Rhizoctonia solani and Bacterial Inoculants Stimulate Root Exudation of Antifungal Compounds in

17 Pages·2017·2.53 MB·English

agronomy
Article
Rhizoctonia solani and Bacterial Inoculants Stimulate
Root Exudation of Antifungal Compounds in Lettuce
in a Soil-Type Specific Manner
SaskiaWindisch1,*,SebastianBott1,Marc-AndreasOhler1,Hans-PeterMock2,
RicoLippmann2,RitaGrosch3,KorneliaSmalla4,UweLudewig1andGünterNeumann1
1 DepartmentofNutritionalCropPhysiology,InstituteofCropScience(340h),UniversityofHohenheim,
70599Stuttgart,Germany;[email protected](S.B.);[email protected](M.-A.O.);
[email protected](U.L.);[email protected](G.N.)
2 Leibniz-InstitutfürPflanzengenetikundKulturpflanzenforschung,06466Gatersleben,Germany;
[email protected](H.-P.M.);[email protected](R.L.)
3 DepartmentPlantHealth,LeibnizInstituteofVegetableandOrnamentalCropsGroßbeeren/Erfurte.V.,
14979Großbeeren,Germany;[email protected]
4 JuliusKühn-Institut-FederalResearchCentreforCultivatedPlants,InstituteforEpidemiologyandPathogen
Diagnostics,38104Braunschweig,Germany;[email protected]
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:RobertJ.Kremer
Received:4May2017;Accepted:12June2017;Published:18June2017
Abstract: Previous studies conducted on a unique field site comprising three contrasting soils
(diluvialsandDS,alluvialloamAL,loessloamLL)underidenticalcroppinghistory,demonstrated
soiltype-dependentdifferencesinbiocontrolefficiencyagainstRhizoctoniasolani-inducedbottomrot
disease in lettuce by two bacterial inoculants (Pseudomonas jessenii RU47 and Serratia plymuthica
3Re-4-18). Disease severity declined in the order DS > AL > LL. These differences were
confirmed under controlled conditions, using the same soils in minirhizotron experiments.
Gaschromatography-massspectrometry(GC-MS)profilingofrhizospheresoilsolutionsrevealed
benzoicandlauricacidsasantifungalcompounds;previouslyidentifiedinrootexudatesoflettuce.
Pathogeninoculationandpre-inoculationwithbacterialinoculantssignificantlyincreasedtherelease
ofantifungalrootexudatesinasoiltype-specificmanner;withthehighestabsolutelevelsdetected
ontheleast-affectedLLsoil. Soiltype-dependentdifferenceswerealsorecordedforthebiocontrol
effectsofthetwobacterialinoculants;showingthehighestefficiencyafterdouble-inoculationonthe
ALsoil. However,thiswasassociatedwithareductionofshootgrowthandroothairdevelopment
andalimitedmicronutrientstatusofthehostplants. Obviously,diseaseseverityandtheexpression
of biocontrol effects are influenced by soil properties with potential impact on reproducibility of
practicalapplications.
Keywords: lettuce;soilmicrobiome;rootexudates;planthealth
1. Introduction
Pathogen-relatedyieldlossesareamongthemostprominentlimitationsincropproduction[1].
Theuseofresistantcultivars,applicationofchemicalpesticides,aswellascroprotation[2]represent
approachesaimedtoreducetheincidenceandseverityofpathogenattacks[1,3].However,particularly
forsoil-bornepathogenicfungiwithawidehostspectrum,suchasRhizoctonia,Fusarium,orPythium,
thedevelopmentofefficientcontrolstrategiesremainsahighlychallengingtask. Longpersistence
of the pathogen in soils as achieved by the formation of sclerotia [4,5] and saprophytic growth
stages, as well as restricted availability of resistant crop cultivars or fungicides are major limiting
Agronomy2017,7,44;doi:10.3390/agronomy7020044 www.mdpi.com/journal/agronomy
Agronomy2017,7,44 2of17
factors[6]. However,distinctsoil-microbialpopulationscaninducesuppressionofpathogens[7],and
thegeneralsuppressivenessofsoilsagainstplantpathogens[8]isacharacteristicofsoilqualityand
health [9]. This implies that biological control of pathogens via microbial antagonists is a realistic
strategy,providedthatitispossibletoidentifyspecificcontrolstrains,whichcanbetransferredto
pathogen-affectedsoilstoachieveaspecificsoilsuppressiveness[8]byharnessingthebeneficialsoil
microbiomesagainstpathogens[10]. However,theexploitationofmicrobialinoculantsasbiocontrol
agentsincropproductionsystemsisfrequentlyhamperedbyinconsistentresultsatthefieldscale[3],
likelylinkedtoplantperformance. Inmanycasesthereisstillasubstantiallackofknowledgeabout
thefactorsdeterminingthesuccessfulestablishmentofbiocontrolsystems.
The rhizosphere of the host plants is the major site for plant-microbial interactions, with
rhizodeposition,i.e.,thereleaseoforganiccarbonthroughplantroots,asthemajordrivingforce[11,12].
