View of Effect of soil-spraying time on root-colonization ability of antagonistic Streptomyces griseoviridis

Effect of soil-spraying ^{time on} root-colonization ability of antagonistic Streptomyces

Hanna Kortemaa

Departmentof^{PlantBiology, PlantPathology,POBox28,FIN-00014 University}of^{Helsinki,Finland.}

Currentaddress: KTTK,Seed Testing Department,POBox111.^FIN-32201 Loimaa, Finland, e-mail: hanna.kortemaa@mmm.fi

Kielo Haahtela

DepartmentofBiosciences,Divisionof^GeneralMicrobiology,POBox56,FIN-00014 Universityof

Helsinki,Finland

Aino Smolander

Vantaa ResearchCentre.Finnish Forest ResearchInstitute,POBox 18,FIN-01301 Vantaa,Finland

The root-colonization ability ofStreptomyces griseoviridisAnderson et al. wastestedonturniprape (Brassica rapa subsp. oleifera^{DC.) and carrot(}Daucus carota L.) bythe sand-tube method. Non- sterile sandwassprayedwithamicrobialsuspension immediately or7 days after the seed had been sown.Resultsexpressed aspopulation frequenciesand densities indicated that S.griseoviridis effec- tivelycolonizes therhizospherewhen the microbe isapplied immediatelyaftersowingbut less effec- tively when it is applied 7 days later. Detection values of S. griseoviridis werehigherforturniprape than for carrot. Insterile sand,S. griseoviridis invariblycolonized therhizosphere ofturniprape after each of the twoapplications. Thesefindings indicate that S.griseoviridis cancompete withindige-

noussoil microbesintherhizosphere ifit issufficientlyabundantinthe soil before the seed emerges.

Ifapplied later, however, it competes ratherpoorly. Inroot-free nonsterilesand, S.griseoviridisdis-

persedandsurvived well. ^r

Keywords:actinomycetes, biological control,Brassica rapa ssp.oleifera^,Daucus carota,plant growth- promotingrhizobacteria (PGPR),rhizosphere

ntroduction

S. griseoviridis,abiocontrolagentused against some seed-borneand soil-borne plant pathogens (Tahvonen 1988), produces the auxin indole-3-

acetic acid (IAA).The concentration of lAA pro- duced on solid media by S. griseoviridis is of the _same magnitude _as that reported to havea growth-promoting effect (Tuomi et al. 1994).

Mycostop (Kemira Oy, Finland) is abiofungi- cide produced by fermentation of the spores and

©Agriculturaland Food ScienceinFinland Manuscriptreceived March 1997

Kortemaa, H. etal. Effect ofsoil-spraying timeon Streptomyces griseoviridis mycelium of aS. griseoviridis strain isolated

frompeat by Tahvonen(1982).

Several isolates ofStreptomyces spp., includ- ing the S. griseoviridis isolated from peat, pro- duce polyene antibiotics. Incontrast tononsup- pressive isolatesmostof the suppressive isolates produce a candicidin-type antibiotic (Raatikai- nen etal. 1993).Scanning electronmicroscopy studies show that S. griseoviridis isa hyperpar- asite of various plant pathogenic fungi (Tapio and Pohto-Lahdenperä 1991).Besides antibiosis and parasitism, competition is often mentioned_{as a} mechanism ofbiocontrol. AccordingtoSivan and Chet (1989), the inhibition of germination of chlamydospores might be due to competition between Trichoderma harzianum Rifai and Fusariumoxysporum Schlecht.: Fr. Rothrock and Gottlieb (1984), on the otherhand, showed that the antagonism of S. hygroscopicusvar. gelda- nus was due to the antibiotic production, not to competition for nutrients.

Microbes that colonize^rootsareideal foruse asbiocontrol agentsagainst soil-borne diseases (Weller 1988).Because soil spraying, like seed dressing is, an application method that hasre- sulted in goodbiocontrol, our objectivewas to study howroots of turnip rape and carrot are colonized after soil-spraying treatmentand to test whether application time has any effect on root colonization. We tested the root-coloniza- tion ability in both nonsterile and sterile sandto establish the effect of microbial competition in the rhizosphere ^onthe colonization potential. As well_asthe growth ofS. griseoviridis in the rhizo- sphere,_weexamined the dispersal of thisantag- onist in root-free sand.

