns of the DGGE pro?les were analysed by Quantity One software (version4.5,Bio-Rad Lab,Hercules,CA,USA).The position and intensity of each band were determined automatically.The density value of each band was divided by the average band density of the lane to minimize the in?uence of loaded DNA concentrations among samples.Normalized data were used for
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3350
20040060080010001200(a)
(b)
1
3
5
7
9
Number of croppings
P l a n t t o t a l b i o m a s s / m g p l a n t -1 D
W
501001502002503000
1
357
9
Number of croppings
T o t a l p h e n o l i c c o m p o u n d s c o n t e n t
/ μg f e r u l a i c a c i d e q u i v a l e n t s g -1 s o i l D W Figure 1Cucumber total dry biomass (a)and soil total phenolic content (detected by the Folin-Ciocalteau method)(b)in the continuously mono-cropped system.Data are expressed as means of triplicates with standard errors shown by vertical bars.
principal component analysis (PCA)with Canoco for Windows 4.5software as described previously (Zhou et al .,2011).The richness (S),evenness (E)and diversity (H )indices of soil bacterial and fungal community structures were calculated as described previously (Liu et al .,2007).
Results
Cucumber plant biomass
In the glasshouse experiment,cucumber plant total dry biomass was not statistically different among the ?rst ?ve croppings (Figure 1a),but signi?cantly decreased in the seventh cropping,and then signi?cantly increased in the ninth cropping (P <0.001,LSD =244.4).
Phenolics in the soil
Total phenolic contents in all cucumber-cultivated soils were larger than in the ?eld soil (Figure 1b).Soil total phenolic contents signi?cantly decreased in the seventh cropping,and signi?cantly increased in the ninth cropping (P <0.001,LSD =4.02).
Six phenolic compounds were detected by the HPLC method in both the ?eld soil and the cucumber-cultivated soils (Figure 2).
p -hydroxybenzoic acid was the most abundant in the ?eld soil sample.In cucumber-cultivated soil samples,p -coumaric acid content was the most abundant,followed by p -hydroxybenzoic acid >syringic acid >ferulic acid >vanillin >vanillic acid.Content of each phenolic compound identi?ed was signi?-cantly larger in cucumber-cultivated soils than in the ?eld soil.p -coumaric acid had the largest increase,the content of which in cucumber-cultivated soils was about six to ten times larger than that in the ?eld soil.Generally,in cucumber-cultivated soils,each phenolic compound tended to decrease in the seventh cropping,and to increase in the ninth cropping.
Cucumber radicle elongation and seedling growth
In the Petri dish experiment,both soil extracts and the phenolic compounds mixture signi?cantly inhibited cucumber radicle elongation (P <0.001,LSD =0.59),and the effect of the phenolic compounds mixture was more pronounced (cucumber radicle length was 7.7±0.13,6.6±0.09and 5.9±0.25cm for the control (water),soil extracts and the phenolic compounds mixture,respectively,data not shown).Likewise,in the seedling growth experiment,the phenolic compounds mixture signi?cantly reduced cucumber leaf area,plant height and dry mass (P <0.01,P <0.05and P <0.001,respectively)(Table 1).
Soil dehydrogenase activity,MBC content and bacterial and fungal abundances
Soil dehydrogenase activity,MBC content and bacterial and fungal abundances were signi?cantly increased by amendments of phenolics (P <0.01,P <0.001,P <0.001and P <0.001,respectively)(Table 1).Soil dehydrogenase activity and MBC content were increased by 142and 62%,respectively,as compared with the soil treated with water.Soil bacterial and fungal abundances of the phenolics-amended soil were 1.6and 2.9times that of the soil treated with water,respectively.
Soil bacterial and fungal community structures
DGGE analyses showed that amendments of phenolics changed soil bacterial (Figure 3)and fungal (Figure 4)community struc-tures.DGGE banding patterns of bacterial (Figure 3a)and fungal (Figure 4a)communities were different before planting,in the soils treated with water and the phenolics-amended soil,as dif-ferences in DGGE band number,position and density could be observed.DGGE pro?les were subjected to PCA to demonstrate the relative position of the individual soil samples.The analysis resulted in a clear separation of the three soils (triplicate sam-ples for each treatment were grouped together:Figures 3b and 4b),suggesting that different soil samples had different bacterial and fungal community structures.The number of visible bands,Shannon diversity index and evenness index of bacterial commu-nities were signi?cantly smaller in the phenolics-amended soil than in the soil treated with water and the soil before plant-ing (P <0.001);however,these indices of fungal communities showed the opposite trend (Table 2).