Duringtheseinteractions,bothbeneficialmicroorganismsandunfavorablepathogensareattractedby
theplantroots[12–14]withrhizodepositionsactingassignalsandascarbonandnitrogensources,but
alsoascomponentsofplantdefenseagainstpathogens.Constantmicrobiome–rootinteractionsmediate
nutrient turnover in the rhizosphere, which enables the plants to acquire nutrients and to receive
benefitsduringgrowthphases[15,16],whilesoil-bornepathogensadapttotherhizosphere,compete
withbeneficialsoilmicrobesfornutrientavailability,andcanharmthehostplant. Rhizodeposition
ishighlyvariableandinfluencedbymanyabioticandbioticfactors. Itcomprisespassivelossesof
organiccompounds,aswellashighlyregulatedsecretoryprocesseswithadaptivefunctions[17,18].
Due to the central role of rhizodeposits in shaping rhizosphere-microbial communities, a more
detailedunderstandingofthefactorsdeterminingrhizodepositionwithimpactonplantpathogens
andmicrobialantagonistsintherhizosphere,willprovideimportantinformationontheconditions
required for successful establishment of biocontrol systems. As an example, biological control of
Rhizoctoniasolani(Kühn)teleomorphThanatephoruscucumeris(A.B.FrankDonk)[6]bybacterialstrains
ofPseudomonasjesseniiandSerratiaplymuthicahasbeenproposedasapromisingapproachagainst
bottomrotdiseaseinlettuce(Lactucasativa)[2,19–23].
Torevealdifferencesinbiocontrolefficiencylinkedtothesoiltypeandnotinfluencedbylocal
climateorcroppinghistory,Schreiteretal.[23]usedauniqueexperimentalfieldplotsystem,withthree
differentsoiltypesstoredatthesamefieldsitefor10yearsunderthesameagriculturalmanagement.
Clearsoiltype-dependentdifferencesinthebacterialcommunitystructureofthebulksoilsandthe
corresponding lettuce rhizospheres were detected. This was associated with distinct quantitative
patternsoflow-molecularweightcompoundsintherhizospheresoilsolutions,mainlyrepresenting
rhizodeposits, detected by microsampling and gas chromatography-mass spectrometry (GC-MS)
profilinginaparallelminirhizotronstudyconductedwiththesamesoils[16].Particularly,benzoicacid
andlauricacidwereofspecialinterestbesidesotherorganicacids,variousaminoacids,amines,sugars,
andsugaralcohols. Itisdocumentedthatthesetwocompoundsexhibitantifungalactivityagainst
Rhizoctoniasolani,Pythiumultimum,Sclerotiniasclerotiorum,andothers[24,25],andhavebeenpreviously
identifiedinrootexudatesoflettucegrowninsoil-freesystemsinhydroponicculture[26]. Inoculation
ofthethreesoilswithRhizoctoniasolaniAG1-IB(R.solani)revealedastrongsoiltype-dependenteffect
ondiseaseseverityunderfieldconditions,whiletherhizospherecompetenceandthebiocontrolactivity
ofthepre-inoculatedbacterialbiocontrolstrainsPseudomonasjesseniiRU47andSerratiaplymuthica
3Re-4-18weremuchlessaffectedbysoiltypedifferences[3].
Based on this background information, the present study addressed the question of whether
similarplant-pathogeninteractionsandbiocontroleffectsarereproducibleusingthesamefieldsoilsin
aminirhizotronstudytoenablethecharacterizationofantifungalrootexudatesintherhizosphere
soilsolutionandtheirpotentialrelationshipswiththepresenceofthepathogenand/orthebacterial
inoculants. Lettuceplants(LactucasativaL.cv. Tizian)weregrowninminirhizotrons,equippedwith
removablerootobservationwindowsfornon-destructivemicro-samplingofrhizospheresoilsolution
with sorption filters applied to the surface of different root zones, followed by re-extraction and
GC-MSanalysis[16].
Agronomy2017,7,44 3of17
2. Results
2.1. PlantBiomassProductionisAffectedbyRhizoctoniasolaniAG1-IB
TheseverityofR.solani-inducedbottomrotdiseasewasclearlyinfluencedbythesoiltypeand
increasedintheorderloessloam(LL)<alluvialloam(AL)<diluvialsand(DS)(Table1),asindicated
bydecliningshootbiomassproduction. WhileshootbiomassoflettuceplantsgrownontheLLsoil
wasnotsignificantlyaffectedbyR.solaniinoculation,shootbiomasssignificantlydeclinedby38%on
theALsoil. Thelowestbiomassofnon-inoculatedcontrolplantswasrecordedonDSsoil,wherethe
plantsdiedwithinthefirstweekafterpathogeninoculation(Table1).
Agronomy 2017, 7, 44 3 of 16
Table1.S2h.1o. oPtladntr yBiommaassss P(rgodpulcatniotn− i1s )AofffecLteadc tbuy cRahsizaotcitvoaniLa .soclva.niT AizGia1-nIBg rownonthreedifferentsoilswithout
(control)andTwhei tshevienroitcyu olfa Rti. osonlaonif-iRndhuizcoecdt obnotitaomso lraont idAiseGas1e- wIBas( +clReahrilzyo icntfoluneian)c.ed(+ b+y Pthlea nsotisl tdyipeed anwdi thinthe
firstweekinacrfeteasreRd hiinz otchteo noiradesro llaoneissi nlooacmul a(LtiLo)n <) .alluvial loam (AL) < diluvial sand (DS) (Table 1), as
indicated by declining shoot biomass production. While shoot biomass of lettuce plants grown on
the LL soil was not significantly affected by R. solani inoculation, shoot biomass significantly
Treatment DiluvialSand AlluvialLoam LoessLoam
declined by 38% on the AL soil. The lowest biomass of non-inoculated control plants was recorded
on DCSo nsotirlo, wlhere the plants d0ie.d2 2w±ith0in.2 the first week af1te.r1 3pa±tho0g.1ena inoculation (Ta1b.l3e3 1)±. 0.5a
+Rhizoctonia ++ 0.70±0.3b 1.45±0.2a
Table 1. Shoot dry mass (g plant−1) of Lactuca sativa L. cv. Tizian grown on three different soils
Means±standwairthdoeurt r(ocorsntorofl)f oaunrd iwndithe pineoncduelantitorne opfl iRchaitzeosct.oDniaif sfoelraenni tAcGh1a-IrBa c(+teRrhsizionctdoniciaa)t. e(+s+i gPnlainfitcsa dnitedd ifferencesfor
agivensoiltypweit(ht-inte tshte, fpir=st 0w.0ee5k) .after Rhizoctonia solani inoculation).
