FZB24® Bacillus subtilis – mode of action of a microbial agentkilian_2000

Pflanzenschutz-Nachrichten Bayer 1/00, 1 72–93

1 Introduction

The development of biological productsbased on beneficial micro-organisms can extend the range of options formaintaining the health and yield ofcrops. Targeted research into the prin-ciples of biological control of microbialpathogens began in the early twentiethcentury (Cook and Baker 1983).As earlyas 1897 a “bacteriological fertilizer forthe inoculation of cereals” was marketedunder the proprietary name Alinit by„Farbenfabriken vorm. Friedrich Bayer& Co.“ of Elberfeld, Germany, today’sBayer AG. The product was based on aBacillus species now known by the taxo-nomic name Bacillus subtilis. Accordingto contemporary literature sources theuse of Alinit raised yields by up to 40%.In the mid-1990s in the USA, Bacillussubtilis started to be used as a seed dressing, with registrations in more thanseven crops and application to morethan 2 million ha (Backmann et al. 1994).This was the first major commercial success in the use of an antagonist. InGermany, FZB24® Bacillus subtilis hasbeen on the market since 1999 and is usedmainly as a seed dressing for potatoes.Although activity and effects have beenreported for a number of antagonits, theunderlying mechanisms are not fully understood.This deficiency in our knowl-edge often still hinders attempts to opti-

mize the biological activity by employ-ing tailored application strategies.Accordingly, the present article sum-marizes current knowledge about themode of action of FZB24® Bacillus subtilis and the biotic and abiotic environmental factors that influence itsaction.

2 Microbiological activity of soils

Bacteria are the most abundant micro-organisms in the soil, with an average of6 x 108 cells per g of soil and a weight ofapproximately 10,000 kg/ha. Bacterialmass thus accounts for approximately5% of the total organic dry weight ofsoils. The number of bacteria dependsstrongly on the season, the type of soil,the moisture content, the oxygen supplyin the soil, as well as the tillage and fer-tilization of the soil, and also on the penetration of the soil by plant roots andthe depth from which the soil sampleswere taken. The micro-organism popu-lation density and the make-up of thepopulation in terms of species can varyby up to a factor of 50 only as a result of soil tillage or organic fertilization(Scheffer and Schachtschabel 1979;Lynch 1983).Next to the genera Pseudomonas,Arthrobacter, Clostridium, Achromo-bacter, Micrococcus, and Flavobacterium,Bacillus species are the most common

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FZB24® Bacillus subtilis – mode of action of a microbial agentenhancing plant vitality

M. Kilian, U. Steiner, B. Krebs, H. Junge, G. Schmiedeknecht, R. Hain

Pflanzenschutz-Nachrichten Bayer 1/00, 1 73

types of bacteria isolated from soil samples (Darbyshire and Greaves 1973;Hallmann et al. 1998) and can accountfor up to 36% of the bacterial popu-lations. Like the total number of micro-organisms, this amount varies accordingto environmental factors, plantation, typeof fertilization, and the other factorsmentioned above (Lynch 1983, Mahaf-fee and Kloepper 1996; Darbyshire andGreaves 1973).Bacteria having the ability to form anti-fungal metabolites can be isolated easilyfrom soil samples. However, there havebeen only little systematic studies of theabundance of such micro-organisms as apercentage of the total population. Leynset al. (1990) and Lievens et al. (1989)found that about 30% of all bacteria isolated from soils were able to produceantifungal inhibition zones in vitro.About 3% of these isolates were assign-ed taxonomically to the genus Bacillus.

The rhizosphere, which comprises theregion close to the surfaces of roots,and the root surface itself, the rhizo-plane, are colonized more intensively bymicro-organisms than the other regionsof the soil. Rhizobium bacteria, pseudo-monads, and mycorrhiza fungi areamong the best-known colonizers of thisregion. Many micro-organisms from therhizosphere can influence plant growthand plant health positively, and are therefore often referred to as “plant growth promoting rhizobacteria” (Schip-pers 1992). However, their effects mustbe seen as the complex and also cumu-lative result of various interactions be-tween plant, pathogen, antagonists, andenvironmental factors (Schippers 1992).The various effects produced by Bacillussubtilis and the mechanisms proposedfor these effects as well as the inter-actions between them will be discussedin more detail below (Fig. 1).

Fig. 1: Overview of the modes of action of FZB24® Bacillus subtilis and the interaction betweenthe bacillus, the plant, and the pathogen

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3 Competition through colonizationof the rhizosphere and the rhizo-plane by Bacillus subtilis

Micro-organisms in the rhizosphere andthe rhizoplane live on discarded anddead epidermis cells and root hairs andas well from metabolites such as assi-milates and amino acids excreted by theroots. In total, up to 20% of the energygained through assimilation in the leavesof a plant may be lost again via its roots(Martin 1971, Lynch 1983). Especiallythe so-called border cells which are eliminated into the surroundings fromthe periphery of the root above the rootcap are significant for plant – microbeinteractions (Hawes et al. 1998). Undercontrolled conditions, border cells andthe metabolites formed by them ac-counted for 98% of the carbohydrate-rich material released by the plant as anexudate (Griffin et al. 1976). They havechemotactic effects on micro-organismsand stimulate their sporulation and growth.Though Bacillus subtilis is generally characterized as less competitive in therhizosphere than e.g. pseudomonads,colonization of the root by variousstrains of this species has been found.Relatively high population densitieshave been isolated from root surfacesand from the rhizosphere (Curl and Truelove 1986, Leyns et al. 1990; Bergeret al. 1996; Hallmann et al. 1998). As for other bacteria that colonize the rhizosphere, for Bacillus subtilis, colo-nization of the roots and “co-growth”during their further development ap-pears to require the presence of a thinfilm of water on the root surface (Bowen and Rovira 1976, Liddel andParke 1989).

