Defoliation and fertiliser influences on the soil microbial community associated with two contrasting Lolium perenne cultivars

Defoliation and fertiliser influences on the soil microbial community associated with two contrasting Lolium perenne cultivars
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  Defoliation and fertiliser influences on the soil microbial communityassociated with two contrasting  Lolium perenne  cultivars Lynne M. Macdonald a,b, * , Eric Paterson a , Lorna A. Dawson a , A James S. McDonald b a  Macaulay Institute, Craigiebuckler, Aberdeen AB15 8QH, UK  b  Department of Plant and Soil Science, University of Aberdeen, Aberdeen AB24 3UU, UK  Received 26 October 2004; received in revised form 13 June 2005; accepted 20 June 2005Available online 26 July 2005 Abstract The influence of repeated defoliation on soil microbial community (SMC) structure and root turnover was assessed in two contrasting  Lolium perenne  cultivars (AberDove and S23) grown in fertilised ( C F) and non-fertilised (NF) soil. BiOLOG sole carbon source utilisationprofiles (SCSUPs) indicated consistently greater potential carbon utilisation in defoliated ( C D) compared to non-defoliated (ND) soilsregardless of cultivar and fertiliser, and was accounted for in a variety of substrate groups (sugars, carboxylic, amino and phenolic acids).Potential carbon utilisation was also stimulated in C F compared to NF soils, primarily through increased potential utilisation of carboxylicacids. PLFA indicators for the bacterial biomass did not significantly differ between cultivar, soil fertilisation, or defoliation. Defoliatedswards grown in fertilised soil ( C F C D) had a higher fungal:bacterial ratio and a greater bacterial stress index (cy19:0/18:1w7c), comparedto that of  C F ND, NF ND and NF C D, and regardless of cultivar. Overall SMC structure (canonical variate (CV) analysis of PLFAs)discriminated based on cultivar, defoliation and soil fertilisation. Primary discrimination of the SMCs could be related to differences in rootdensity and total plant biomass, and in the case of NF soils, secondary community shifts, evident with defoliation, related to rootdisappearance over the growing season. Despite the strong common effects of defoliation, and to a lesser extent soil fertilisation, cultivarspecific drivers of the soil microbial community were maintained, resulting in consistent, but subtle, discrimination of the SMC associatedwith the contrasting  L. perenne  cultivars. q 2005 Elsevier Ltd. All rights reserved. Keywords:  Cultivar; Defoliation; Grazed ecosystem; Phospholipid fatty acids; Root turnover; Soil microbial community 1. Introduction Roots release a wide range of carbon (C) substrates(rhizodeposits) ranging from passive leakage of simplesugars and amino acids (exudates) to deposition of morerecalcitrant C in the form of whole cells on root death. Thequantity and quality of rhizodeposition defines an importantsubstrate and energy source in determining the structure andfunction of the soil microbial community (SMC) (Kuzyakovand Cheng, 2001). Consequently, rhizodeposition is animportant factor driving microbial mediated nutrientcycling processes. In low input grassland ecosystems plantgrowth is often limited by the availability of mineralnutrients (Tisdale and Nelson, 1975), and is heavilydependent on the activities of the SMC to liberate mineralsfrom soil organic matter and litter returns. To understandnutrient cycling efficiency in plant–soil systems, it isimportant to determine the factors governing root C-flowand consequences for the SMC.Defoliation represents a strong selective pressure intemperate grassland ecosystems, altering C and energy flowthrough the SMC. Carbon partitioning following defoliationis dependent on a variety of factors including plant species,severity/frequency of cutting, and environmental factors(Fulkerson et al., 1994; Thornton and Millard, 1996).Preferential C allocation to shoots following defoliationoccurs at the expense of below-ground allocation oftenresulting in reduced root biomass and increased rootmortality (Guitian and Bardgett, 2000; Dawson et al.,2000). Despite reduced below-ground C allocation andinhibited root growth, a transient increase in root exudation Soil Biology & Biochemistry 38 (2006) 674–$ - see front matter q 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.soilbio.2005.06.017 *  Corresponding author. Address: CSIRO Entomology, PMB 2 GlenOsmond, SA 5064, Australia. Tel.: C 61 618 830 38498; fax: C 61 618 830308550. E-mail address: (L.M. Macdonald).  