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336X.Zhou et al .
20406080100
120p-hydroxybenzoic
acid
syringic acid
vanillic acid
vanillin
p-coumaric acid
ferulic acid
Phenolic compound
C o n t e n t / μg g -1 s o i l
D W
Figure 2Soil phenolic compounds and their contents (detected by high performance liquid chromatography)in the continuously mono-cropped system.Data are expressed as means of triplicates with standard errors shown by vertical bars.
Table 1Cucumber seedling growth,soil dehydrogenase activity,microbial biomass carbon (MBC)content and bacterial and fungal abundances as affected by amendments of phenolics
Phenolic compounds mixture
Water P a LSD b Leaf area /cm 2plant ?1159±8210±6**28.21Plant height /mm
63±375±1*9.19Dry mass /mg plant ?1DW
509±6638±13***0.04Dehydrogenase activity /μg TPF g ?1soil 24hour ?1 2.93±0.22 1.21±0.04**0.62MBC content /mg C kg ?1soil
245.38±5.41151.92±0.84***15.20Bacterial abundance /1011copies g ?1soil 7.92±0.12 4.29±0.10***0.44Fungal abundance /108copies g ?1soil 3.72±0.04 1.30±0.02***0.12Fungi-to-bacteria ratio /10?4
4.70±0.12
3.03
±0.12
***
0.46
a P from one-way ANOV A,***,**,*<0.001,0.01,0.05,respectively.
b Least
signi?cant difference (P =0.05).
Data are expressed as means with standard errors.
Discussion
Phenolic compounds identi?ed in the soil
In this study,phenolic compounds could not be detected in soil water and so 2m NaOH solution was used to extract soil phenolics (Dalton et al .,1987).After phenolics are released into the soil,factors such as moisture regime,nutrient status,soil temperature and OM content may affect the availability and action of phenolic compounds (Inderjit &Duke,2003;Inderjit,2005).The protonated phenolic compounds could be sorbed by soil OM and/or polymerized into humic substances in the soil (Blum,1998).Soil OM contributes most to the sorption of phenolics in the soil matrix,and there was a positive relationship between phenolic compounds sorption and OM content (Tharayil et al .,2006).Thus,the failure to ?nd phenolic compounds in soil water might be attributed to the large OM content in the ?eld (3.7%)and cucumber-cultivated soils (ranging from 6.2to 8.3%at different cucumber croppings).Yu &Matsui (1994)identi?ed nine organic acids (benzoic,p -hydroxybenzoic,2,5-dihydroxybenzoic,3-phenylpropionic,cin-namic,p -hydroxycinnamic,myristic,palmitic and stearic acids,as well as p -thiocyanatophenol and 2-hydroxybenzothiazole)in the root exudates of hydroponic cultured cucumber.Politycka et al .(1984)identi?ed seven phenolic compounds (ferulic,p -hydroxybenzoic,p -coumaric,protocatechuic,salicylic,syringic and vanillic acids)in the cucumber-cultivated peat-bark substrate.The number of and speci?c phenolic compounds detected in our experiment were different from those in these previous studies.An et al .(2001)also reported that fewer allelochemicals were detected in the mix of soil and Vulpia myuros residues than in the plant residues alone.One possible explanation would be the differences in the substrates used to cultivate cucumber.Natural soils,which contained a large abundance of microorganisms,were used in our experiment.It is known that processes such as trans-port,retention and transformation may in?uence the quantitative and qualitative availability of phenolics in the soil.Phenolic
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(a)
(b)
Figure3Denaturing gradient gel electrophoresis pro?le(a)and principal component analysis analysis(b)of bacterial communities in soil before cucumber planting(B),soil treated with water(W)and soil amended with the phenolic compounds mixture(M).
compounds could be sorbed to the soil,and the sorption is compet-itive among different phenolic compounds(Tharayil et al.,2006). Moreover,soil microorganisms could consume and decompose these compounds(Inderjit,2005;Jilani et al.,2008).The pref-erential microbial utilization of one phenolic compound over another has been shown in plant–microbe–soil systems(Blum, 1998).Thus,some phenolic compounds might not be detected in natural soils because they have been sorbed and/or used
by (a)
(b)
Figure4Denaturing gradient gel electrophoresis pro?le(a)and principal component analysis analysis(b)of partial fungal communities in soil before cucumber planting(B),soil treated with water(W)and soil amended with the phenolic compounds mixture(M).
microorganisms:other compounds could appear as the microbial by-products of the original compound.
