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Mechanisms involved in biocontrol by microbialinoculants

C Dunne, I Delany, A Fenton, F O’Gara

To cite this version:C Dunne, I Delany, A Fenton, F O’Gara. Mechanisms involved in biocontrol by microbial inoculants.Agronomie, EDP Sciences, 1996, 16 (10), pp.721-729. �hal-00885771�

agronomie: plant genetics and breeding

Mechanisms involved in biocontrol

by microbial inoculants

C Dunne, I Delany, A Fenton, F O’Gara

Department of Microbiology, University College Cork, Cork, Ireland

(Received 30 July 1996; accepted 23 September 1996)

Summary — Biological control offers alternative environmentally friendly strategies for the control of phytopathogensin agriculture and horticulture. Biocontrol metabolites are designed so that they do not have any adverse effects onhost plants or on indigenous microflora and, in addition, resistance to these metabolites does not appear to develop. Aspromising alternatives to chemical pesticides, some biocontrol agents have been found to produce a variety of antifun-gal secondary metabolites and lytic enzymes. The 2,4-diacetylphloroglucinol is a secondary metabolite produced byPseudomonas fluorescens F113, a strain capable of protecting sugar beet against the causal agent of ’damping off’,Pythium ultimum; environmental and genetic factors involved in 2,4-diacetylphloroglucinol production are discussed.Stenotrophomonas maltophilia strain W81 (P) produces chitinase and protease enzymes and is capable of conferringplant protection against the disease-causing activity of Pythium ultimum in vitro; transposon mutagenesis and subse-quent in vivo assays have demonstrated that the biocontrol ability of W81 (P) is mediated by lytic enzyme production.

Pseudomonas fluorescens / 2,4-diacetylphloroglucinol / Stenotrophomonas maltophilia / hydrolytic enzymes /Pythium ultimum

Résumé — Mécanismes impliqués dans le biocontrôle par des inoculants microbiens. Le contrôle biologiqueoffre une stratégie alternative et respectueuse de l’environnement pour le contrôle d’agents phytopathogènes. Lesmétabolites de biocontrôle sont conçus de façon à ne pas avoir des effets négatifs sur les plantes hôtes et sur lamicroflore indigène ; de plus, ces métabolites ne semblent pas développer des phénomènes de résistance à leuraction. En tant qu’alternative encourageante aux pesticides chimiques, des agents de biocontrôle peuvent produire unevariété importante de métabolites secondaires et d’enzymes lytiques. Le 2,4 diacéthylfluoroglucinol est un métabolitesecondaire produit par Pseudomonas fluorescens F113, une souche capable de protéger la betterave à sucre vis-à-visde Pythium ultimum, agent de fonte de semis ; les facteurs environnementaux et génétiques conditionnant la produc-tion du 2,4 diacéthylfluoroglucinol sont discutés. Stenotrophomonas maltophilia strain W81 (P) produit des chitinases etdes protéases capables de conférer une protection vis-à-vis des maladies induites par le Pythium ultimum. Aprèsmutagenèse par transposon et des essais in vivo, il a été démontré que l’activité de biocontrôle de Stenotrophomonasmaltophilia se fait effectivement via la production d’enzymes lytiques.

Pseudomonas fluorescens / 2,4-diacétylphloroglucinol / Stenotrophomonas maltophilia / enzymes hydroly-tiques / Pythium ultimum

*

Correspondence and reprints

INTRODUCTION

The fact that consumers of agricultural produceare now demanding foods which are free fromchemical contamination, in particular pesticideresidues, has been recognized both by food pro-ducers and legislators. As a direct result, theagrifood industry is now facing directives limitingthe use of and, in some cases, banning certainagrichemicals. Therefore, in order to maintain

current food production levels, alternatives mustbe exploited for the safe control of crop

pathogens and pests. Presently, the control ofsoil-borne diseases of food crops is mediated

