CSIRO PUBLISHING Keynote Paper

www.publish.csiro.au/journals/app Australasian Plant Pathology, 2007, 36, 95–101

Development of a biocontrol agent for plant disease control with specialemphasis on the near commercial fungal antagonist Clonostachys roseastrain IK726

D. F. JensenA,B, I. M. B. KnudsenA, M. LubeckA, M. MamarabadiA, J. HockenhullA

and B. JensenA

ADepartment of Plant Biology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40,DK1871 Frederiksberg C, Copenhagen, Denmark.

BCorresponding author. Email: dfj@life.ku.dk

Abstract. Numerous experiments demonstrating potential biocontrol effects on soilborne diseases have been reportedin the scientific literature. However, from the lists of approved and registered biocontrol agents, it is striking how fewhave been commercialised and are used in practise for plant disease control. The main hindrances are often claimed tobe legislative aspects and the costs involved in the registration. Although this is in many respects true, there is a rangeof both biological and technical problems which must be considered when developing an effective biocontrol agent forcommercial use.

Among the success stories for control of seed- and soilborne diseases are fungal biocontrol agents based onTrichoderma harzianum, Clonostachys rosea and Conithyrium minitans, and bacterial biocontrol agents based on strainsof Agrobacterium, Pseudomonas and Streptomyces. We have developed C. rosea strain ‘IK726’, which has proved to be aneffective antagonist in several crops against seed- and soilborne diseases. Although a biocontrol agent based on C. rosea‘IK726’ is not yet commercialised, this paper will be used to address some of the biological and technical aspects thatmust be dealt with in such a development.

Background

Research on biological control of plant diseases was intensified∼30 years ago when it was realised that the biased useof chemical pesticides could have adverse effects on theenvironment, and that chemical residues could affect quality andsafety of food and feed. Thus, there was a need to develop newbiological disease control measures with less adverse impacts.The change to focus on biological control was, for severalresearch groups, stimulated by important contributions such asthe text books on biological disease control by Baker and Cook(1974) and Cook and Baker (1983) as well as the basic conceptsof ecology of soilborne plant pathogens presented in the booksof S. D. Garrett (Garrett 1970) and others.

Numerous experiments demonstrating potential biocontroleffects on plant diseases are reported in the scientific literature.However, upon going through the lists of registered biocontrolagents (BCAs), it would seem that only a few are commercialisedat present. The main hindrances are often claimed to belegislative aspects and the costs involved in registration.However, there is also a range of other commercial, biologicaland technical problems that must be solved when developingan effective BCA. Examples of successful commercialisation ofBCAs are given in Table 1. A more extensive list can be foundin Fravel (2005). Further information on marketed productscan be found by searching the internet using the trade namesof the BCAs.

The success of BCAs based on Trichoderma spp. can,among others, be attributed to the early scientific contributionof groups lead by R. Baker (e.g. Ahmad and Baker 1987a,1987b), I. Chet (e.g. Chet 1987; Sivan and Chet 1989), Y. Elad(e.g. Elad et al. 1982), C. R. Howell (e.g. Howell and Stipanovic1983), R. Lumsden (e.g. Lumsden et al. 1992), G. E. Harman(e.g. Sivan and Harman 1991) and, later, M. Lorito (e.g. Loritoet al. 1994) and A. Stewart (e.g. McLean et al. 2005). Thebooks by Harman and Kubicek (1998) and Kubicek andHarman (1998) also contain major contributions relevant tothe success of BCAs based on Trichoderma spp. Sutton andcoworkers (e.g. James and Sutton 1996; Sutton et al. 1997)have made a major contribution to the understanding of thebiology of Clonostachys rosea and its use in biocontrol ofgrey mould (Botrotinia cinerea and Botrotinia squamosa).J. Whipps (e.g. Jones and Whipps 2002) is one of the maincontributors to the understanding of the biology of Coniothyriumminitans in controlling sclerotium-forming pathogens. Kerr(e.g. Jones and Kerr 1989) is known for his unique workwith biocontrol of crown gall caused by Agrobacteriumtumefaciens and Tahvonen (1982) undertook most of thebasic research, which lead to the commercial exploitationof strains of the bacterium Streptomyces griseoviridis in theproduct Mycostop. Contributions of groups lead by J. M. Cook,D. Weller and L. Thomashow (e.g. Weller and Cook 1985;Thomashow et al. 1990; Weller et al. 2002), G. Defago