Antagonists for the control of plant diseases are also selected according totheir ability to colonize the rhizosphere(Parke 1991).As a result of the colonization by the applied antagonist, the naturally occurr-ing micro-organisms are faced with acompetition situation, both for spacethat offers favorable possibilities for development, such as attachment sitesor regions into which plant exudatesemerge, and for nutrients and essentialgrowth factors. Roots evidently haveonly a limited capacity to provide a cer-tain population size and a certain speciesof micro-organism (Handelsmann andStabb 1996).

3.1 Colonization of the rhizosphereand the rhizoplane by FZB24® Bacillussubtilis

The ability of FZB24® Bacillus subtilis to colonize roots has been demon-strated in in vitro experiments, in whichtomato seeds that had been treated withFZB24® were cultivated on Gelrite-Murashige and Skoog-medium. The absolutely clear medium also made it possible to observe root growth as well as bacteria development. FZB24®

Bacillus subtilis colonized the root fromthe treated seed and closely followed its growth in the rhizosphere region,so that a 0.4 – 0.8mm thick film of bacteria was formed around the root(Figs. 2, 3).It was also possible to confirm the colonization of the roots with the aid ofscanning electron microscopy. The closeassociation of Bacillus subtilis with pearoots was shown by the fact that the bacteria attached themselves directly tothe rhizodermis (Fig. 4).

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Fig. 2+3: Tomato roots (cv ‘Minibell’) on gelrite medium colonized by FZB24®-Bacillus subtilis.Application of the bacteria was done by seed dressing (Photo: Dr. Thomzik, Bayer AG)

Fig. 4: Scanning electron micrograph of a pea root with adhering FZB24® Bacillus subtilis cells(Photo: Dr. Schmiedeknecht, Humboldt University Berlin)

grain

Bacillus subtilis

radicle

Bacillus subtilisroot tip

Bacillus subtilis

Bacillus subtilis


3.2 Population development of FZB24®

Bacillus subtilis in the soil and on theroot

The highest and most durable coloni-zation rates of Bacillus subtilis in the rhizosphere were attained in artificialsubstrates or if the substrate had beensterilized before the application of thebacteria (Batinic et al. 1998; Krebs et al.1998; Grosch et al. 1996, Zimmer et al.1998a). In all investigations, the numberof FZB24® Bacillus subtilis bacteria as apercentage of the total micro-organismpopulation showed a distinct decrease inthe course of time. The colonization ofthe root and of the rhizosphere was influenced by a number of environmentalfactors, such as plant species, soil type,and application technique. This wasshown by pot experiments with maize invarious substrates (Table 1).

(Zimmer et al. 1998b). The root tips,which are the most physiologically activepart of the root and release the greatestamounts of root exudate, are preferenti-ally colonized by Bacillus subtilis (Fig. 6).The different factors that influence thecolonization of plant roots by micro-organisms may therefore also be respon-sible for the differences in the re-isolationrates of FZB24® Bacillus subtilis, whichare of the order of 1x103 to 1x107 CFU/gof fresh root weight. However, it has notalways been possible so far to demon-strate a clear relation between the inten-sity of colonization and the effects onplant health and plant productivity (Bullet al. 1991, Handelsmann and Stabb1996, Tutzun and Kloepper 1994).Investigations on the harvested crophave also confirmed the decrease in thepopulation of FZB24® Bacillus subtilisa few weeks after application. Potatoes

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Table 1: Population development of FZB24® Bacillus subtilis on maize roots and inthe soil after seed treatment.

Number (10x) of spores and cells/g afterSoil type isolation site

7 days 21 days 40 days

Clayey humusroot 11 3.2 0.1

substrate 0.4 0.4 0.2

Loamy sandroot 16 3.3 1

substrate 0.2 1.7 0.09

The metabolic activity of cells increaseswith rising temperature, and this increaseis often accompanied by a higher multi-plication rate. This leads to a distinctly increased colonization of the root by Bacillus subtilis, as has been shown inmodel experiments with peas (Fig. 5).Clear differences in the colonization ofdifferent parts of the root were found

from three practical field trials in 1998were harvested to determine the num-bers of Bacillus sp. present. At the end ofthe vegetation period, the counts of Bacillus sp. found on the potatoes fromthe plots in which the seed tubers hadbeen treated with FZB24® Bacillus subtiliswere not higher than on those yieldedfrom untreated seed tubers.