Changes in cucumber total biomass in the continuous
mono-cropping system
Cucumber plant biomass signi?cantly decreased in the seventh cropping(Figure1a),which was consistent with the?ndings that
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Table2Number of visible bands(S),Shannon diversity index(H)and Evenness(E)of bacterial and fungal community structures as affected by amendments of phenolics
Bacterial community Fungal community
Treatment S H E S H E
Before planting61.00±0.58 3.80±0.010.87±0.0027.00±1.15 2.87±0.040.76±0.01 Water65.00±1.73 3.81±0.050.87±0.0126.67±1.20 2.85±0.030.76±0.01 Phenolic compounds mixture47.33±0.33 3.31±0.000.75±0.0034.67±0.67 3.28±0.020.87±0.01 P a*****************
LSD b 3.710.110.02 3.590.110.03
a P from one-way ANOV A,***,**,*<0.001,0.01,0.05,respectively.
b Least signi?cant difference(P=0.05).
Data are expressed as means with standard errors.
continuous mono-cropping of the same crop in the same land can cause a reduction in crop(soil sickness)(Yu et al.,2000).Various factors are thought to contribute to soil sickness,including build-up of soil-borne pathogens,deterioration of soil physico-chemical properties,changes in nutrient availability,changes in soil biological properties,and accumulation of autotoxic substances; however,the main causes of soil sickness of cucumber are still not clear(Yu et al.,2000).In this study,we only monitored the dynamics of soil phenolic contents in the continuously mono-cropped cucumber system and the effects of other factors involved in soil sickness are not known.
Impact of phenolic compounds amendments on soil microbial communities
It has been well established that phenolic compounds have phytotoxic effects on cucumber(Yu&Matsui,1994;Blum et al., 2000),as shown in the present experiment(Table1).Stimulatory effects of phenolic compounds on soil microorganisms were also observed,as soil dehydrogenase activity,soil MBC content and bacterial and fungal abundances were greater in the phenolics-amended soil than in the soil treated with water(Table1).Our results were in accordance with the view that phenolic compounds at appropriate concentrations could stimulate the growth of indigenous soil microorganisms(Blum et al.,2000;Qu&Wang, 2008).Blum et al.(2000)suggested that the induction of phenolic acid-utilizing(PAU)bacterial communities in the soil could mitigate the adverse effects of phenolics on cucumber.Inverse relationships were found between PAU bacteria in the rhizosphere of cucumber seedlings and absolute rates of leaf expansion and shoot biomass,and the decline in seedling growth and the simultaneous increase in PAU rhizosphere bacteria could not be attributed to resource competition between the seedlings and the PAU bacteria.Soil bacterial abundance was larger(Table1) and bacterial community structure diversity indices were smaller (Table2)in the phenolics-amended than in the water-treated soil. Certain bands disappeared and others appeared in the DGGE pro?le of the phenolics-amended soil(Figure3).These results suggested that amendments of phenolics stimulated certain species of soil bacteria and inhibited other species.Previous studies also found that a wide range of taxa(88–1043)responded positively to selected root exudate solutions(organic acids and sugars);fewer (<24)responded negatively(Shi et al.,2011).Thus,phenolic compounds can not only act as substrates of soil bacteria,but also as detrimental allelochemicals.
Different responses of soil bacterial and fungal communities to amendments of phenolics
In the present study,soil bacteria and fungi responded differently to amendments of phenolics:the number of visible bands and diversity indices of the bacterial community increased,while those of the fungal community decreased in the phenolics-amended soil.The increase in soil bacterial abundance was smaller than that in soil fungal abundance in the phenolics-amended soil,and amendments of phenolics increased the fungi-to-bacteria ratio (Table2).These results were consistent with the observation of de Graaff et al.(2010),who reported that the relative increase in fungal gene copy numbers in response to labile carbon additions was generally greater than the relative increase in bacterial gene copy numbers.The different responses of soil bacteria and fungi to amendments of phenolics might explain their preferential use of labile carbon compounds(de Graaff et al.,2010).For example, bacteria have a greater metabolic reactivity than fungi in their ability to use readily available organic compounds,whereas fungi are able to express enzymatic activities,enabling them to use complex organic compounds for their metabolism(Rinnan& B?a?a th,2009).