© Australasian Plant Pathology Society 2007 10.1071/AP07009 0815-3191/07/020095

96 Australasian Plant Pathology D. F. Jensen et al.

Table 1. Examples of commercialised biocontrol agents (BCAs) basedon either bacteria or fungi

Fungal BCAs Bacterial BCAs

Root Shield (Trichoderma NoGall (Agrobacterium radiobacterharzianum) K84)

Supresivit (T. harzianum) Mycostop (Streptomyces)BinabT (T. harzianum + Kodiak (Bacillus sp.)

T. polysporum) Cedomon (Pseudomonas chlororaphis)Trichodex (T. harzianum) Spot-Less (Pseudomonas aureofaciens)Tricho-Dry (Trichoderma sp.)GlioMix (Gliocladium

catenulatum)ContansWG (Coniothyrium

minitans)

(e.g. Stutz et al. 1989; Keel et al. 1990) and B. Gerhardson(e.g. Hokeberg et al. 1997) have been important for thedevelopment of BCAs based on pseudomonads. The workof bringing potential antagonists further along the road tocommercial exploitation has often been driven by enthusiastic,dedicated persons from, or in close collaboration with,industry. Examples of such activities crucial for the successof biocontrol are developments of methods of large-scaleproduction, formulation and delivery in addition to registeringand marketing.

We are developing Clonostachys rosea strain ‘IK726’ into aBCA for plant disease control. The strain ‘IK726’ has provedto be an effective antagonist in several crops against diseasescaused by a range of pathogens, e.g. Alternaria spp. (Jensen et al.2004), Bipolaris sorokiniana and Fusarium culmorum (Knudsenet al. 1995), Pythium spp. (Møller et al. 2003) and Tilletia tritici(Jensen et al. 2001). Although a product based on ‘IK726’ isnot yet commercialised, work with this BCA will be used hereto illustrate aspects that must be dealt with in such a development(Table 2). The programme was initiated in 1990, when Nordicresearch collaboration in biocontrol was introduced.

The Nordic project 1990–1993

Research groups from Finland, Norway, Sweden and Denmarktook part in this collaboration. The central idea was that thebest chance for success in biocontrol would be to focus onseedborne and seedling diseases. For controlling such diseases,the BCA could be placed in close proximity to the pathogen andcould be active at the right time for efficient control. The BCAsCedomon and GlioMix, which have now been commercialised inthe European Union (EU), are both based on antagonists foundin the Nordic project and our aim is that C. rosea ‘IK726’ beadded to the list in the near future (Table 1).

Commercial considerations

Market size and how easy market introduction will be,are important commercial considerations. A BCA can becommercially attractive if it is specific for a few diseasesin small but high value crops (i.e. control of crown gall inorchards, disease control in golf courses or disease controlin protected crops) or for specific use in large field crops(i.e. control of damping-off in cotton or seedling diseases inwheat and barley). Other successful BCAs will have a broader