Fig. 5: Re-isolation of FZB24® Bacillus subtilis from pea roots as a function of the temperatureand the application technique. Model experiment with peas, sterile quartz sand substrate, 30 days after sowing and treatment. (Seed treatment: Dip treatment of seeds in suspension of1g FZB24WG/l. Soil treatment: Drench treatment with 1,2x107 spores /ml substrate)

Fig. 6: Re-isolation of FZB24® Bacillus subtilis from pea roots as a function of the re-isolationlocation and the application technique. Model experiment with peas, 20°C, sterile quartz sandsubstrate, 30 days after sowing and treatment. (Seed treatment: Dip treatment of seeds in suspension of 1g FZB24WG/l. Soil treatment: Drench treatment with 1,2x107 spores/ml sub-strate)


4 Formation of antibiotic metabolites

Various antibiotics can be produced byBacillus subtilis; of these, bacilysin is regarded as taxonomically relevant forthe group because of the regularity of itsoccurrence (Loeffler et al. 1990). In liquidcultures, FZB24® Bacillus subtilis alsoproduces iturin-like lipopeptides such asthose described by Krebs et al. (1996).The efficacy of purified lipopeptides ofthis type against various phytopathoge-nic fungi is in the range of 5–100µg/ml,which is similar to that of fungicidalagents.The formation of secondary metabolitesby micro-organisms in synthetic culturemedia and the quantity and compositionof these secondary metabolites dependsstrongly on the culture conditions andthe growth phase of the culture (Loeffleret al. 1990; Krebs et al. 1996, Krebs et al.1998; Gupta and Utkede 1987), which isalso true in the case of Bacillus subtilis.For the production of large quantities ofthese metabolites it is therefore neces-sary to optimize the growth conditionsand culture media during the fermenta-tion process.Additionally the productionof such metabolites also depends on thestage of development of the bacteria.The lipopeptides formed by Bacillussubtilis are released into the mediumonly at the time of endogenous sporeformation during the stationary phase ofthe culture (Loeffler et al. 1990).Investigations have been carried out todetermine the significance of the meta-bolites being effective against fungi invitro, especially of the lipopeptides, forthe efficacy of FZB24® Bacillus subtilis.Maize seedlings were planted in sterilequartz sand in small pots and drenchedwith 107 spores per ml of substrate, cor-

responding to the recommended appli-cation rate for horticultural crops.Appli-cations of lipopeptides added directly tothe substrate were used for comparison.Whereas the added lipopeptides couldbe partly re-extracted and quantitativelydetermined, no lipopeptides were detec-ted in the substrates and roots treatedwith FZB24® Bacillus subtilis.The definiteproof of the formation of these antibio-tically active metabolites in non-sterilehumous substrates was impossible be-cause of the complex matrix and the metabolic activity of the accompanyingmicroflora. The assignment of the lipo-peptides found to a distinct organism ishindered by the fact that organisms thatproduce lipopeptides of this type occurvery widely in soil and plant samples under natural conditions, as was shownby Lievens et al. (1989).Moreover, it is probable that due to thecompetition between the micro-organismsin the soil, only very small amounts offree nutrients are present, so that second-ary metabolites of the type in questionare formed only in extremely smallquantities. Where lipopeptides were actually detectable, their concentrationswere less than the minimum inhibitoryconcentration for phytopathogenic soilfungi.Further confirmation that the formationof antifungal metabolites does not con-tribute significantly to the effect emer-ges from a comparison of different iso-lates of Bacillus subtilis. No correlationis found between e.g. the ability to formmetabolites that are effective againstFusarium oxysporum on various mediain vitro and the observed effects on thecourse of the Fusarium wilt disease ingreenhouse experiments with ornamen-tals (Grosch et al. 1999).

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It is therefore difficult to judge whetherthe lipopeptides play any part in the reduction of the incidence and severityof plant diseases achieved by the ap-plication of FZB24® Bacillus subtilis. Incontrast the formation of compoundspossessing antibiotic activity appears tobe a more basic factor for the effectivityof pseudomonads.The importance of theantibiotics, like phenazine, formed bythese micro-organisms, for the suppres-sion of plant diseases has been demon-strated with mutants deficient for phena-zine-production (Pierson and Thomas-how 1992). Moreover, the antibiotic wasdetected on roots in the soil (Mazzola etal. 1992), and a quantitative dose-effectrelationship between antibiotic forma-tion and the disease-suppressing effectof Pseudomonas fluorescens against Pythium sp. has been demonstrated incucumbers (Maurhofer et al. 1992).

5 Plant resistance induced by Bacillus subtilis

All plants have evolved defense mecha-nisms against pathogens. The efficacy ofthese resistance reactions is modified as afunction of the ontogenetic developmentof the plants and the influence of bioticand abiotic environmental factors. Thus,contact with non-pathogenic micro-orga-nisms or limited infections leads to a de-crease in the susceptibility of the plants.Thisincreased resistance due to exogenousfactors with no alteration of the plant genome is known as induced resistance.Induced resistance can be triggered bothby pre-inoculation with non-pathogens,pathogens, symbionts, and saprophytesand by application of so-called abiotic inducers such as salicylic acid or microbialmetabolites (Schönbeck et al. 1993).