Treatment Diluvial Sand Alluvial Loam Loess Loam
2.2. BiocontrolActiviCtyonotrfoPl seudomonasj0e.s2s2e ±n 0i.i2 RU47andS1e.r1r3a ±t i0a.1p al ymuthica3R1.3e3- 4±- 01.58 a
+Rhizoctonia ++ 0.70 ± 0.3 b 1.45 ± 0.2 a
2.2.1. EffectsonSMheoanos t±G straondwartdh errors of four independent replicates. Different characters indicate significant
differences for a given soil type (t-test, p = 0.05).
OntheDSsoil,nobiocontrolactivityofthebacterialinoculantsagainstR.solaniwasdetectable
2.2. Biocontrol Activity of Pseudomonas jessenii RU47 and Serratia plymuthica 3Re-4-18
duetorapidseedlingdecayduringthefirstweekafterpathogeninoculation(Table1). Thedeclineof
shootbiomas2s.2o.1n. EthffeecAts Lons Sohioloitn Gdruowcethd byR.solaniinoculation(Table1)wasmitigatedbypre-inoculation
withP.jessenii,reOanc thhien DgS dsoriyl, nmo baitotceorntprorlo adctuivcittyi oonf thneo btacstiegrinali fiincoacunlatnlyts dagiaffinesrte Rn. tsoflarnoim watsh deetcecotanbtlreo ltreatment
withoutR.sodlaune itoin raopcidu slaeetdiolinng (dFeicgayu dreur1inbg) t.hVe fiisrsut awlesekc oafrtienr gpaothfopgelna nintocdualamtioang (eTa(bFlei g1)u. Trehe2 d)ercleinvee oafl edthemost
shoot biomass on the AL soil induced by R. solani inoculation (Table 1) was mitigated by
prominentbiocontroleffectbydouble-inoculationwithP.jesseniiandS.plymuthica(+P.jess./S.ply.),
pre-inoculation with P. jessenii, reaching dry matter production not significantly different from the
indicatedbycsounrtvroilv traelatomfeanlt lwpitlhaonutts Ri. nsolraenis ipnoocnuslaetitoon (RFi.gsuorela 1nbi). iVniosucaul lsacotriionng .ofD pelasnpt idtaemhaigge h(Fibgiuorec o2)n trolactivity
(Figure2),threevleoawlede stthes hmooostt bprioomminaesnst pbiroocodnutrcotl ioefnfecwt abys rdeocuobrled-iendocuinlatitohne wdiothu bP.l eje-sisnenoiic uanladt eSd. variantsin
plymuthica (+P. jess./S. ply.), indicated by survival of all plants in response to R. solani inoculation.
both treatments with and without R. solani inoculation (Figure 1a,b). Single inoculation revealed
Despite high biocontrol activity (Figure 2), the lowest shoot biomass production was recorded in the
thatthiseffecdtoucboleu-ilndocbuleatmed avianrilayntas tintr bibotuh tteredatmtoentths ewpithr easnedn wciethoouft SR.. spollaynmi iunothcuiclaatio(nF i(gFiugurere 11ab,b).). Bycontrast,
ontheLLsoiSli,nngloe isniogcunliafiticona nretvetraeleadt tmhaet nthtise effffeecct tcsouwlde bree mraeicnolyr adtteridbuatendd tos thheo portesbeinocem oaf Ss.s plpymroudthuicac tionranged
betweenmin(.F1ig.3urge 1pbo). tB−y1 caonntdrasmt, aonx .th1e. 7LLg spoiol,t −no1 s(iSgnuipficpanlet mtreeantmtaenryt eFffiegctus rweeSre1 r)e.corded and shoot
biomass production ranged between min. 1.3 g pot−1 and max. 1.7 g pot−1 (Supplementary Figure S1).

Figure1.Cont.
Agronomy 2017, 7, 44 4 of 16
Agronomy 2017, 7, 44 4 of 16
Agronomy2017,7,44 4of17

Figure 1. Habitus (a) and shoot and root dry mass (g pot−1) (b) of Lactuca sativa L. cv. Tizian. The
Figure1.Habitus(a)andshootandrootdrymass(gpot−1)(b)ofLactucasativaL.cv .Tizian.Theplants
plants were grown on alluvial loam without bacterial inoculation (w/o Bac.), pre-inoculated with
wFeirgeugrreo 1w. nHoanbaitlulusv (iaa)l laonadm swhoitohto uantdba rcoteorti adlriyn omcualsast i(ogn p(wot/−1o) B(ba)c .)o,fp Lrea-citnuoccau slaattievda wL.i tchvP. sTeiuzdioamn.o nTahse
Pseudomonas jessenii RU47 (+P. jess.), Serratia plymuthica 3Re-4-18 (+S. ply.), a combination of both (+P.
jepslsaenntiis RwUe4r7e (g+rPo.wjenss .o)n,S aerllruavtiiaalp llyomamut hwiciath3oRuet- 4b-1a8ct(e+riSa.l pilnyo.)c,ualcaotimonb i(nwa/toio nBaocf.)b, optrhe(-+inPo.cjeuslsa/teSd. pwlyi.t)h,
jess/S. ply.), with and without (Control) subsequent inoculation with Rhizoctonia solani AG1-IB
with and without (Control) subsequent inoculation with Rhizoctonia solani AG1-IB (+Rhizoctonia).