Direct phytotoxic effects of phenolic compounds in a continuous mono-cropping system
Our results showed that soil phenolic content increased after cucumber was planted in the?rst cropping,indicating a strong release of phenolics into soil by the plant.Both phenolic content and cucumber total biomass were the least in the seventh cropping(Figure1).This indicates that the small content of soil phenolics in the seventh cropping might be more a consequence of poor plant growth than a cause.Amendments of phenolics at the concentration detected in the soil under
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Autotoxicity of phenolics in a mono-cropping system339
mono-cropped cucumber inhibited cucumber growth and changed soil microbial communities.These results suggested that the observed phytotoxicity of amendments of phenolics might not account for the poor growth performance of the seventh cropping. Previous studies also showed that although phenolic acids affected germination and seedling growth on germination paper,they had no effect on seed germination,seedling growth or early plant growth in soil even when the amounts applied were much greater than the amounts detected in soil(Krogmeier&Bremner,1989). Allelopathy is usually the result of the joint action of several compounds released by a donor plant(Inderjit&Duke,2003). The interactions of compounds in soil are potentially complex, and there is the potential of antagonistic or synergistic interactions with other compounds(Duke et al.,2009).For example,Inderjit et al.(2002)found that the binary mixture of two phenolic acids (p-hydroxybenzoic and ferulic acids or p-hydroxybenzoic and p-coumaric acids or p-coumaric and ferulic acids)was generally antagonistic.Seal et al.(2004)found that abietic acid could decrease the inhibitory effect of a phenolic mixture(caffeic, p-hydroxybenzoic,vanillic,syringic and p-coumaric acid),and they suggested that abietic acid had a‘buffering’role and counteracted the typical negative effects of allelopathy on germination and plant growth of the other compounds present in the root exudates.Phenolic compounds are only a fraction of the large variety of compounds released from live plant roots or decaying plant debris(Jones et al.,2004;Broeckling et al.,2008).Thus,one might hypothesize that there are other toxic root-exudates and/or non-toxic compounds,which could act antagonistically with phenolics in the soil.
Conclusions
Proof of allelopathy requires demonstration of accumulation of a putative phytotoxin(s)at concentrations proven to cause harm to the target species in the soil(Inderjit&Duke,2003).In this study,soil phenolic contents increased after cucumber was cultivated in the?rst cropping,but did not accumulate during croppings on natural soils.Though amendments of phenolics at the concentration detected in the soil obviously inhibited cucumber growth,direct phytotoxic effects of phenolics on cucumber probably did not happen in our continuous mono-cropping system;other root exudate compounds might act antagonistically with phenolics in the soil.Future work should focus on the identi?cation of a more detailed composition of cucumber root exudates,and further on the determination of the joint action of multiple compounds as quanti?ed in the exudates.
In the soil-microorganism-plant system,soil microbial com-munities are critical to soil biological processes necessary for maintaining a healthy and fertile soil and suppressing plant dis-eases(Paul,2007).Changes in soil microbial communities may lead to changes in the functions performed(Sharma et al.,2004). There is the possibility that phenolics could in?uence cucumber performance indirectly through a changing soil microbial com-munity.Moreover,phenolic compounds might play an important role in regulating soil microbial communities in the continuously mono-cropped cucumber system:this needs some further studies, including the monitoring of soil microbial properties to con?rm this hypothesis.
Acknowledgments
This work was supported by the National Basic Research Program of China(2009CB119004-05),the National Staple Vegetable Industrial Technology Systems of China(CARS-25-08)and the National Natural Science Foundation of China(30971998).The authors also gratefully acknowledge Professor Diego Alejandro Sampietro for valuable comments on this manuscript. Supporting Information
The following supporting information is available in the online version of this article:
Figure S1.The greenhouse experiment set up.The greenhouse experiment was conducted in plastic pots(30-cm diameter,25-cm height)from April2005to July2009.Two croppings of cucumber (April to July,July to October)were grown in every year.There was one cucumber plant per pot.Fresh soil was taken each year (April25)from the upper soil layer(0–15cm)of a?eld under grass for more than15years and used to set up a series of pots (8kg per pot)for new plantings.Soil in other previously cropped pots was retained with no further soil added.Cucumber was sown on April25and July25in each of5years.In the?nal year,the plants grown on soils which had one,three,?ve,seven and nine previous crops of cucumber were harvested.
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