The induction of resistance has frequentlybeen described and discussed in the lite-rature as an ability of micro-organisms.Established phytopathological tests forinduced resistance are based on the se-paration in space and/or in time betweenthe application of the inducing agentand the inoculation of the plants. Biotro-phic fungal pathogens such as powd-ery and downy mildews or Phytophthorainfestans are better controlled with resi-stance inducers.It is assumed that the enhanced resist-ance of the plants is due to altered geneexpression. In many cases the inductionof resistance is accompanied by induc-tion of various so-called PR proteins(pathogenesis-related proteins). Someof these are 1,3-�-glucanases and chiti-nases having the ability to lyse fungalcell walls. Other PR proteins are lesswell characterized or exhibit antimicro-bial activities (van Loon and van Strien1999). On the one hand, PR proteins areregarded as markers of induced resist-ance, while on the other, these proteinsthemselves appear to be involved in theincreased resistance of the plants.

5.1 Changes in the gene expression ofthe plants after application of FZB24®

Bacillus subtilis

A test system for the identification of resistance inducers, based on the follow-ing principle, was developed by Hain etal. (1995). Genes in plants are combinedwith promoters that regulate their geneactivity. The genes responsible for de-fense reactions in plants were regulatedalso with promoters, which can be swit-ched on by various “stimuli” (induciblepromoters). Transgenic tobacco plantswith the gene for herbicide resistance (in


this case BASTA® = phosphinothricinN-acetyltransferase = pat) combined withvarious inducible promoters were there-fore cultivated for the test system. Pro-moters that switch on the genes involvedin defense reactions against pathogenswere used for this purpose. The promo-ters used were the following:• prp1 – promoter of the proteins that

accompany pathogenesis develop-ment from potatoes

• chit2a – promoter of a chitinase genefrom peanuts

• Vst1 – promoter of the stilbene synt-hase gene from vines

When the promoter is activated by the treatment of the test plants, the gene

for herbicide resistance is expressed as a consequence. 1–5 days after the ap-plication of an inducing agent, the plants are insensitive to a spray treat-ment of 5 –15l of Basta/ha. Effective resistance inducers lead to plants withno herbicide damage. Resistance induc-ers described in the literature, like Na-salicylate and the commercial prod-uct Oryzemate were detected with thistest system.FZB24® Bacillus subtilis was tested inrepeated experiments in this test screen-ing system. All three promoters were activated by the treatment with FZB24®,though partly with different intensities.The prp1 promoter responded partic-ularly strongly (Fig. 7). Both soil treat-

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Fig. 7: Transgenic tobacco plants with the prp1/PAT gene fusion for identification of resistanceinducers. Plants 120 h after treatment with FZB24® (left) or with blank formulation (right). Testplants were treated with 15l of Basta/ha. Photograph taken 9 days after herbicide treatment.The activation of the prp1 promoter by FZB24® is clearly demonstrated by the reduction ofherbicide damage on the left of the photograph


ment by drenching and leaf treatment by spraying resulted in the activation ofthe promoters. These experiments pro-vide direct evidence of the involvementof resistance-inducing mechanisms inthe biological efficacy of FZB24® Bacillussubtilis.The experiments show that the bacteriaquickly trigger a signal that can besystemically translocated within theplant, so that an altered gene expressionand hence herbicide resistance were also induced in the above-ground parts of theplant. It has not been established whetherthe resistance-inducing metabolites them-selves act as signals, or whether theytrigger the formation of yet unknown systemically translocatable signals. Nosystemic colonization of the plants byFZB24® Bacillus subtilis was found.As well as the induction of PR proteins,the application of plant growth promotingrhizobacteria were also followed by ac-tivation of other defense genes in plants.Podile and Lami (1998) demonstrated asystemic increase in the phenylalanineammonium lyase (PAL) activity in pigeon-pea seedlings after treatment of the seedswith the Bacillus subtilis strain AF1.

5.2 Demonstration of induced resistanceby FZB24® in phytopathological tests

Many plants, such as tomatoes, beans,and tobacco produce pathogenesis-related proteins, which are described asmarkers of induced resistance. Rauscheret al. (1999) were able to show that theapplication of resistance inducers wasfollowed by the formation of PR-1 pro-teins inhibited the differentiation of infection structures of bean rust in theapoplastic space of bean leaves. Theroots of various plants were treated with

FZB24® and infected with fungal patho-gens on the leaves. Five days after theapplication of FZB24® Bacillus subtilisto the roots, tomato plants showed distinctly less attack by Phytophthora infestans and by Botrytis cinerea (Fig. 8).Disease severity by P. infestans was found to be reduced by up to 50% in laboratory tests. The infestation of B. cinerea is generally much more diffi-cult to reduce by induced resistance.Only with a higher concentration of bac-teria a reduction of 20% was achieved.The application of the abiotic resistanceinducer salicylic acid reduced the infec-tion density of P. infestans by 30 %,whereas it was found to be ineffectiveagainst B. cinerea.The use of a reference strain of Bacillussubtilis did not lead to any changes inthe susceptibility of the plants.Further evidence of increased resistanceof the plants came from experimentsthat showed a reduction of 25% of disease severity of powdery mildew onwheat (Fig. 8).A number of metabolites of bacteria areunder consideration as triggers of indu-ced resistance; among others, these in-clude lipopolysaccharides (Newmann etal. 1995), enzymes (Palva et al. 1993),and siderophores (Leeman et al. 1996),and also salicylic acid (Meyer and Höfte1997). The resistance systemically induc-ed in tobacco by extracellular pectinasesand cellulases of Erwinia carotovora isprobably due to the release of cell wallfragments as signals for the activation of defence genes (Palva et al. 1993).Bacillus subtilis forms mainly serine-specific endopeptidases (Kula 1982).From plant cell walls, proteases cleavemainly hydroxyproline-rich glycoproteins(Showalter 1993). These are derived