has been reported following defoliation (Paterson and Sim,2000). Thus, defoliation alters both the supply of simplecompounds (root exudates) that are quickly and easilyutilised by the microbial community, and of recalcitrantC compounds (root production/quality) decomposed overthe longer-term.A positive relationship between grazing intensity and thesize and activity of the SMC is often reported, and the SMCtypically has a lower fungal:bacterial ratio under grazedswards (Bardgett et al., 1996). As a result, nutrient cyclingin grazed ecosystems is dominated by fast-cycling throughbacterial based food-webs compared to the slower fungalbased cycles of non-grazed grassland systems (Bardgettet al., 1998). Defoliation induced changes in the structureand activity of the SMC, and hence nutrient cyclingefficiencies, are likely to result from the cumulativedifferences in C partitioning over the growing season.Studies examining the influence of defoliation on theSMC are commonly made only at the plant species level,despite recognition that cultivars of a plant species cancontrast in patterns of C allocation (Thornton and Millard,1996; Humphreys, 1989a,b). Consequently, it may beunwise to make species level generalisations about theeffect of defoliation on the SMC. This study aimed todetermine the influence of repeated defoliation and soilfertiliser amendment on the structure of the rhizosphereSMC and C inputs via root turnover, in two  Lolium perenne cultivars. The  L. perenne  cultivars selected were AberDoveand S23, previously described in Macdonald et al. (2004).These two cultivars have been reported as having differentpotential to tolerate defoliation (Humphreys, 1989a,b;Smith et al., 2001). It was hypothesised that: (i) the SMCassociated with the two  L. perenne  cultivars would differ instructure due to differences in below-ground C allocation;(ii) repeated defoliation would alter the SMC due tomodified below-ground C partitioning, likely to relate tocultivar specific re-growth ability; (iii) the response of theSMC to defoliation would be fertiliser dependent, as aconsequence of impacts on plant re-growth and availabilityof C and N to soil microorganisms. 2. Materials and methods 2.1. Soil and sward establishment  The soil was collected from Fasset Hill, Sourhope(Scottish Borders: NT 852207), an un-fertilised perma-nently grazed  Agrostis - Festuca  grassland, classed U4A(Rodwell, 1992). The soil, a brown ranker (Hapumbrept;FAO/UNESCO, 1994) derived from old red sandstone(Dry, 1993) (7.46% C, 0.61% N, pH 3.55 (CaCl 2 ), 23% clay,13.7% organic matter), was sampled to a depth of 30 cm andsieved moist (6 mm). Non-fertilised (NF) soil was packedinto 12 boxes (556 ! 356 ! 222 mm deep), to achieve a bulk density of 1 g cm K 3 . A further 12 boxes were prepared withsoil supplemented with NPK fertiliser at a rate equivalent to80:40:40 kg ha K 1 , termed fertilised soil treatment ( C F).Monoculture swards of   L. perenne  AberDove and  L. perenne  S23 were established in the non-fertilised (NF)and fertilised ( C F) soil boxes over an 8 month period(August 1999–April 2000), in a completely randomiseddesign. In order to account for differences in germinationrate AberDove was seeded at a rate of 5 g m K 2 , and S23 at arate of 7 g m K 2 . The moisture content was maintainedgravimetrically at 45% water-holding capacity on a twice-weekly basis. Two clips (October 1999 and January 2000) toa height of 4 cm, were carried out over the establishmentperiod in order to keep the sward in a manageable condition. 2.2. Defoliation treatments and sampling At the beginning of the growing season (April 2000), andweekly thereafter, defoliation (clipping to a height of 4 cm,termed C D) was carried out on three replicate swards of each cultivar-fertiliser treatment. A further 3 replicate boxesof each cultivar-fertiliser treatment remained non-defoliated(ND). In order to maintain a manageable non-defoliatedsward, three management clips were carried out on NDswards over the 24-week experimental period.Soil sampling occurred 24 weeks after the initialdefoliation event. Three soil cores (31 mm diameter) wererandomly sampled from each box to a depth of 5 cm andbulked to provide sufficient soil for analysis. The shootmaterial was excised at the soil surface, roots carefullyremoved, and the soil sieved ( ! 