Material and methods

Root colonization in nonsterile sand

The dispersal of S. griseoviridis in the rhizo- sphere _was tested by the sand-tube methodas describedbyAhmad and Baker (1987) and Kor-

temaa etal.(1994).Seeds of turnip rape, Brassi- ca ^rapa subsp.

oleifera

^{cv. Kulta, and carrot,}

Daucus carotacv.Nantes Fancy,were surface- sterilized with ethanol and sodium hypochlorite (NaOCl) asdescribed by Kortemaa etal.(1994).

PVC-plastic tubes 20cmlong and 3.2 cmin di- ameter werelongitudinally sliced and fastened together with rubber bands. The tubes were blockedatthe bottom with cottonwool and filled withnonsterile, sieved (0.5-1.2 mm) sand (pH 6.2).Thesand, waterand water-soluble fertiliz- er were mixed_as described earlier,resulting in a water potential of^- 1 kPa (Kortemaa et al.

1994).One surface-sterilized seedwas sown in each tube,and the tubeswereplaced vertically, five per each plasticpotcontaining thesamesand mixture. A microbial suspension was prepared of Mycostop biofungicide as a 0.01% suspen- sion inwater. The average colony-forming unit (cfu) value of S. griseoviridis of the suspension detectedonsemi-selectivewateragar withglyc- erol(Kortemaa etal. 1994)was 8^x 10⁴ml

1

^;sml

of this microbial suspensionwas spread evenly with apipette on the sand surface in each tube immediately after sowing (day0)or was applied 7 days after sowing. Thepots werecovered with plastic bags, and no waterwas added aftersow- ing. The pots were incubated for4 weeks in a growth chamber (16 h light period, light inten- sity 150 mMols'm 2at 20°C and 8 h dark period at 18°C).

After 4weeks, the tubes were opened, and the^rootswere ^cutinto2cm segments.The sand adheringtotherootsegments wasconsidered_as rhizosphere soil. For population-density counts, the cfu values were determined by a dilution- plating method on ^water-agar plates, although this method did notpermit population densities lower than 10²cfu g ^] soil tobe detected. Sand- free root segments and above-ground portions of seedlings, i.e. stemsand leaves, wereplaced on ^water-agarplates^to isolate S. griseoviridis.

Root-colonization frequencies were counted forroot segments and the rhizosphere, and the population densitywas countedascfug'

1

^rhizo-

sphere soil. Each experiment comprised ¹⁰plants of turnip^rapeandcarrotand thetwoapplication

times for the suspension. Five control plants were nottreated. The experimentwasconducted three times.

Root colonization in sterile sand

For this experiment, turnip rape was grown in sterile sand in a large glass pot containing four longitudinally sliced tubes. The sand _was steri- lized for I h and other material for 20 min ^at

121°C in anautoclave. Awatersuspension of S.

griseoviridis, which had grown for 7 days on glucose-yeast-malt agar(GYM) (Kortemaaetal, 1994), was prepared for the spraying treatment instead of Mycostop suspension. The average population density of the suspension, which mainly consisted of spores of 5. griseoviridis, was 6.5 x 10⁶ cfu ml

1

^. For these experiments, the sterile seed was sown, the sand-tubeswere incubated, and S. griseoviridis was applied and isolatedasdescribed above. The only exception was that for population density counts the mi- crobe was isolated in the same mannerforone tube in each pot on water-agar and GYM-agar platestoensure that the sand was not contami- nated with other microbes. Each experimentcon- tainedone untreated controlpot and threepots with the twoapplication times of the S. griseo- viridis suspension. The experimentwasconduct- ed twice.

Dispersal in nonsterile sand without plants

For this experiment alarge plastic box(40 cmx 60cm)and32 plastic tubes werefilled with the nonsterile sand-water-fertilizer mixture de- scribed above. The tubes wereplaced randomly in the box in_avertical position. Five millilitres of 0.01% Mycostop suspension was pipetted evenly ontothe surface of each sand-tube;after this treatment, the tubes _{were not} watered. The averagepopulation density of the suspensionwas 3 ^{x I04}cfu ml 1^. The box with the sand ^tubes,

whichwascovered^toavoid loss ofmoisture, was incubated inagrowth chamber under the condi- tions described above for 4 weeks. Each week (7, 14,21 and 28 days aftertreatment), eight tubeswererandomly takenas asample formeas- urements of population frequencies and densi- tiesatdifferent depths of sand. The soil for these

countswassampledatevery2cmbetween⁰and 12cmfrom thetop of the tube. The population density wascounted by the dilution-plate meth- od on water-agar plates. The experiment was conducted twice.