from the most important cell wall protein extensin, which is present in allhigher plants (Showalter 1993). It is pos-sible that through these interactionswith the cell wall, the proteases releasedby FZB24® Bacillus subtilis also causethe release of fragments that act as sig-nal substances inducing resistance.In contrast to biological control of pathogens, which is based only on com-petition or antibiosis, the protection ofplants by induced resistance can be effective even when the inducing bacterialpopulation has already decreased. Thereason may be that defense mechanisms,once activated, increase the defensivecapacity of the plants against various pathogens for a long time, or that evenlow population densities act continuouslyas signal sources.

6 Promotion of root growth

A larger and healthier root system, suchas has been observed in a number of greenhouse and field experiments withFZB24® Bacillus subtilis, also leads toimproved uptake of water and nutrients.In a greenhouse experiment with kohl-rabi plants, the soil was drenched imme-diately after sowing and again 4 weekslater with 0.2 g FZB24®WG/l water at arate of 2 l/m2. The treatment led to a 5%increase in the dry root weight (Fig. 9).In addition to an improved germinationof the seeds, the yield of the plants at theend of the cultivation time was up to12% higher, depending on the variety.The root development of potato plantswas determined in a field trial in 1998.The potatoes were planted in mid-May

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Fig. 8: Induced resistance by FZB24® Bacillus subtilis against leaf diseases in wheat and tomatoes in laboratory experiments, in comparison with another strain of Bacillus subtilis andthe resistance inducing agent Na-salicylate


and treated during planting with a liquidseed treatment in the recommended do-sage of 10g of FZB24® WG/100 kg of seedpotatoes. With the beginning of tuberformation in early August, the root freshweight of the plants treated with FZB24®

was 6% higher than that of untreatedplants.The yield of the plants after treat-ment with FZB24® in this experimentwas increased at harvest time in Septem-ber by about 8%.Enhanced root formation of infectedplants has been described by Garett(1956) as a disease escape mechanism.Increased root growth enables the plantto grow out of contaminated regions ofthe substrate and to replace infectedroot sections more easily, and at thesame time enables the plant to reachearlier growth stages in which it is lesssusceptible. The intensified root forma-tion after application of FZB24® may

therefore also be a reason for a reduc-tion of plant damage due to infections of Rhizoctonia solani or Fusarium oxy-sporum.

7 Effect on plant growth and yield

Another reason that has been proposedfor the promotion of plant growth bybacteria that colonize the rhizosphere is the production of phytohormones and phytohormonally active metabolites(Kloepper et al. 1991).Dolej (1998) was able to show that thegrowth-promoting effect of culture fil-trates of FZB24® Bacillus subtilis is notdue to lipopeptides having an antibioticaction.This is supported by investigationswith Bacillus subtilis mutants that nolonger had the ability to form antibio-tics, but still led to increased yields frompeanut plants (Backmann et al. 1994).

Fig. 9: Promotion of root growth of kohlrabi (cv ‘Rogli’) by two drench treatments with FZB24®

WG. Left: untreated; Right: plant treated with FZB24®


The phytohormonal activity of the meta-bolites formed by FZB24® Bacillus subtilis in liquid cultures has been demon-strated by biological test methods. Com-plex culture filtrates and fractions derivedfrom them led, like cytokinines, to enhanc-ed growth of radish cotyledons, and likeauxins, increased the elongation of thecells of wheat coleoptiles. These effectsappear to be initiated by mixtures of se-veral proteins, while further separationand purification of the culture filtrates ledto the loss of the effects (Alemayehu1998). According to Tang (1994), a num-ber of Bacillus subtilis isolates have theability to form phytohormones such aszeatin, gibberellic acid, and abscisic acid.A culture filtrate of a Bacillus subtilis isolate that was used as a resistance indu-cer against biotrophic fungal plant patho-gens has also been found to contain thecytokinins zeatin and zeatin riboside. Thesenescence-delaying effect of them couldbe a cause of the reduced damaging effectof pathogens and the increased yield ofplants in which resistance has been in-duced (Steiner 1990).Enhanced root growth is often accom-panied by increased branching and ahigher number of root tips. Their meri-stems are the most important sites for the synthesis of free cytokinins (Torrey 1976). These are presumablytransported into the shoot via the xylem.Intensified and prolonged synthesis ofthese phytohormones may be regardedas a cause of delayed senescence and improved yields (Mengel 1973).Since the application of Bacillus subtilisleads to stronger root growth, there mayalso be an increased synthesis of plantcytokinins, which also cause delayed senescence and higher yields, as describedabove. These effects on the phytohor-

mone balance of the plants can explainwhy increased yields are found even forplants that show no visible attack bysoil-borne or root diseases.An increase in the yield was also achiev-ed by leaf applications of FZB24®. Inthree field trials in 1998 with potatoes, aleaf treatment was carried out in com-bination with the application of leaf fungicides to control P. infestans. Fourapplications of FZB24® with a dosage of0.4% were carried out at intervals of 10 to 14 days beginning in mid-June. Theeffects were compared with a dry seedtreatment with FZB24®. Because of thefungicide treatment, the plants were largely protected against attack by P. infestans. The application of FZB24®