5 mm). Soil samples werestored overnight in a cold room (4  8 C). Root and shootmaterial was washed, freeze dried, weighed, and finelyground for analysis of total C and N (CHN analyser, Carlo-Erba Strumentazione, Milan). For the defoliated treatments,the weekly shoot clippings were dried and weighed in orderto calculate total shoot production. 2.3. Sole carbon source utilisation profiles (SCSUPs) Sole carbon source utilisation profiles (SCSUPs) weredetermined using BiOLOG MT microplates (BiOLOG, Inc.,Hayward, California), customised to contain a range of compounds (mono-saccharides, oligo-saccharides, car-boxylic acids, acidic, neutral, basic, aromatic, andsecondary amino acids and phenolic acids) typical of rootexudate C sources (detailed in Macdonald et al., 2004).A sub-sample (10 g) of the sieved soil was shaken in 100 ml1/4 strength Ringers solution (Oxoid) for 10 min to extractsoil micro-organisms. A 10 K 4 dilution (50 ml) wascentrifuged at 750 g  for 10 min to remove particulatesbefore inoculation of 150  m l aliquots into the BiOLOGwells. The microplates were incubated at 25  8 C for 7 d andthe colour development was measured as absorbance at590 nm every 24 h using a microplate reader (Emax,Molecular Devices, Oxford, UK).  L.M. Macdonald et al. / Soil Biology & Biochemistry 38 (2006) 674–682  675  2.4. Phospholipid fatty acid (PLFA) profiles The biomass and structure of the SMC was assessed bythe ester-linked PLFA composition of the soil. Lipidextraction and phospholipid fatty acid (PLFA) analysiswere carried out using the modified method (White et al.,1979) based on that of  Bligh and Dyer (1959). The separated fatty acid methyl-esters were identified and quantified bychromatographic retention time and mass spectral compari-son using a standard qualitative bacterial acid methyl estermix (Supelco, Supelco UK, Poole, Dorset, UK) that rangedfrom C11 to C20. Only compounds present at concen-trations greater than the limit for quantitative detection(0.01  m g g K 1 ) were reported. For each sample the abun-dance of individual fatty acid methyl-esters was expressedas nmol PLFA g K 1 soil. Fatty acid nomenclature used wasthat described by Frostega˚rd et al. (1993a,b). The fatty acidsi15:0, a15:0, 15:0, i16:0, 17:0, cy17:0, 18:1 u 7 and cy19:0were chosen to represent bacterial PLFA (Federle, 1986;Tunlid et al., 1989; Frostega˚rd et al., 1993a,b). Thepolyenoic, unsaturated PLFA 18:2 u 6 was used as anindicator offungal biomass (Federle, 1986). It was assumedthat the primary source of this eukaryotic PLFA was soilfungi (Zogg et al., 1997). The ratio offungal:bacterial PLFAwas used as an indicator of changes in the relativeabundance of these two microbial groups (Bardgett et al.,1996). The ratios between the bacterial fatty acids cy17:0and cy19:0 and their metabolic precursors, 16:1 u 7c and18:1 u 7c, as indicators of physiological stress (Grogan andCronan, 1997), referred to as ‘stress 1’ and ‘stress 2’,respectively; and the  trans  /  cis  ratio of 16:1 u 7 as anotherwidely observed response to various stresses (Heipieperet al., 1996), ‘stress 3’. 2.5. Root turnover  Mini-rhizotron tubes (320 mm long, 20 mm internaldiameter) were installed at an angle of 60 8  to the horizontalin all planted soil boxes at the time of soil packing. Clearplastic inserts, with ladders consisting of 0.5 cm squaresaligned to the top, left, and right of the tube surface, wereinserted and secured, to allow repeated image capture fromprecise soil locations. Digital images were captured with aboroscope camera (Olympus, Keyneld, Southend-on-sea,Essex) from nine locations (3, 5 and 7 cm depths, on theupper, left and right, surface of the tube) at 2-week intervalsbetween April and October 2001. The images were analysedusing ‘RooTracker’ software (version 1, Duke University,USA), for the appearance and disappearance of roots. 2.6. Statistical analyses Three-way analysis of variance (cultivar–fertiliser–defoliation) was carried out on the measured plantparameters and microbial characteristics to determinesignificant differences between the experimental factors(Genstat 5.3, NAG Ltd, Oxford, UK). For the BiOLOG datathe average well colour development (AWCD) of the 45carbon sources for each sample were calculated and used totransform well values to eliminate variation in well colourdevelopment caused by different cell densities (Garland,1996). The AWCD of different substrate groups wascalculated prior to performing the three-way ANOVA.The PLFA data was additionally analysed by multivariateanalysis following transformation to mol% to discountdifferences due to total PLFA extracted and arcsintransformed to normalise the dataset. PLFA data (mol%)was firstly analysed by principal components analysis(PCA) to reduce the dimensionality in the data arisingfrom having more variates than samples and then bycanonical variate analysis (CVA) using Genstat 5.3 (NAGLtd, Oxford, UK). CVA differentiated samples based ontheir overall microbial community structure. 