Pseudomonas jessenii RU47 (+P. jess.), Serratia plymuthica 3Re-4-18 (+S. ply.), a combination of both (+P.
(+Rhizoctonia). Means ± standard errors of four independent replicates. Different characters indicate
Means ± standard errors of four independent replicates. Different characters indicate significant
jess/S. ply.), with and without (Control) subsequent inoculation with Rhizoctonia solani AG1-IB
significant differences (one-way ANOVA, Holm-Sidak test, p = 0.05).
differences(one-wayANOVA,Holm-Sidaktest,p=0.05).
(+Rhizoctonia). Means ± standard errors of four independent replicates. Different characters indicate
significant differences (one-way ANOVA, Holm-Sidak test, p = 0.05).

Figure2.VisualscoringofdiseaseincidenceofLactucasativaL.cv.Tizian,expressedaspercentageof
Figure 2. Visual scoring of disease incidence of Lactuca sativa L. cv. Tizian, expresse d as percentage of
healthy,weak(growthdepressionandleafnecrosisduetofungalinfection),anddeadplants.Theplants
healthy, weak (growth depression and leaf necrosis due to fungal infection), and dead plants. The
wFeirgeugrreo 2w. nViosnuaall lsucvoirailnlgo aomf dwisiethaoseu tinbcaicdteenricael oinfo Lcaucltautcioa nsa(twiv/ao LB. cavc..) T,pizriea-nin, oecxuplraetessdewd iatsh pPesrecuednotmaognea osf
plants were grown on alluvial loam without bacterial inoculation (w/o Bac.), pre-inoculated with
jesseniiRU47(+P.jess.),Serratiaplymuthica3Re-4-18(+S.ply.),acombinationofboth(+P.jess/S.ply.),
healthy, weak (growth depression and leaf necrosis due to fungal infection), and dead plants. The
Pseudomonas jessenii RU47 (+P. jess.), Serratia plymuthica 3Re-4-18 (+S. ply.), a combination of both (+P.
withandwithout(Control)subsequentinoculationwithRhizoctoniasolaniAG1-IB(+Rhizoctonia).
plants were grown on alluvial loam without bacterial inoculation (w/o Bac.), pre-inoculated with
jess/S. ply.), with and without (Control) subsequent inoculation with Rhizoctonia solani AG1-IB
Pseudomonas jessenii RU47 (+P. jess.), Serratia plymuthica 3Re-4-18 (+S. ply.), a combination of both (+P.
(+Rhizoctonia).
2.2.2. EffectsonRootGrowthandMorphology
jess/S. ply.), with and without (Control) subsequent inoculation with Rhizoctonia solani AG1-IB
2.2.2.N (E+ofRfsehiicgztonsc itofionnc iaRan)o.t ottr eGartomwetnht aenffdec Mtsowrpehreorloegcoyr dedforrootbiomassoflettuceplants,neitheronthe
AL (Figure 1), nor on the LL soil. A trend for lower root biomass production was detectable for
No significant treatment effects were recorded for root biomass of lettuce plants, neither on the
2.2.2. Effects on Root Growth and Morphology
theALsoilinthetreatmentswithR.solaniinoculation(Figure1). However,inalltreatmentswith
AL (Figure 1), nor on the LL soil. A trend for lower root biomass production was detectable for the
S. plymNuot hsiigcaniifnicoacnutl atrtieoantmoenntth eeffAecLtss woiel,rer oroectohradierdle fnogr trhoo(Ft ibgiuormea3sbs )owf laesttsuicgen pifilacanntst,l ynerietdhuerc eodn bthye
3 A0–L5 0(F%iginurteh e1)p, rneosern ocne othfeR .LsLo lsaonii.l.S Aim tirleanrldy ,frooro ltohwaeirr dreonosti tbyio(Fmigaussr ep3rco)dduecctliionne dwaafste dreptreec-tianbolceu floarti othne
Agronomy 2017, 7, 44 5 of 16
AL soil in the treatments with R. solani inoculation (Figure 1). However, in all treatments with S.
Agronomy2017,7,44 5of17
plymuthica inoculation on the AL soil, root hair length (Figure 3b) was significantly reduced by 30–
50% in the presence of R. solani. Similarly, root hair density (Figure 3c) declined after pre-inoculation
wwitihthS S.p. lpylmymuuthtihciac,a,b booththi nint htheec oconntrtorlotl rteraetamtmenentta nandda fatfetrerin ioncouculaltaitoinonw witihthR R.s. osloalnain.iN. Nooc ocmompparaarbablele
efeffefcetcstsw wereered edteetcetcatbalbeloe nonth tehLe LLLso silo,iwl, hwehreerreo orotohta hiraliern lgetnhgrthan rgaendgebde tbweetwene0e.n7 10.m71m mamnd a0n.d87 0m.87m minm
ailnlt arella ttrmeaetnmtse(nStus p(SpulepmpelenmtaernytaFrigyu FrieguS2re). S2).