WG to the leaf did not produce any visible improvement in the protectionagainst the leaf pathogen, but led to anincrease in the yield of 8.5%, which washigher than the yield increase achievedwith the dry seed treatment in these experiments (Fig. 10).In addition to the effects on the phyto-hormone balance of the plants, an im-provement of the tolerance of the plantsmay also contribute to increased yields.Tolerance is defined as the ability of theplant to survive attack by pathogens orthe action of abiotic stress factors withsmaller losses of viability and produc-tivity than another plant subjected to thesame exposure intensity (Aust et al.1991). Possible factors that lead to to-lerance of plants towards pathogens (according to Clarke 1986) are:

• reduced sensitivity of the plants to-wards toxic metabolites produced bythe pathogens,

• ability of infected and uninfectedparts of the plants to compensate

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through increased metabolic activity,e.g. photosynthesis,

• delayed senescence,• less influence on sink-and-source

relationships.

Tolerance was described to be influenc-ed e.g. by culture filtrate fractions ofFZB24® Bacillus subtilis having cyto-kinin-like and auxin-like activities.In vitro tests with callus cultures of to-matoes showed increased stress toler-ance towards the Fusarium toxin fusaricacid, with the result that the browningsymptoms were reduced (Alemayehu1998).Culture filtrates of a Bacillus subtilis isol-ate, used as a resistance inducer againstbiotrophic leaf pathogens, induced chan-ges in the sink-and-source relationshipsin barley after attack by powdery mil-dew. This was related both to the trans-port of assimilates from the host to thepathogen and to the translocation of assimilates in the whole plant. It couldbe proved that the application of culture

filtrates led to increased movementof assimilates from the flag leaf into theears, which then also had a higher starchcontent (Kehlenbeck et al. 1994).

8 Conclusions

Numerous mechanisms seem to be in-volved in the effect of FZB24® Bacillussubtilis. These mechanisms can con-tribute in different degrees to the re-duction of disease and the enhance-ment of yields, depending on the plant,the environmental conditions, the appli-cation form, and the time of application.The most obvious effects of the bacteriaon diseases have been found in the caseof attack by root pathogens. This couldbe due to the fact that all of the mecha-nisms mentioned, i.e. competition, anti-biosis, resistance induction, and diseaseescape as a result of growth effects, areable to operate in the root area, whereasonly resistance induction can operate inthe leaf area, since no translocation ofthe bacteria takes place.

Fig. 10: Effect of FZB24® Bacillus subtilis on the yield of potatoes after seed tuber treatment(20g FZB24 DS/100kg) and leave applications (0,4% FZB24 WG)


In contrast with biological methods forcontrolling pathogens, which are basedonly on competition or antibiosis, theprotection of plants by resistance in-duction can be effective even when thebacterial population that triggered theseeffects has already declined. This may be because defense mechanisms, onceactivated, increase the defensive capa-city of the plant against various patho-gens over a long period, or because evenlow population densities function contin-uously as inducing agents.However, the observed biological effects are also due to changes in thephysiology of the plant. In the firstplace, the tolerance towards abiotic andbiotic stress factors is improved becausethe root system of the plant is strength-ened, and hence also the uptake of water and nutrients. In addition, manyresults indicate that the application ofBacillus subtilis changes the phytohor-mone balance in the plant in such amanner that greater quantities of reservesubstances are incorporated into stor-age organs.The large number of mechanisms invol-ved may be one reason why FZB24®

Bacillus subtilis can be used for a widespectrum of crops with their differentculture conditions. However, none of theobserved mechanisms has any curativeeffect, so that early treatment, prefer-ably right from the beginning of culti-vation, is advisable.

9 Summary

The present article summarizes currentknowledge about the mode of action ofFZB24® Bacillus subtilis and the bioticand abiotic environmental factors thatinfluence its action.

Bacteria are present in the soil in an aver-age content of 6x108 cells/g of soil, andwith a live weight of about 10,000kg/ha,they are the most abundant micro-orga-nisms in soil samples. However, the num-bers of bacteria vary by up to a factor of50, depending on biotic and abiotic en-vironmental factors. Bacillus species areamong the most common organisms isolated from soil samples. Within thismicrobiological context FZB24® Bacillussubtilis must temporarily establish itself inthe rhizosphere of the cultivated plant.A number of mechanisms that couldcontribute to increased yields andreduced attack by pathogens followingthe application of Bacillus subtilis havebeen described in the literature. The following mechanisms and effects haveso far been demonstrated experimen-tally for FZB24® Bacillus subtilis:

• Competition by temporary coloni-zation of the rhizosphere and rhizoplane.