3. Results 3.1. Plant parameters Shoot production, and C:N ratio, were unaffected bycultivar and soil fertilisation (Table 1a), but weresignificantly ( P ! 0.001) lower in defoliated ( C D)compared to non-defoliated (ND) swards (Table 1b). Thestanding root biomass was significantly ( P ! 0.05) greater inNF ND swards compared to other soil–defoliation treat-ments, and significantly ( P ! 0.05) greater in AberDove NFcompared to other cultivar–fertiliser treatments. The C:Nratio of root material was significantly ( P ! 0.001) lower in C D compared to ND treatments (Table 1b). Soilimprovement did not alter root C:N in AberDove swards,but was significantly ( P ! 0.05) greater in S23 C F comparedto S23 NF and AberDove swards.Root density was significantly ( P ! 0.001) greater inAberDove ND compared to S23 ND regardless of fertiliser,and significantly ( P ! 0.001) reduced by defoliation in bothcultivars (Table 1b). Over the growing season, cumulativeroot appearance and cumulative root disappearance did notsignificantly differ with cultivar, were significantly( P ! 0.05) greater in  C F compared to NF soil, andsignificantly ( P ! 0.001) reduced by defoliation (Fig. 1). 3.2. Sole carbon source utilisation profiles Cultivar specific differences in potential C utilisationwere evident in several substrate groups (Table 2), beinggreater in S23 swards compared to AberDove swards:AWCD ( P ! 0.05), oligo-sugars ( P ! 0.001), secondaryamino acids and neutral amino acids ( P ! 0.05). Soilfertiliser resulted in a greater AWCD ( P ! 0.05), mainlydue to greater potential utilisation of carboxylic acids( P ! 0.001). Most evident in the BiOLOG SCSUPs wasthe strong stimulation of potential C utilisation (AWCD,  L.M. Macdonald et al. / Soil Biology & Biochemistry 38 (2006) 674–682 676  P ! 0.001) in defoliated treatments, accounted for in oligo-saccharides ( P ! 0.01), carboxylic acids ( P ! 0.001), sec-ondary amino acids ( P ! 0.001) and phenolic acids( P ! 0.001) (Table 2). Additionally in AberDove  C Dpotential utilisation of aromatic amino acids was signifi-cantly ( P ! 0.01) greater. The potential utilisation of neutral( P ! 0.001) and basic ( P ! 0.001) amino acids was greater in C D treatments compared to ND treatments with theexception of AberDove  C F. In ND swards, AWCD wassignificantly ( P ! 0.01) influenced by fertiliser and cultivar:greater in S23 NF ND compared to S23 C F ND, and lowerand unaffected by fertiliser in AberDove ND. Significantcultivar–fertiliser ( P ! 0.05) and cultivar–defoliation( P ! 0.01) interactions were observed in the potentialutilisation of acidic amino acids: greater in S23 C Fcompared to AberDove  C F, and lower in S23 ND thanother plant–defoliation treatments. 3.3. Soil microbial community structure Indicators of the size of the total microbial biomass(240.01 nmol g K 1 soil  G 7.22), and the bacterial biomass(101.77 nmol g K 1 soil G 3.51) did not significantly differ,with cultivar, fertiliser or defoliation (Tables 3a and 3b).The fungal signature was significantly ( P ! 0.05) greaterin  C F C D, compared to other fertiliser–defoliationtreatments. As a result, the fungal:bacterial ratio wassignificantly ( P ! 0.05) greater in C F C D compared to NF C D treatments, but unaffected by defoliation in NF soils.S23 NF C D and S23 C F ND had significantly ( P ! 0.05)lower total cyclopropyl PLFAs compared to AberDoveNF C D. No difference was observed in ‘stress 1’, however‘stress 2’ was significantly greater ( P ! 0.05) in  C F C D Table 1aPlant parameters associated with  Lolium perenne  cultivars AberDove and S23, grown in non-fertilised (NF) or fertilised ( C F) soil with ( C D) or without (ND)defoliation. Data are means of 3 replicates G standard errors, significant differences at the 95% level are indicated with different lettersTable 1bAnalysis of variance for the plant parameters associated with plant, soil fertilizer, and defoliation treatmentsd.f.Plant ParametersShootBiomassShoot C:N RootBiomassRoot C:N Root Density Total PlantBiomassRoot:ShootPlant 1 NS NS NS NS NS NS NSFertiliser 1 NS NS NS NS NS NS NSDefoliation 1 *** *** NS *** *** *** NSPlant ! Fertiliser 1 NS NS * * NS NS *Plant ! Defoliation 1 NS NS NS NS *** NS NSFertiliser ! Defoliation 1 NS *** NS NS NS NS NSPlant ! Fertiliser ! Defoliation1 NS NS NS NS NS NS NSSignificant treatment effects and interactions are indicated: *** P ! 