Figure3. Roothairdevelopment(a),roothairlength(mm)(b)androothairdensity(mm−1)(c)of
Figure 3. Root hair development (a), root hair length (mm) (b) and root hair density (mm−1) (c) of Lactuca
LactucasativaL.cv. Tizian. Theplantsweregrownonalluvialloamwithoutbacterialinoculation
sativa L. cv. Tizian. The plants were grown on alluvial loam without bacterial inoculation (w/o Bac.),
(w/o Bac.), pre-inoculated with Pseudomonas jessenii RU47 (+P. jess.), Serratia plymuthica 3Re-4-18
pre-inoculated with Pseudomonas jessenii RU47 (+P. jess.), Serratia plymuthica 3Re-4-18 (+S. ply.), a
(+S.ply.),acombinationofboth(+P.jess/S.ply.),withandwithout(Control)subsequentinoculation
combination of both (+P. jess/S. ply.), with and without (Control) subsequent inoculation with Rhizoctonia
withRhizoctoniasolaniAG1-IB(+Rhizoctonia).Means±standarderrorsoffourindependentreplicates.
solani AG1-IB (+Rhizoctonia). Means ± standard errors of four independent replicates. Different characters
Differentcharactersindicatesignificantdifferences(one-wayANOVA,Holm-Sidaktest,p=0.05).
indicate significant differences (one-way ANOVA, Holm-Sidak test, p = 0.05).
22.2.2.3.3..P Plalannt-tN-NuutrtirtiitoionnaallS Statatutuss
ForthemacronutrientsN,P,K,andMgsuppliedtothesoilsasfertilizersinsufficientamounts
priortoplantcultivation,nosignificanttreatmenteffectsorcharacteristicdeficiencysymptomswere
re corded for the lettuce plants grown on the AL and the LL soil (not shown). However, on AL
Agronomy 2017, 7, 44 6 of 16
For the macronutrients N, P, K, and Mg supplied to the soils as fertilizers in sufficient amounts
prior to plant cultivation, no significant treatment effects or characteristic deficiency symptoms were
recorded for the lettuce plants grown on the AL and the LL soil (not shown). However, on AL soil,
micronutrient shoot concentrations of Zn, Mn, and Fe (Figure 4a–c) of the treatments with R. solani
inoculation showed a trend of decline in the order w/o Bac. > P. jessenii > S. plymuthica > P. jessenii +S.
Apglyromnoumtyhi2c0a1,7 ,7f,in44ally reaching critical values close to the deficiency thresholds [27] for6 ofth17e
double-inoculation. In the control treatment without R. solani inoculation, this effect was restricted to
lettuce plants with double-inoculation with P. jessenii and S. plymuthica (+P. jess./S. ply.).
soil, micronutrient shoot concentrations of Zn, Mn, and Fe (Figure 4a–c) of the treatments with
For lettuce plants grown on LL soil, no significant treatment effects in shoot micronutrient
R.solaniinoculationshowedatrendofdeclineintheorderw/oBac. >P.jessenii>S.plymuthica>P.
concentrations were recorded. Shoot concentrations of Mn ranged between 29.4 mg kg−1 DM and
jessenii+S.plymuthica,finallyreachingcriticalvaluesclosetothedeficiencythresholds[27]forthe
53.4 mg kg−1 DM, and Zn between 20.4 mg kg−1 DM and 29.5 mg kg−1 DM (Supplementary Figure S3).
double-inoculation. InthecontroltreatmentwithoutR.solaniinoculation,thiseffectwasrestrictedto
No nutrient analysis was conducted for the plants on DS soil due to the rapid decay already during
lettuceplantswithdouble-inoculationwithP.jesseniiandS.plymuthica(+P.jess./S.ply.).
the first week after R. solani inoculation.

FFiigguurree 44.. MMicicroronnuutrtireiennt tcocnocnecnetnrtartaiotinosn osfo ZfnZ (na)( Ma)nM (bn) (abn)da Fned (cF)e in(c s)hionotssh (omotgs k(gm−1g DkMg−) 1ofD LMac)tuocfa
Lsaactitvuac aL.s actviv. aTiLz.iacnv.. TThiez ipanla.nTtsh weeprlea ngtrsowwne roeng arlolwuvniaol nloaalmlu vwiaitlhloouatm bawctiethrioaul tinboaccutelartiiaolni n(owc/uol aBtaioc.n),
(w/o Bac.), pre-inoculated with Pseudomonas jessenii RU47 (+P. jess.), Serratia plymuthica 3Re-4-18
(+S.ply.),acombinationofboth(+P.jess/S.ply.),withandwithout(Control)subsequentinoculation
withRhizoctoniasolaniAG1-IB(+Rhizoctonia).Means±standarderrorsoffourindependentreplicates.
Thedottedlinesindicatethethresholdlevelsformicronutrientdeficiency.Differentcharactersindicate
significantdifferences(one-wayANOVA,Holm-Sidaktest,p=0.05).
Agronomy2017,7,44 7of17
For lettuce plants grown on LL soil, no significant treatment effects in shoot micronutrient
concentrations were recorded. Shoot concentrations of Mn ranged between 29.4 mg kg−1 DM
and53.4mgkg−1 DM,andZnbetween20.4mgkg−1 DMand29.5mgkg−1 DM(Supplementary
FigureS3). NonutrientanalysiswasconductedfortheplantsonDSsoilduetotherapiddecayalready
duringthefirstweekafterR.solaniinoculation.