through the application of fungicides and soilfumigants such as methyl bromide (MeBr).However, fungicides traditionally used for thecontrol of plant pests, for example metalaxyl andbenomyl, are now widely perceived as environ-mentally detrimental. MeBr, used as a soil fumi-gation agent, is a significant contributor tostratospheric ozone depletion as greater than60% of the applied pesticide may be releasedfrom the plant bed and enter the atmosphere(Noling and Becker, 1994). These chemical pesti-cides may, in some cases, have also lost their

antifungal effectiveness due to the emergence ofresistant pathogenic strains. Furthermore, theapplication of chemical fungicides and fumigationof soil may be lethal to many soil insects and to

key microorganisms (eg, mycorrhizal fungi) thatunderpin biological mechanisms that ensure soilfertility. The resulting agricultural produce mayoften contain higher levels of pesticide residuethan the legal limit set by several countries. In anattempt to overcome these problems the EUCommission has limited the production and useof MeBr to 1991 levels, with an aim to furtherdecreases within a few years. An international

treaty (USEPA 1993) further restricts MeBr use.

Biological control offers significant advantagesover the use of such chemical treatments.Biocontrol strategies, based on the negativeinteractions among microbial communities andbetween microbes and higher organisms(Weller, 1988; Atlas et al, 1993), tend to be highlyselective. These strategies do not have anyadverse effects on the host plant or on theindigenous beneficial microbial population and, inaddition, resistance to biopesticides does notappear to develop.

Biocontrol strategy

Suppressive soils in which the development ofsoil-borne plant diseases are impeded are welldocumented (Weller, 1988). Such soils may pro-vide a rich and valuable reserve of plant-benefi-cial microorganisms. These natural examples ofbiological control are often due to the presence ofhealth-promoting rhizosphere microflora. In par-ticular, it is recognized that some pseudomon-ads, especially strains of fluorescent

Pseudomonas, play a positive role in pest inhibi-tion in established suppressive soils. Thisobserved protection of plants against attack byfungal and other pests is often due to the produc-tion of a variety of secondary metabolites(reviewed in O’Sullivan and O’Gara, 1992). Plantprotection against pathogen attack conferred byapplication of strains of fluorescentPseudomonas has been shown to be mediated

by compounds such as phenazines, pyrrol-typeantibiotics and 2,4-diacetylphloroglucinol (Phl)produced by these microbes (O’Sullivan andO’Gara, 1992; Thomashow and Weller, 1992;Dowling and O’Gara, 1994; Voisard et al, 1994;Cronin et al, unpublished results). In addition,niche exclusion, competition for nutrients, pro-duction of iron scavenging siderophores, cyanideand lytic enzymes have all been implicateddirectly in plant protection by pseudomonad iso-lates (O’Sullivan and O’Gara, 1988, 1992; Loritoet al, 1994; Kobayashi et al, 1995; Cronin et al,unpublished results; Dunne et al, unpublishedresults). A more direct mode of action involvesthe stimulation of the plant’s own defence mech-anisms through the induction of systemicacquired resistance (Smith et al, 1991; Van Peeret al, 1991; Leeman et al, 1995; Pieterse et al,1996).

In an effort to improve or create novel biocon-trol agents as an effective alternative to chemicaltreatments, extensive studies have resulted in

the isolation and identification of a number of the

secondary metabolites involved in plant protec-tion. Many effective biocontrol strains producehydrogen cyanide (HCN) and Phl which is largelyresponsible for the protection of sugar beetagainst ’damping off’ caused by the fungusPythium ultimum (reviewed in Dowling andO’Gara, 1994; Shanahan et al, 1992). The role ofPhl in the protection of agronomically importantcrops has been demonstrated against a wide

variety of fungal pathogens. However, the controlof certain diseases may require the production ofmore than one antifungal metabolite. Forinstance, the control of black root rot of tobacco,caused by Thielaviopsis basicola, requires theproduction of both Phl and HCN by thePseudomonas fluorescens strain CHAO (Keel etal, 1992).

Alternatively, the inhibition of phytopathogenicfungi in soil by lytic enzyme producing bacteriahas previously been proven as a relatively suc-cessful biocontrol strategy. Chitinases and glu-canases have a proven ability to degrade thechitin and glucan matrix integral to the structureof many fungal cell walls (Lorito et al, 1994).Successful cloning of the biosynthetic genesresponsible for the production of these enzymeshas resulted in the development of transgenicmicrobes and plants with improved abilities tocombat fungal disease due to the production andexport of microbial enzymes.