Statistical analysis

Cfu values were logarithmically transformed before analyses of variance ^[PROC GLM(SAS Institute Inc. ^1988)].Tukey’s Studentized Range (HSD)Test _was used to compare significantly differentmeans.

Results

Root colonization ⁱⁿ nonsterile and sterile sand

When Mycostop suspensionwassprayed imme- diately (day 0) after sowing, the root-coloniza- tion frequencieson the^rootsand rhizosphere of turnip rape andcarrotwerehigher than when the suspension wasapplied7 days after sowing(Ta- ble 1).The frequency valueswerehigher fortur- nip rape than ^carrot. The stems and leaves of turnip rape seedlings were colonized 100% af- ter0 and 73% after 7 days oftreatment.Forcar- rots, the corresponding values were 77% and 54%.

The differences in population density values between the ^two applications were significant (P=0.05) atallrootdepths in therhizosphere soil (Table 2). At adistance of0-2cmfrom theseed, the differences_were significant between both treatmentsand plant species.

Kortemaa, H. etal.

Effect of

soil-spraying timeonStreptomycesgriseoviridis

Table 1.Root-colonization frequencyof Streptomycesgriseoviridisonroot segments andinrhizosphereof turniprape (Brassica rapasubsp.^oleifera)and carrot (Daucus carota) after two differentsprayingtimes of Mycostop suspension innonsterile sand.

Root-colonizationfrequency^(%)

Root^a Rhizosphere^b

day^O

Depth^(cm) day^O day 7^d day 7

Brassica

0-2 100(30)' 100(30)

47⁽³⁰⁾ 10(27) 18(19) 0(15)

100(30) 100(29) 100(24) 83(19) 85(14)

100(30)

2-4 ^100(29) 47⁽³⁰⁾

4-6 100(24) 10(27)

6-8 83(19) 23(19)

8-10 85(14) 0(15)

Daucus

0-2 97(29) 51(28)

4(25) 0(24) 0(11) 0 (8)

97(29) 83(29) 82(25) 64(17) 22(10)

61(28)

2-4 83(29) 7(25)

4-6 82(25) 0(24)

6-8 65(17) 0(11)

8-1 ²³⁽¹⁰⁾ o ⁽⁸⁾

aS.griseoviridisisolated from root segments.

bS.griseoviridisisolated from root segments and/or fromrhizosphere soil.

cSuspension sprayedonday 0aftersowing.

dSuspension sprayedonday 7 aftersowing.

•Number ofsamplesstudied (n).

The rhizosphere ofturnip rapewaseffectively colonized by S. griseoviridis in sterile sand. The results for the dilution series on GYM agar showed that the sand remained uncontaminated during the experiments. The root-colonization frequencies were 100%. Population densities

were high, and therewere nosignificant differ- ences betweentreatments(Table 3). The stems and leaves of turnip rape seedlings were colo- nized66% and 75% after the 0 day and the 7- daytreatments.

Table2. Population densities (cfu values) ofStreptomyces griseoviridis in rhizosphereofturniprape (Brassica rapasubsp.^oleifera)and carrot(Daucus carota) after two differentsprayingtimes ofMycostop suspen- sioninnonsterile sand.

Population density (cfu) 10²g 1^{of soil}

Depth^(cm)

0-2 2-4 4-6 6-8

Treatment 8-10

Brassica day 0 day 7

11000*

3 100^b

2800* 5 300“

1 100^h

I OOO1 3^b

350 nd 5l0^b

Daucus day 0 day 7

530 ^< 1

< 1

270*

nd

<1 ^< 1

nd nd

2201

Means withinacolumn followedbythe sameletterwerenotsignificantlydifferentaccordingtoTukey’s Studentized Range (HSD) Test (P=0.05). nd: not detected

Table3. Populationdensities (cfu values) ofStreptomycesgriseoviridisinrhizosphereofturniprape (Brassica rapasubsp. oleifera)after twosprayingtimes of sporesuspensiononsterile sand.