2.2.4. AntifungalCompoundsintheRhizosphereSoilSolution
The antifungal compounds benzoic acid and lauric acid [24,25], previously identified in root
exudatesoflettuce[26],weredetectedbyGC-MSanalysis(Table2)aftercollectionbymicro-sampling
withsorptionfilters[16]fromlettuceplantsin1–2cmsubapicallateralrootzonesandinmorebasal
partsoftherootzones(8–9cmbehindtheroottip). OntheALsoil,R.solaniinoculationsignificantly
increasedtheconcentrationsofbenzoicacid(Figure5a)collectedfromsubapicalrootzonesby234%
andinthebasalrootzonesby296%. Lauricacidconcentrations(Figure5b)tendedtobeincreased
only in the basal parts of the root zones, as compared to the control treatment without R. solani
inoculation(Table2). Incontrast,ontheLLsoil,onlytrendsforincreasedrootexudationofbenzoic
acid(11%subapical,29%basal)andlauricacids(+22%subapical)wereinducedbyR.solaniinoculation
butnosignificanteffectsincomparisontothecontroltreatmentwithoutR.solaniinoculationwere
observed. Ingeneral,lauricacidconcentrationsinsamplescollectedontheLLsoilwerehigherthan
onALsoil.
Table 2. Benzoic and lauric acid concentrations in rhizosphere soil solutions collected by
micro-sampling with sorption filters in 1–2 cm subapical regions of young roots and from older
basalrootzones(8–9cm)ofLactucasativaL.cv.Tizian.Theplantsweregrownonalluvialloamtreated
without(control),withRhizoctoniasolani(+R.solani),withPseudomonasjessenii(+P.jess.)andSerratia
plymuthica(+S.ply.),withRhizoctoniasolaniandonebacterialinoculant(+R.solani+P.jess;+R.solani
+S.ply.),withacombinationofbothbacterialinoculants(+P.jess/S.ply.)andwithRhizoctoniasolani
andbothbacterialinoculants(+R.solani+P.jess./S.ply.). Relativevaluesbasedonpeakareas(gas
chromatography-massspectrometry(GC-MS)analysis)aftersubtractionofbackgroundlevelsinbulk
soilsamples.
BenzoicAcid LauricAcid
Treatment
Subapical(Young) Basal(Old) Subapical(Young) Basal(Old)
AL LL AL LL AL LL AL LL
Control 3.92a 5.00a 2.77a 6.30a 1.76a 4.29a 0.14a 14.00a
+R.solani 13.07b 5.53a 10.97b 8.12a 0.85a 5.25a 1.36ab 9.56a
+P.jess. 12.84b 10.54b 12.98b 13.58ab 1.00a 10.55b 1.52b 21.80a
+S.ply. 13.08b 10.49b 12.75b 9.58ab 0.51a 10.66b 1.01ab 9.40a
+R.solani
10.75b 9.62b 13.65b 15.53b 0.92a 9.29b 1.34ab 25.03a
+P.jess.
+R.solani
11.71b 8.63b 12.68b 15.79b 1.00a 7.82ab 0.94ab 29.87a
+S.ply.
+P.
jess./S. 12.84b N.d. 12.63b N.d. 0.85a N.d. 0.93ab N.d.
ply.
+R.solani
+P.jess./S. 12.14b N.d. 11.31b N.d. 0.43a N.d. 1.09ab N.d.
ply.
Meansofthreeindependentreplicates.Ineachcolumndifferentcharactersindicatesignificantdifferences(one-way
ANOVA,Holm-Sidaktest,p=0.05).N.d.=notdetermined.DS:Diluvialsand,AL:alluvialloam,LL:loessloam.
Agronomy2017,7,44 8of17
Agronomy 2017, 7, 44 8 of 16

Figure 5. Concentrations and relative changes (% compared to the untreated controls) of benzoic acid (a)
anFdi glauureric5 .aCciodn c(ben) tirna trihoinzsoasnpdherreel astoivile scohlauntigoenss( %colcloemctepda rferdomto 1th–e2 ucmnt rseuabteadpiccoanl trreoglsi)oonfs boefn yzoouicnagc irdoots
an(da )fraonmd loaludreirc baacsidal( rbo)oitn zrohnizeos s(p8h–9e rcemso bialssaoll urotiootnss) ocof lLleaccttuedcaf sraotmiva1 –L2. ccvm. Tsuizbiaanp.i cTahler epgliaonntss owfeyroeu gnrgown
onr oalolutsvaianld lofaromm (AolLd)e arnbda lsoaelsrso lootazmo n(LeLs)( 8tr–e9actemd bwaistahloruoto btas)ctoefriLaal citnuoccauslaattiivoanL (w.c/vo. BTaizci.a),n w.iTthh eRphilzaoncttsonia
solwaneir eAgGro1w-IBn o(+nRa. llsuovlainail)l,o wamith( AbLa)ctaenrdiallo iensoscluolaamnt(sL (L+)Btarce.a tinedocwuliatnhtosu =t bPascetuedrioamloinnoacs ujleastsieonnii (wRU/o47B aacn.d),/or
Sewrraitthia Rphlyizmoucttohniciaa 3soRlean-4i-A18G) 1a-nIBd (w+iRth. sao lcaonmi),bwiniathtiobna cotef rbiaalctienroiacul ilnanotcsu(la+nBtasc .anindo cRuhlaiznotcsto=nPias esuoldaonmi AonGas1-IB
(+Bjeascs.e niniioRcuUla4n7tsa n+dR/. osrolSaenrir)a. tRiaeplalytmivuet hviacalu3eRs eb-4a-s1e8d) aonnd pweiatkh aarceoam (bGinCa-tMioSn aonfablaycstiesr)i aalftinero csuulbatnrtascatinodn of
baRckhgizroocutonndi alesvolealns iinA Gbu1l-kIB so(+ilB saacm. ipnloecsu. lManetasn+sR ±. ssotalanndia).rdR eelrartoirvse ovfa 3lu–9e sinbdaesepdenodnenpte arekpalirceaate(sG. CFo-Mr eSach
soailn taylypsei,s )daifffteerresnutb ctrhaacrtaioctnerosf binadckicgartoeu snidgnleifvicealsnti ndbifuflekresnociless a(monpele-ws.aMy eAanNsO±VsAta, nHdoarlmd-eSrirdoarsk otfe3st–,9 p =
0.05).