The role of 2,4-diacetylphloroglucinol (Phl)in biocontrol

While Phl is seen to have a broad inhibitory spec-trum and may effectively inhibit such phytopatho-genic fungi as Pythium, Gaeumannomyces andThielaviopsis (Keel et al, 1990, 1992; Haas et al,1991; Vincent et al, 1991; Fenton et al, 1992),the exact mechanism of action of this compoundhas yet to be fully elucidated.

P fluorescens strain F113 is a soil isolate that

originated from the rhizosphere of sugar beetplants grown in a suppressive soil capable ofconferring protection against the causal agent of’damping off’, P ultimum, both in the laboratoryand in vivo. This strain produces a number ofsecondary metabolites including protease, HCNand Phl (Shanahan et al, 1992). Current studiesinvolving F113 are concentrating largely on regu-lation and synthesis on Phl through biochemicaland genetic techniques.

Factors affecting 2,4-diacetylphloroglucinolproduction

A high performance liquid chromatography(HPLC) assay has been developed for the deter-mination of Phl and its monoacetylated precur-sor, monoacetylphloroglucinol (MAPG), in growthculture media (Shanahan et al, 1993). This assayis used to monitor production of the two sec-ondary metabolites by the Pseudomonas strainF113 in both qualitative and quantitative fashion.This facilitates the investigation of physiologicalas well as regulatory parameters influencing theirproduction.

Microorganisms often display a preference forspecific carbon sources for the production of par-ticular secondary metabolites. It has previouslybeen reported that carbon and nitrogen sourcescan influence such metabolite production (Fenget al, 1994). P fluorescens strain HV37a pro-

duces three distinct antifungal metabolites, andthe biosynthesis of these was seen to be differ-entially regulated by glucose (Douglas andGutterson, 1986). The production of Phl andMAPG by F113 is greatly dependent on the car-bon and nitrogen source on which it is grown.This effect is most apparent when the strain is

grown with either sucrose or succinate as thesole carbon source (fig 1). This holds major impli-cations for effective biological control as antifun-gal secondary metabolite production will bedependent on the components of seed and rootexudates. A knowledge of the nutrient compo-nents of these plant exudates will help predict theproduction of Phl in situ.Phl production in F113 is also influenced by

varying growth conditions. We have previouslyreported that the optimum temperature for Phlproduction is 12 °C and that F113 only producesPhl in small quantities unless the ratio of surfacearea to volume was increased. This is an indica-

tion that the physical parameters of soil could beconducive to maximum metabolite production(Shanahan et al, 1992).The production of various secondary metabo-

lites, including siderophore, HCN and protease,has been seen to be affected by the iron status ofthe cell (Adams et al, 1994). The concentration ofavailable Fe3+ ions enhances Phl production in

F113. In a plate inhibition assay, in which

overnight cultures of F113 were grown on SAmedia amended with increasing concentrationsof ferric chloride, increased Phl production result-ed in increased inhibition of the Bacillus test cul-ture (Dowling et al, 1996) (fig 2).

Genetic factors in 2,4-diacetylphloroglucinolsynthesis

Mutants of P fluorescens strain F113 defective inPhl production have been isolated and character-ized. These mutants were shown to be incapableof inhibiting Pythium using an in vitro plate bioas-say and through HPLC analysis demonstrated asunable to produce Phl or MAPG. Further pheno-typic characterization allowed the mutants to beclassed as either biosynthetic or regulatorymutants. G22 has been previously reported as amember of the Phl biosynthetic class of mutantsof F113 and is a near isogenic mutant defectiveonly in Phl synthesis (Fenton et al, 1992). Thesecond class of mutants belong to the regulatoryclass and exhibit pleiotropic phenotypes in thatthey are unable to produce Phl, protease orHCN.