Population density(cfu) 10²g1^{of soil}

Depth^(cm)

Treatment 0-2 2-4 4-6 6-8 8-10

day 0 39 000 23 000 5 600 7 700 920

day 7 79 000 42 000 13 000 4 000 1900

Meanswerenotsignificantlydifferentaccordingto Tukey’sStudentized Range (HSD)Test (P=0.05).

Dispersal ⁱⁿ nonsterile sand

without plants

S. griseoviridis dispersed well in root-free sand when5 ml of Mycostop suspensionwassprayed onthe surface of the sand. The isolation frequen- cies of S. griseoviridis had already reached al- most 100%atadepth of 0-6 cm from the top7 days after inoculation (Fig. 1), and continuedto accumulate over the next 2 weeks (14 and 21 days aftertreatment).

The population densities of S. griseoviridis isolated from different depths in the sand tube werehighestatthetopand decreased with depth (Fig. 2). The population of S. griseoviridis was stable during the experiment; differences in pop-

ulation densities between sampling dayswere^not significant (P=0.05) for any depthcategory.

Discussion

Root colonizationwas more effective when S.

griseoviridis wasappliedto nonsterile sand im- mediately after sowing than when itwasapplied 7 dayslater,probably because during those days the rhizosphere was colonized by other soil mi- crobes with which S. griseoviridis was unable

to compete. The results of sterile-sand experi- ments, in which the rhizosphere was 100% col-

Fig. I.Accumulation of isolation frequenciesofSlreptomycesgri- seoviridis at different soil depths during 28 days aftersprayingof Mycostop suspensiononroot-free sand.

Kortemaa, H. etal.

Effect of

soil-spraying timeonStreptomycesgriseoviridis

onizedby S. griseoviridis after both treatment times, supportthis contention. The higher popu- lation densities in sterile than in nonsterile sand areprobably due tothe higher inoculation den- sities and lack of competition with other mi- crobes in sterile sand. The number of micro-or- ganisms in nonsterile sand was probably rather low, and the absence of many of the microbes, both plant-growth-promoting and deleterious strains,tobe found in soil with abundant organ- ic material most likely affected competition.

Scheretal. (1984) noted that microbial compe- tition negatively affects the root-colonization capacity of fluorescent pseudomonads in non- sterile soil.

Isolation frequencies and population densi- ties were ^greaterfor turnip rape than forcarrot because theroot exudates of turnip rape were probablymoreabundantor more available than those ofcarrot. The sameresults were obtained inourprevious study(Kortemaa atel. 1994)us-

ing the plate test. Sterile plants of turnip rape and^carrotinoculated with S. griseoviridis were grown on water-agar plates. The difference in rootcolonization between thetwo plant species wasclear. Without the effect of othermicrobes, the difference between plant species was sug- gestedtobe of plant origin, i.e. dueto root exu- datesormorphological differences.

In both plant species the upper part of the rootwas morefrequently colonized by S. griseo- viridis than the lower parts. The population densities in the rhizosphere of turnip rape were higher than in the root-free sand experiment but population densities were lowest in the carrot rhizosphere. The results are similarto those of ourpreviousstudies,whichweredone using oth- erapplication methods(Kortemaa etal. 1994,

1997).All these differences_were probably due

to root exudates(Curl and Truelove 1986) The soil-sprayingtreatmentmethod used here resulted in better dispersal in sand than did seed

Fig. 2.^Populationdensities (cfu values) of Streplomyces griseoviridis at different^soildepths during28daysafter spraying ofMycostop suspensiononroot-free sand. Differencesinpopulationdensities betweensampling dayswerenotsignificant (P=0.05) for any depth category.

treatment (Kortemaa etal. 1994).Root-coloni- zation was best when the S. griseoviridis sus- pension wasmixed into the sand before sowing (Kortemaa etal. 1997).After seedtreatment,the antagonistmustactively colonize the rhizosphere with the aid of therootand seed exudates. After the soil-spraying treatment the antagonist dis- persed wellasshown by the experiment without plants, the antagonist being available all around theroot. Seven days after seed sowing, the rhizo- sphere was mainly colonized by other soil mi- crobes and S. griseoviridiswasnotabletocolo- nize the rhizosphere effectively.