0.001, ** P ! 0.01, * P ! 0.05. 0100200300400500   A   b  e  r +   D o  v  e    N   F    N +   D  A   b  e  r +   D o  v  e    N   F  +   D  A   b  e  r +   D o  v  e  +   F    N +   D  A   b  e  r +   D o  v  e  +   F  +   D  S  2  3    N   F    N +   D  S  2  3    N   F  +   D  S  2  3  +   F    N +   D  S  2  3  +   F  +   D    R  o  o   t  a  p  p  e  a  r  a  n  c  e   /   d   i  s  a  p  p  e  a  r  a  n  c  e   #  r  o  o   t  s   /  m    2 appearancedisappearance Fig. 1. The cumulative appearance and disappearance of roots over thegrowing season in  Lolium perenne  AberDove and S23 grown in non-fertilised (NF) or fertilised ( C F) soil, with ( C D) or without (ND)defoliation.Dataaremeansofthreereplicates,barsindicatestandarderrors.  L.M. Macdonald et al. / Soil Biology & Biochemistry 38 (2006) 674–682  677  compared to other treatments, and ‘stress 3’ significantly( P ! 0.05) greater in C F compared to NF soil treatments(Tables 3a and 3b).Multivariate analysis of the 44extracted PLFAs was usedto discriminate microbial communities based on their broad-scale structure (Fig. 2). Cultivar discrimination was evidentin all fertiliser–defoliation pairings, with the S23 SMCconsistently orientating in a more positive direction on theprimary canonical axes compared to the equivalentAberDove pairing. Cultivar based discrimination wasstronger (as evidenced by a larger Mahalanobis distance),in NF compared to C F soil in both C D and ND pairings.Similarly, orientation of  C F soil lay consistently in a morepositive direction on the primary CV axes compared to theequivalent NF treatment. Separation due to soil improve-ment was greater in AberDove compared to S23 swards.The impact of defoliation was evident in all treatments,causing a positive shift along the primary CV axes, and in Table 2Analysis of variance of sole carbon source substrate utilisation potential associated with plant, fertiliser and defoliation treatmentsd.f. Substrate groupsAWCD Sugars CarboxylicacidsAmino acids PhenolicacidsMono Oligo Acidic Neutral Basic Aromatic SecondaryPlant 1 * NS *** NS NS * NS NS * NSFertiliser 1 * NS NS *** NS NS NS NS NS NSDefoliation 1 *** *** ** *** *** *** * *** *** ***Plant ! fertiliser 1 NS NS NS NS * * *** ** NS NSPlant ! defoliation 1 NS NS NS NS ** * NS NSFertiliser ! defoliation 1 NS NS NS NS NS NS *** NS NS NSPlant ! fertiliser ! defoliation1 ** *** NS NS NS *** *** NS NS NSSignificant treatment effects and interactions are indicated: *** P ! 0.001, ** P ! 0.01, * P ! 0.05.Table 3aPLFA indicators in the soil microbial community associated with  Lolium perenne  AberDove and S23 grown in non-fertilized (NF) or fertilized ( C F) soil, with( C D) or without (ND) defoliation. The stressindicatorswerecalculatedas ratiosofthe followingPLFAsignatures: Stress1 Z cy17:0/16:1 u 7c, stress2 Z cy19:0/18:1 u 7c, stress 3 Z 16:1 u 7 trans/cis. Data are means of 3 replicates, G standard errors, significant differences at the 95% level are indicated with differentlettersThe stress indicators were calculated as ratios of the following PLFA signatures. Stress 1 Z cy17:0/16:1w7c, stress 2 Z cy19:0/18:1w7c, stress 3 Z 16:1w7 trans  /  cis . Data are means of three replicates, G standard errors, significant differences at the 95% level are indicated with different letters.Table 3bAnalysis of variance for the PLFA signature groups and ratios associated with plant, soil fertilizer, and defoliation treatmentsd.f.Indicator Groups: nmol g K 1 soil Physiological indicatorsActiveBiomassBacterial Fungal fungal:bacterialCyclopropyl Stress 1 Stress 2 Stress 3Plant 1 NS NS NS NS NS NS NS *Fertiliser 1 NS NS NS NS NS NS NS NSDefoliation 1 NS NS NS NS NS NS NS NSPlant ! Fertiliser 1 NS NS NS NS NS NS NS NSPlant ! Defoliation 1 NS NS NS NS NS NS NS NSFertiliser ! Defoliation 1 NS NS * * NS NS * NSPlant ! Fertiliser ! defoliation1 NS NS NS NS * NS NS NSSignificant treatment effects and interactions are indicated: *** P ! 0.001, ** P ! 0.01, * P ! 0.05.  L.M. Macdonald et al. / Soil Biology & Biochemistry 38 (2006) 674–682 678
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