independentreplicates.Foreachsoiltype,differentcharactersindicatesignificantdifferences(one-way
ANOVA,Holm-Sidaktest,p=0.05).
3. Discussion
3.1. Plant Pathogen Interactions
Agronomy2017,7,44 9of17
Comparedtothenon-inoculatedcontroltreatment,pre-inoculationwithP.jessenii,S.plymuthica
andacombinationofboth(+P.jess/S.ply.) increasedrootexudationofbenzoicacid(Figure5a)inthe
subapicalandbasalrootzonesontheALsoilbetween197%and361%andontheLLsoilbetween83%
and149%. Sincenosignificantdifferencesbetweenthebacterialinoculantswererecorded(Table2),
Figure 5 summarizes the effects of all bacterial inoculants. An increased exudation of lauric acid
wasobservedinthebasalrootzonesonALsoil(730%and743%)andadditionallyonLLsoilinthe
subapicalrootzones(97%and147%). Thehighestlauricacidconcentrationsandthehighestlevelsof
antifungalrootexudatesingeneralwererecordedonLLsoil(Figure5).
3. Discussion
3.1. PlantPathogenInteractions
Similartopreviouslyreportedfieldobservations[3],soiltype-dependentdifferencesinseverityof
bottomrotdiseaseinlettuce,inducedbyinoculationwithR.solaniAG1-IB(predominantinfield-grown
lettuceinGermany[28]),wereconfirmedinthepresentminirhizotronstudy. Theexperimentwas
conductedundercontrolledconditionsonthreedifferentsoils(DS:diluvialsand,pH6.1;AL:alluvial
loam,pH6.7;LL:loessloam,pH7.1)withthesamecroppinghistoryduringthelast10years. Shoot
biomass production of R. solani-inoculated lettuce plants declined in the order LL > AL > DS soil
(Table1),reflectingthenegativeimpactofthepathogenonlettucegrowthaspreviouslyreportedon
thesamesoilsunderfieldconditions[23]. However,inminirhizotronculture,diseasesymptomson
lettucegrownonDSsoilappearedmuchfaster,andincontrasttothefieldexperimentsallplantsdied
alreadyduringthefirstweekafterpathogeninoculation. Thehigherconducivenessforbottomrot
diseaseoftheDSsoilobservedinallexperimentsmaybecausedbythebiggerporesizesandbetter
oxygenavailabilityinsandysoils,whichenablesmorerapidhyphalgrowthofR.solanitowardsthe
hostplant[3,29]. Themoreseverediseasesymptomsintheminirhizotronstudymaybeattributedto
higherrootingdensitiesinthelimitedsoilvolumeoftheminirhizotronsandduetoshorterspatial
distances between the pathogen and the host plant in comparison to field conditions with larger
soil volume. Moreover, the constant temperature conditions in the growth chamber (23–25 ◦C) in
theoptimumrangeforhyphalgrowthofR.solaniAG1-IB[28]couldfurtherpromotetheinfection
process,particularlyontheDSsoilwiththeweakestplantdevelopmenteveninthenon-inoculated
control(Table1). Accordingly,Schreiteretal.[3]reportedacertainbackgroundinfectionpotentialfor
bottomrotdiseaseinallinvestigatedsoils,evenwithoutartificialinoculationwithR.solaniAG1-IB,
withthelowestdiseaseseverityonLLsoil,incomparisontodiseaseseverityforplantsgrownonDS
andALsoil.
OntheALsoil,R.solaniinoculationresultedina60%reductioninshootbiomass(Table1)and40%
oftheplantsfinallysurvived(Figure2). Incontrast,noapparentbottomrotsymptomsweredetected
ontheLLsoil(Table1)andsupportedtheobservedresultsofhighersoilsuppressivenessinthefield
experiment[3],reflectedalsobythelowestinfectionpotentialonthenon-inoculatedcontrolsoils[3].
Accordingly,ontheALsoil,pathogeninoculationsignificantlyincreasedtheconcentrationsofbenzoic
andlauricacids(Table2,Figure5)intherhizospheresoilsolutions(234%toca. 900%),previously
identifiedasrootexudatesoflettucewithdocumentedantifungalactivityagainstRhizoctonia,Fusarium,
andotherpathogenicfungi[24,25].Incontrast,onlyanon-significanttrendforincreasedrootexudation
oftheantifungalcompoundsbyamaximumof30%wasdetectedintherhizosphereontheLLsoil
with antifungal suppressive potential (Table 2; Figure 5). These findings suggest that the release
of the antifungal root exudates is part of the pathogen defense response in lettuce, as previously
reported also for Fusarium-resistant peanut cultivars [30]. Rhizoctoniasolani-affected plants on the
ALsoilobviouslyrespondedwithincreasedexudationofantibioticcompounds,whileonlyaweak
response was triggered in the R. solani inoculated plants grown on the LL soil with low R. solani
infectionpotential.