Through complementation analysis three dis-tinct loci were implicated as necessary for Phlproduction in F113. These loci include a biosyn-thetic locus consisting of 6kb of DNA, and tworegulatory loci which correspond to the gacA andlemA two-component sensor/regulator system(Hrabak and Willis, 1992; Laville et al, 1992). In

addition, a third distinct regulatory region hasbeen identified which can complement either ofthe regulatory mutations. Further detailed analy-sis of other regulatory components has alsoimplicated two sigma factors, sigma 38 andsigma 70, either directly or indirectly and a loci-linked regulator in the regulation of Phl produc-tion (Sarniguet et al, 1994; Cook et al, 1995;Schnider et al, 1995). A complete structuredmodel for the complex regulation of Phl produc-tion in P fluorescens F113 has yet to be fully elu-cidated.

Genes involved in the biosynthesis of Phl havebeen cloned from different strains (Fenton et al,1992; Bangera et al, 1996). Sequence analysishas resulted in the identification of a number of

putative genes involved in the biosynthetic path-way (Cook et al, 1995; Delany et al, unpublisheddata). These genes show similarity to chalconeand stilbene sythases of plants; sterol carrier pro-teins of eukaryotes; drug and sugar transportersand transcriptional repressors (Cook et al, 1995;Delany et al, unpublished). To date, at least sixputative open reading frames have been implicat-ed in Phl biosynthesis. However, the precise roleof each in the biosynthetic pathway remainsunclear.

Biochemical approaches to studyingthe biosynthesis of 2,4-diacetylphloroglucinol

A putative pathway for the synthesis of MAPGhas been documented by Mann (1987). MAPG isalso produced by the P fluorescens strain F113and has been implicated as a Phl precursor. Theactivity of a transacetylase enzyme, responsiblefor the conversion of MAPG to Phl, is proposedas the final step in the biosynthetic pathway. In abiochemical assay investigating the conversion ofMAPG to Phl, the MAPG acetyltransferase activi-ty of whole cell extracts of F113 can be demon-strated and quantified. On boiling the cell extractsthis activity was lost, an indication that this activi-ty is enzymatic (Shanahan et al, 1993). G22, thebiosynthetic mutant of F113, has no such activity.However, on introduction of the pCU203 biosyn-thetic clone, MAPG acetyltransferase activity is

restored. This demonstrates that the MAPG

acetyltransferase activity is encoded on the

pCU203 biosynthetic clone. The precise generesponsible for this activity has yet to be identi-fied in F113. However, the combination of bio-chemical and genetic approaches used in our

study permits the assignation of enzymatic activi-ty to specific genetic loci (Dowling et al, 1996).

Ecological implications of2,4-diacetylphloroglucinol in biocontrol

The cloning and characterization of genesinvolved in the production of a biocontrol metabo-lite offers possibilities for the development ofimproved biocontrol agents. We have previouslyshown that the introduction of the Phl biosynthet-ic genes directly confers biocontrol ability on Pfluorescens strain M114 (Fenton et al, 1992).

However, the large-scale use of soil inoculantsas biopesticides poses important ecologicalquestions. The direct inhibition or antagonism oftarget pathogens is beneficial but the disruptionof indigenous microbiota in the rhizosphere couldresult in long-term deleterious effects. In an

experiment monitoring the persistence of F113and G22 following ten resowings over a period of270 days, there was no significant deleteriouseffect to G22 under the experimental conditions(Carroll et al, 1995). In addition, this experimenthas demonstrated that there was no lasting per-turbation of the resident bacterial microflora,although the biocontrol ability of the appliedstrain is dependent on its establishment in the

rhizosphere.

In order to monitor the fate of genetically modi-fied microorganisms following release into theenvironment, a chromosomally integratablelacZY cassette was developed in our laboratory(Fedi et al, 1996). This facilitates the marking ofpotential biocontrol pseudomonads without theuse of antibiotic resistance markers that are gen-

erally perceived as undesirable in organismsdesigned for release in large quantities. In micro-cosm experiments, a lacZY marked strain ofF113 was unaltered with respect to ecological fit-ness and enabled monitoring of its in situ perfor-mance. Interestingly, lateral transfer of the lacZYmarker, although detected in vitro, could not bedetected in rhizosphere microcosms.