• FZB24® has the ability to form anti-biotic metabolites in vitro. However,this depends very strongly on theculture medium. It was not possibleto confirm the formation of thesemetabolites on the root and in thesubstrate in vivo.

• Induced resistance by activation ofdefense genes in plants, which hasbeen demonstrated both by mole-cular-biological methods and byphytopathological tests.

• Promotion of plant and root growth.The formation of substances andmixtures having cytokinin-like andauxin-like effects by B. subtilis hasbeen demonstrated in vitro. Howe-ver, the enlarged and more highlybranched root system of the plant

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also alters the endogenous phyto-hormone balance. The stronger rootsystem ultimately also leads to animproved uptake of water and nutri-ents, and hence to faster growth andgreater dry stress tolerance. Moreo-ver, the growth promotion leads tothe possibility of disease escape,since the plants can grow out of sen-sitive stages more quickly and en-hanced root growth allows a bettercompensation for diseased parts ofthe roots.

Zusammenfassung

FZB24® Bacillus subtilis – Wirkungs-weise eines mikrobiellen Pflanzenstär-kungsmittels

In dieser Arbeit wird der aktuelle Wis-sensstand zur Wirkungsweise von FZB24®

Bacillus subtilis und zu den biotischenund abiotischen Umweltfaktoren, diedie Wirkung beeinflussen, zusammenge-fasst.Bakterien kommen im Boden im Mittelmit 6x108 Zellen/g Boden vor und sindmit einem Lebendgewicht von ca.10.000 kg/ha die häufigsten Kleinlebe-wesen in Bodenproben. Die Bakterien-zahl variiert um den Faktor 50 in Abhän-gigkeit von biotischen und abiotischenUmweltfaktoren. Bacillus-Arten gehörenzu den häufigsten Gattungen, die ausBodenproben isoliert werden. In diesemmikrobiologischen Umfeld muss sichFZB24® Bacillus subtilis temporär in derRhizosphäre der Kulturpflanze etablie-ren.In der Literatur sind eine Reihe mög-licher Wirkmechanismen beschrieben,die zu den immer wieder beobachtetenErtragssteigerungen und Reduktionen

des Befalls mit Pathogenen nach einerBacillus subtilis Anwendung beitragenkönnen. Für FZB24® Bacillus subtiliskonnten bisher die folgenden Mechanis-men und Effekte experimentell demon-striert werden:• Konkurrenz durch vorrüberge-

hende Besiedelung der Rhizosphäreund Rhizoplane durch Bacillus subtilis.

• FZB24® ist in vitro in der Lage, anti-biotische Stoffwechselprodukte zubilden. Dies ist aber stark von derZusammensetzung der Nährmedienabhängig. In vitro konnte eine Bil-dung dieser Stoffwechelprodukte ander Wurzel nicht bestätigt werden.

• Resistenzinduktion durch Aktivie-rung von Abwehrgenen in Pflanzen,was sowohl mit molekularbiologi-schen Methoden wie auch mit phyto-pathologischen Tests nachgewiesenwerden konnte.

• Förderung des Pflanzen- und Wur-zelwachstums. In vitro konnte dieBildung von Substanzen und Sub-stanzgemischen mit cytokinin- bzw.auxinartigen Wirkungen durch B.subtilis festgestellt werden.Aber auchdas vergrößerte und stärker ver-zweigte Wurzelsystem der Pflanzeverändert deren endogene Phytoh-ormonbalance. Das stärkere Wurzel-werk führt letztlich auch zu einerverbesserten Aufnahme von Wasserund Nährstoffen und damit zuschnellerem Wachstum und größererTrockenstresstoleranz. Darüber hin-aus ermöglicht die Wachstumsförde-rung ein disease escape, indem diePflanze schneller empfindlichen Stadien entwachsen kann undkranke Wurzelteile besser kompen-siert werden.


Résumé

FZB24® Bacillus subtilis – Mode d’ac-tion d’un stimulateur microbiologiquede vigueur végétale

Ce travail récapitule les connaissancesactuelles relatives au mode d’action duFZB24® Bacillus subtilis et aux facteursenvironnementaux biotiques et abioti-ques qui influent sur cette action.On trouve des bactéries dans le sol, enmoyenne à raison de 6.108 cellules/g, et,avec un poids vif d’environ 10 000 kg/ha,elles représentent les microorganismesles plus fréquents dans les échantillonsde sol. Mais le nombre de bactéries varied’un facteur de 1 à 50, selon les facteursenvironnementaux biotiques et abio-tiques. Les espèces de Bacillus apparti-ennent aux genres les plus fréquemmentisolés des échantillons de sol. Dans cet environnement microbiologique, leFZB24® Bacillus subtilis doit s’établir àtitre provisoire dans la rhizosphère de laplante cultivée.Dans la bibliographie, de très nombreuxmécanismes d’action potentiels sontdécrits, qui, après une utilisation de Bacillus subtilis, peuvent contribuer auxphénomènes toujours et encore obser-vés d’augmentation de rendement et deréduction de l’attaque par les microor-ganismes pathogènes. A ce jour, on a pu montrer à titre expérimental les mécanismes et effets suivants duFZB24® Bacillus subtilis:• Compétition par colonisation provi-

soire de la rhizosphère et des rhizo-plans par Bacillus subtilis.