Agronomy2017,7,44 10of17
BesidesanincreasedreleaseofantifungalrootexudatesinresponsetoR.solaniinfectiononAL
soilinbasalpartsoftherootsystemclosetotheinoculationsites,benzoicacidwasalsodetectedinthe
youngestrootzones(1–2cmbehindtheroottip),upto30cmbelowtheR.solaniinfectionsites(Table2,
Figure5). Rhizoctoniasolaniinfectionsitesareusuallylocatedattheroot-shootjunctionandatthe
lowerleaveswithdirectsoilcontact[28,29]. Thus,releaseofantifungalrootexudates,evenfiveweeks
afterinoculationwiththepathogen,mayindicateasystemicresponseoflettucetoR.solaniinfection.
Accordingly,previousstudieshavealsoreportedthesystemicinductionofbiosyntheticpathwaysfor
aromaticcompoundsinresponsetoR.solaniinfectionandtheirpotentialroleinpathogendefensein
differentcrops,suchaspotatoandrice[29,31].
3.2. DiseaseSuppressionbyBacterialInoculants
Twobacterialstrains,PseudomonasjesseniiRU47andSerratiaplymuthica3Re-4-18[32],characterized
forgoodbiocontroleffectsagainstbottomrotdiseaseinpreviousexperiments[6,21],wereselected
asbiocontrolstrainsusedforpre-inoculationofthelettuceplants. OntheDSsoilwiththeweakest
plantdevelopmenteveninthecontrolswithoutpathogeninoculation(Table1),andwiththemost
severebottomrotsymptoms,noprotectiveeffectsofthebacterialinoculantsagainstR.solaniwere
recorded. In contrast, on the AL soil, particularly pre-inoculation with P. jessenii RU47 tended to
increasebiomassproductionofR.solani-affectedplants(Figure1). However,themostpronounced
biocontroleffectwasevaluatedbyadouble-inoculationwithbothbacterialstrainswhere,incontrast
totheothertreatments,allplantssurvived(Figure2). Theincreasedexpressionofantagonisticeffects
after double inoculation with different biocontrol strains has been similarly reported in previous
studies [33] and may be attributed to additive effects of different antibiotics, cellulolytic enzymes,
and induction of plant defense responses, but also to a broader activity spectrum under different
environmentalconditions[34]. Bycontrast,noadditionalbeneficialeffectsofpre-inoculationwiththe
bacterialinoculantswereobservedontheleastconductiveLLsoil(SupplementaryFigureS1)where
thelettuceplantsdidnotshowanyvisiblesymptomsofbottomrotdisease.
Interestingly,similartoR.solaniinfection,inoculationwiththebacterialinoculantsalsoincreased
theexudationofbenzoicandlauricacidsontheALsoil. Moreover,thesameresponsewasrecorded
onthemoresuppressiveLLsoilwhereR.solaniinoculationhadonlymarginaleffectsonthereleaseof
theantifungalrootexudates(Table2,Figure5). Inaccordancewiththeabsenceofdiseasesymptoms,
thehighesttotallevelsoftheantifungalcompounds(sumofbenzoicandlauricacids)weredetectedin
rootexudatesoflettuceplantsgrownontheLLsoil(Table2,Figure5),whichmayreflectaparticularly
intenseexpressionofpathogendefensemechanisms.Eveninthevariantwithoutpathogeninoculation,
highbackgroundlevelsoflauricandbenzoicacidsweredetectableontheLLsoil(Table2,Figure5).
This raises the question of whether generally high levels of antifungal root exudates contributed
to the particularly low conductivity for Rhizoctonia bottom rot disease on this soil. Interestingly,
Schreiter et al. [3] observed a soil type-dependent rhizosphere effect of the lettuce plants on the
abundanceofbacteriawiththecapacitytodegradearomatichydrocarbons(particularlySphingomonas),
declining in the order DS > AL > LL soil, which follows the soil type-dependent expression in
severityofRhizoctonia-induceddiseasesymptoms(Table1). Thismayreflectadecliningcapacityfor
degradationoftheantifungalrootexudatesassecondarymetabolitesbymembersoftheindigenous
microflora,resultinginthehighestaccumulationonLLsoil,whichmaybealsoresponsibleforthe
lowbackgroundinfectionpotentialforbottomrotdiseaseonthissoil[3]. Apartfrombenzoicand
lauricacids,Neumannetal.[16]alsoreportedparticularlyhighlevelsofsugars,aminoacids,and
organic acids in the rhizosphere soil solutions collected from lettuce plants grown on the LL soil,
associatedwiththehighestshootbiomassproduction(Table1). Thismayindicateagenerallyhigher
capacityforrootexudationduetostrongerplantdevelopmentontheLLsoil,associatedwithahigher
photosyntheticcapacityasamajordrivingforceforrhizodeposition[17].
The exudation of benzoic acid was more strongly induced by bacterial inoculants or by
pathogen–inoculantcombinationsthanbysoleinoculationwithR.solani(Figure5). Thesefindings