Crop protection using lytic enzyme producinginoculants

The isolation of potential biocontrol bacterialstrains from the environment in which they wouldbe required to function may help to ensure thatthe selected strains are suited for delivery asbiopesticide inoculants into that environment(Weller, 1988; Cook, 1993). Many successfulstudies have focused primarily on the isolationand identification of members of the fluorescent

pseudomonad population present in the rhizos-phere due to their abilities to confer plant protec-tion through production of a range of secondarymetabolites (Fenton et al, 1992; Shanahan et al,1992; Dowling and O’Gara 1994). Therefore, ascreening strategy was initiated with the objectiveof isolating plant-beneficial microbes from the rhi-zosphere of crop plants grown in a fungal dis-ease suppressive soil.

Of the isolated bacterial strains,Stenotrophomonas maltophilia strain W81 (P)proved most effective against the causal agent of’damping off’ in sugar beet, P ultimum, when

assayed under in vitro and in soil microcosm con-ditions (fig 3). Frequently isolated from pollutedenvironments, strains of S maltophilia have beenrelatively well studied with regard to their molecu-lar microbial ecology and the exploitation of theirpotential for bioremediation (Blake et al, 1993).However, S maltophilia strains have also beenisolated from the rhizosphere of a range of cropplants (Milus and Rothrock, 1993; Kobayashi etal, 1995), and these almost exclusively root-associated strains (Mclnroy and Kloepper, 1994)have been evaluated as potential biopesticides(Kobayashi et al, 1995).

While capable of fungal inhibition, S maltophil-ia W81 (P) is unable to produce secondarymetabolites such as HCN and Phl frequentlyimplicated in plant protection against pathogensby pseudomonad species. However, using sub-strate-containing solid media W81 (P) was seento produce the extracellular lytic enzymes chiti-nase and protease (Dunne et al, unpublishedresults).

Protection of plants against multiple pests

W81 (P) produces the enzymes chitinase andprotease, both of which have been demonstratedas being directly involved in the biocontrol of

some pathogenic fungi (Mitchell and Hurwitz,1965; Haran et al, 1996). Further studies com-pleted by a number of groups have demonstratedantifungal plant protection mediated throughmulti-enzyme activities, including chitinase andprotease production, by biocontrol agents(Mitchell and Hurwitz, 1965; Oppenheim andChet, 1992; Kobayashi et al, 1995). Extensiveresearch has elucidated the involvement of the

mycolytic properties of microbial chitinases andother enzymes so completely that genes encod-ing for bacterial enzyme production have beenutilized in the development of transgenic plantswith enhanced pest resistance (Oppenheim andChet, 1992).

Other potential targets for biological controlare plant parasitic nematodes. Cyst nematodesare major agricultural pests causing yield lossesthat vary according to soil composition andpathogen densities. Traditional control protocolshave involved crop rotation and the use of envi-

ronmentally detrimental and often toxic chemicalnematicides. However, the use of many of thesecompounds has been limited by internationalagreement and so biopesticides are emerging aspotential alternatives for crop protection (Sikoraand Hoffman-Hergarten, 1994; Cronin et al,unpublished results).

Globodera rostochiensis is a cyst nematodethat effects potato production and is capable ofcausing economic crop failure due to the stuntingof plants and promotion of early plant senes-cence when present in high numbers. The eggshell of the nematode, G rostochiensis, containsa chitin and protein matrix. Previous studies haveshown that commercially available purified micro-bial enzymes, including chitinases and proteas-es, can significantly reduce the hatch of G ros-tochiensis in vitro (Cronin et al, unpublishedresults). The most effective inhibition of nema-tode hatch was observed when the chitin-proteinmatrix of the egg wall was damaged throughincubation of cysts in buffers containing combina-tions of both chitinase and protease enzymes (fig4). A further study has recently demonstratedthat the use of chitinolytic bacterial inoculantsmay provide an effective alternative to chemicaltreatments for the control of potato cyst nema-todes (Cronin et al, unpublished results).