• Le FZB24® est, in vitro, à même deformer des métabolites antibio-tiques. Mais cette production dépendbeaucoup des milieux nutritifs.

Aucune formation de ces métaboli-tes n’a pu être confirmée sur les raci-nes.

• Stimulation des défenses naturellesdes végétaux par activation de leursgènes de défense, ce qui a pu êtremis en évidence tant par des métho-des de biologie moléculaire que pardes essais phytopathologiques.

• Promotion de la croissance végétaleet radiculaire. In vitro, on a pu con-stater sous l’effet de B. subtilis la for-mation de substances et de mélangesde substances ayant une action ana-logue à celle de la cytokinine ou del’auxine. Cependant, líaugmentationdu volume et des ramifications dusystème racinaire des plantes modi-fie également leur bilan phytohor-monal endogène. Enfin, l’appareilradiculaire plus développé permetaussi une meilleure absorption del’eau et des substances nutritives, etdonc une croissance plus rapide etune plus grande tolérance au stresshydrique. En outre, la stimulation dela croissance permet une «fuite enavant» («disease escape»), la plantepouvant croître plus vite pour dépas-ser les stades sensibles, en assurantune meilleure compensation desparties racinaires malades.

Resumen

FZB24® Bacillus subtilis – Mecanismode acción de un vigorizante para plantas

En este trabajo se expone el estado actual de conocimientos científicossobre el mecanismo de acción delFZB24® Bacillus subtilis y sobre los factores medioambientales bióticos yabióticos que influyen en dicha acción.

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Las bacterias están presentes en el sueloen una concentración media de 6 x 108

células/g de suelo y son los microorga-nismos vivientes más frecuentes en lasmuestras de suelo, cifrándose su peso enunos 10.000 kg/ha. Las concentracionesbacterianas pueden oscilar incluso en unfactor de 50, en función de los factoresmedioambientales bióticos y abióticos.Las especies Bacillus son las que se aíslan con mayor frecuencia de las mue-stras de suelo. En este contexto micro-biológico, FZB24® Bacillus subtilis debeestablecerse temporariamente en la rizosfera de las plantas cultivadas.En la bibliografía técnica se describenm˙ltiples mecanismos potenciales de ac-ción que, después de una aplicación deBacillus subtilis, pueden contribuír a losincrementos de rendimientos de cos-echas observados repetidamente y a lareducción de la incidencia de patógenos.Hasta ahora se han podido demostrarexperimentalmente los siguientes meca-nismos de acción o efectos de FZB24®

Bacillus subtilis:

• competencia basada en la coloniza-ción transitoria de la rizosfera y rizo-planos con Bacillus subtilis

• FZB24® «in vitro» es capaz de gene-rar productos metabólicos antibióti-cos. Sin embargo, esto depende delos caldos de cultivo. No se ha po-dido confirmar «in vitro» la forma-ción de estos productos metabólicosen la raíz.

• Resistencia inducida por activaciónde genes defensivos en plantas,detectada tanto por métodos de biología molecular como por en-sayos fitopatológicos.

• Estimulación del crecimiento deplanta y raíz. Se ha observado «in

vitro» la formación de sustancias y mezclas de sustancias debida a B. subtilis, cuyos efectos son simila-res a los de la citoquinina y auxina.También el sistema radicular masamplio y ramificado de las plantascambia su equilibrio fitohormonalendógeno. A fin de cuentas, un sistema radicular mas vigoroso con-duce a una mejor absorción de aguay nutrientes del suelo y, por tanto, aun crecimiento más rápido y unamayor tolerancia a condiciones desequía. Por otro lado, la estimulacióndel crecimiento permite a la planta«escapar de las enfermedades», yaque puede superar con mayor rapi-dez los estadios críticos y compensarmejor las partes enfermas de sus raíces.

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Dr. Ulrike SteinerInstitut für PflanzenkrankheitenUniversität BonnNussallee 9, D-53115 Bonn, Germanyphone: 0049 228 733341fax: 0049 228 732442email: [email protected]://www.uni-bonn.de/pflanzen-krankheiten/

Dr. Michael KilianDr. Rüdiger HainBayer AGLandwirtschaftszentrum MonheimGeschäftsbereich Pflanzenschutz/ForschungD-51368 LeverkusenTel.: O2173-383210 (Dr.Kilian)

email: [email protected].: O2173-384382 (Dr.Hain)

Dr. Helmut JungeDr. Birgit KrebsFZB Biotechnik GmbHGlienicker Weg 185, D-12489 BerlinTel.: 030-67 057 0 Fax: 030-67 057 233email: [email protected]

Dr. Gunter SchmiedeknechtSächsische Landesanstalt für LandwirtschaftFB06 Integrierter Pflanzenschutz, Referat 61, Stübelallee 2, D-01307 DresdenTel.: 0351/4408326Fax: 0351/4408325email: [email protected]

Manuscript received: 14.6.00

FZB24® Bacillus subtilis – mode of action of a microbial agentkilian_2000

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