Our resultssuggestthat S. griseoviridis can compete with indigenous soil microbes in the rhizosphere if it is well established in the sand before the seed emerges. If appliedtothe rhizo- sphere later, however,it competesrather poorly with other microbes. AccordingtoLacey (1973), Streptomyces spp. colonizenewsubstratesmore slowly than do other bacteria and fungi. Our find- ingssuggestthat competition is probablynotthe main mode of action of antagonistic S. griseo- viridis.

S. griseoviridis was isolated in great num- bers in root-free sand with very little organic material. The antagonist survived well in root- free sand after the suspension had been mixed into the sand(Kortemaa etal. 1997). Hatzinger and Alexander (1994) showed that the bacteria which survived well in nonsterile soil were present in the highest population densities in the rhizosphere. Bahme and Schroth (1987) noted that the bacteria which survived in nonrhizo- sphere soil have good potential _as biocontrol

agentsbecause the bacteriacanawait the emerg- ing seed in the soil. AccordingtoWellington et al. (1990), populations of S. lividans and S. vio- laceolatus remainconstant ordecline in natural soil, and after a short mycelial growth phase, sporulation_occursand inoculants survive in the soil as spores.

Mohammadi and Lahdenperä ⁽¹⁹⁹⁴⁾ found that seed dressing controls Rhizoctonia solani Kiihnoncauliflower_moreeffectively than does soil sprayingorthe mixing of Mycostop suspen- sion into the growth substrate. On the otherhand, soil-sprayingtreatmentresulted in bettercucum- ber seed emergence andabetter gerbera flower yield than did other methods. According ^toEl- Abyad etal. (1993) seed-coatingtreatmentwith antagonistic Streptomyces spp.was a more ef- fective way of controlling varioustomatopath- ogens than was soil inoculation. These results together with those of thepresent study support

the idea that both timing and application meth- od mustbe right if effective biocontrol is tobe achieved with S. griseoviridis.

The dispersal of S. griseoviridis after soil- spraying treatment was effective in both the rhizosphere and root-free sand. The root-colo- nization ability of the antagonist dependedon the application time. S. griseoviridis could not compete effectively with indigenous soil mi- crobes, and the rhizosphere waseffectively col- onized only if the sandwastreated immediately after sowing.

Acknowledgements.We thank Tuula Laine andLahjaPe- sonenfor their technical assistance.

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SELOSTUS

Kasvualustan käsittelyhän vaikutus Streptomyces griseoviridis -antagonistin juurten asutuskykyyn

HannaKortemaa, ^KieloHaahtelajaAinoSmolander

Helsingin yliopisto jaMetsäntutkimuslaitos

Mikrobit,jotkaasuttavattehokkaastijuuria,ovatlu- paavia biotorjuntaeliöitä käytettäväksi maalevintäis- ten kasvitautien torjuntaan. Antagonistisen Strepto- mycesgriseoviridis -sädebakteerinkykyäasuttaa ryp- sinja porkkanan juuriatestattiinhiekkaputkimenetel- mällä. Käsittelemätön hiekkakasteltiin mikrobiliuok- sella joko välittömästi tai seitsemän vuorokauden kuluttua siemenen kylvöstä. Tuloksetosoittivat, että S. griseoviridis asuttijuurivyöhykkeen tehokkaasti, jos mikrobikäsittely tehtiin välittömästi kylvön jäl- keen, muttaselvästi heikommin,kunkäsittely tehtiin viikon kuluttuakylvöstä. Mikrobitiheydetolivat ryp- sin juuressa suuremmatkuin porkkanan juuressa.

Antagonisti eristettiin juuren yläosasta useammin

kuin alemmista osista. Steriilissä hiekassa S. griseo- viridis asutti rypsin juuret tehokkaasti molempien käsittelyaikojen jälkeen. Nämä tulokset osoittavat, ettäS. griseoviridispystyykilpailemaan maassaluon- taisestiesiintyvienmikrobienkanssa, jos antagonis- tia onkasvualustassa runsaasti ennen siemenenitä-

mistä. Josantagonisti lisätään maahan myöhemmin, sepystyy suhteellisen huonosti kilpailemaan muiden mikrobien kanssa. Hyvän biotorjuntatuloksen ^saavut- taminenkäytännönkasvintuotannossaedellyttää oi- keankäsittelymenetelmän ja -ajantuntemista. S.gri- seoviridis levisija säilyielävänä hyvin steriloimat- tomassahiekassa, jossaei kasvanutkasveja.