In order to evaluate the effectiveness of

W81 (P) enzyme production in the control of G

rostochiensis, in vitro assays analogous to thepurified enzyme assays were completed. Theseassays involved the incubation of nematode

cysts in the presence of W81(P), followed byperiodic sampling. The preliminary resultsobtained show that W81 (P) treatment of cystsmay provide the basis for an environmentallyfriendly strategy for the control of cyst nematodeswhich is easily integrated into current agriculturalpractices and which is compatible with biocontrolstrategies targeting phytopathogenic fungi.

Elucidation of mechanisms involvedin biocontrol by Stenotrophomonasmaltophilia strain W81(P)

To determine whether lytic enzyme production byS maltophilia strain W81 (P) is directly responsi-

ble for the conferred plant protection observedagainst both nematode and fungal pests, aninsertional mutant, W81A1, deficient in extracel-lular enzyme production was created throughtransposon mutagenesis (Dunne et al, unpub-lished results). A genomic library was construct-ed and enzyme production was restored throughintroduction of a cosmid-borne complementaryregion, pCU800 (Dunne et al, unpublishedresults). Evaluation of extracellular chitinase andprotease production by wild-type W81 (P) and itsmutant derivatives involved assessment on sub-

strate-containing solid media and spectrophoto-metric quantification in colourimetric enzyme

assays (McKellar, 1981).Subsequent evaluation of the biological control

abilities of wild-type S maltophilia W81 (P) and itsmutant derivatives initially involved solid mediabioassays where inhibition of fungal growth is anindication of bacterial antifungal activity. Usingthis bioassay it was observed that W81A1 isunable to inhibit Pythium growth until the comple-menting cosmid-borne clone is introduced

(Dunne et al, unpublished results). In vivo micro-cosm assays clearly demonstrated that both wild-type W81 (P) and the complemented mutant,W81A1pCU800, when applied to sugar beetseeds at an inoculum level of 106 cfu/seed, pro-tect the plant against attack by P ultimum. Theenzyme deficient insertional mutant, W81A1,however, is unable to confer protection on sugarbeet plants which subsequently succumb to theeffects of ’damping off’ (fig 5).

Similar in vitro and in vivo experiments directlyinvestigating the involvement of the lytic enzymes

chitinase and protease in cyst nematode controlby W81 (P) are currently ongoing.

CONCLUSION

Crop protection through the application of biocon-trol agents is compatible with the requirement forthe production of quality agricultural products in

an environmentally friendly way. Extensive stud-ies, such as those described here, have resultedin the elucidation of many factors mediating bio-logical control in pseudomonads. This knowledgehas resulted in the ability to clone biosynthetic orregulatory genes involved in biocontrol and, ulti-

mately, to new strategies for the creation of novelbiocontrol agents. However, alternative biotech-nological strategies have also focused on thedevelopment and evaluation of consortia ormixed microbial inoculants, exhibiting multiplemechanisms of pest control, for enhanced cropprotection. The development of microbial inocu-lants capable of both secondary metabolite andlytic enzyme production, for example, may pro-vide an alternative means of protection for cropsvulnerable to attack by multiple pests.

The ability to deliver viable microbial inocu-lants, or their metabolites, in an active form andat the appropriate time and site of action is

essential for effective biological control.

Therefore, the development of efficient, environ-mentally safe delivery systems, and the evalua-tion of the influences that these inoculants bringto bear on the resident microflora, are importantaspects of biological control strategies. The suc-cessful development and introduction into agri-

cultural practice of reliable biocontrol agents willlead to the reduction of harmful pesticide treat-ment for agricultural produce and a subsequentreduction of chemical pesticide residues in foods.

ACKNOWLEDGMENTS

The authors would like to thank P Higgins for technicalassistance. This work was supported in part by grantsfrom the European Commission (BIO2 - CT92 - 0084,BIO2 - CT93 - 0196, BIO2 - CT93 - 0053, BIO2 - CT94- 3001, BIO4 - CT96 - 0181, BIO4 - CT96 - 0027).

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