Table 2. Aspects to be considered in developing a BCA

Selection of potential antagonists• Isolation of potential biocontrol agents• Screening for selection of antagonistsBasic biology of the selected organism• Ecology of the BCA and antagonist and pathogen interactionsDevelopment of the product• Production (liquid or solid fermentation)• Formulation• Shelf life• Compatibility with existing technologiesDelivery systems• Seed treatment (seed coating, biopriming etc.)• Incorporation in growth substrates• Application to substrate: drench, broadcast, in furrowRegistration of the product• Risk assessment (EU, EPA etc.)• Field performance (GEP efficacy)Commercial aspects• The economic importance of the disease to be controlled• Cost of development, production, registration and marketing• Market size and market introduction• Education and public acceptance

range of target pathogens and can be used for several diseasesboth in protected crops and in field crops (e.g. some of theTrichoderma-based products). An attractive market size canthus be achieved. Biological control is, in general, being seenas a ‘green technology’ with a high public acceptance, whichmight be good for the reputation of the industry involved andbe an asset for market introduction. Biological control differsin many ways from chemical disease control. Commercially,it would be preferable if BCAs were robust like many chemicalswhen they are exposed to biotic and abiotic factors such asplant-, soil- and environmental factors. However, this is notalways the case. For efficient biocontrol, the organisms mustnormally be alive and active at the sites where they are tocontrol the target pathogens. This requires that many biologicalaspects be considered and some of them will be addressed below.Therefore, there will be a need to train extension service officersand people from industry in such a way that they gain thenecessary biological insight into this new technology and thus,can facilitate the market introduction. Other commercial aspectsare costs for development and registration and also costs relatedto technical aspects such as large-scale production, formulationand delivery.

Biological considerations

Isolation, selection and field performance

Two main approaches were followed in the Nordic program.They were as follows:

(1) Isolation of potential antagonists: a potential BCA must befound in an environment or niche similar to where it is goingto be used as a BCA.

(2) Selection of antagonists among potential isolates: ascreening procedure for the selection must simulate theenvironment where the BCA is going to be used for diseasecontrol.

Development of a biocontrol agent for plant disease Australasian Plant Pathology 97

The successful commercial BCAs listed in Table 1 are,except for a few, based on antagonists, which either have theirorigin in suppressive soils (e.g. Mycostop, Root Shield) or wereisolated from plant roots (Cedomon), wounded or infected planttissue (No Gall) or from soil or pathogen propagules in soil(e.g. Contans). It has not been possible for the authors to verifythe origin of all antagonists in the commercialised BCAs. Mostof the antagonists from successful BCAs, however, were isolatedfollowing the first approach, although we cannot rule out thatpotential antagonists with other origins can be found if the rightselection procedure is being used.

In the Danish part of the Nordic project, we concentratedon fungi. Candidates for screening were isolated from eitherbarley field soil or from the barley rhizosphere using proceduresrevealing both fast growing spore producers (r strategists) andslow growing non-spore producers (K strategists). We usedin planta screening for the selection of BCAs with multifactormechanisms of control and adapted to the environment wherethey were to act. The screening procedures in this ecologicalapproach to selection are discussed in detail in Knudsenet al. (1997). In brief, potential antagonists were screened fortheir biocontrol effect on barley or wheat seeds infested withpathogens and planted in pots with sand. The test organisms wereapplied directly to the infested seed at sowing and a disease indexwas scored following 19 days’ growth in a growth chamber at15◦C. The best candidates were selected and then screened for

(a) (b)

(c) (d )

F. culmorum (106) F. culmorum (106) + C. rosea ‘IK726’

Control

‘IK726’

‘IK726’ Control

Fig. 1. The hierarchic in planta screening procedure used for the selection of ‘IK726’. (a) Fungal isolates were screened for antagonistic potential onbarley infested with F. culmorum and planted in pots with moist sand. (b) The disease index was scored 19 days after sowing. (c) Selected candidatesfrom the sand screening test were tested in small field plots. (d) The best candidates were tested for biocontrol performance in large field experiments.Photographs supplied by Inge M. B. Knudsen.

effect in small field plots. The best of these were then testedfor field performance in large field experiments (Fig. 1). Strain‘IK726’ was selected following this hierarchic procedure as oneof the best candidates among more than 400 fungal isolatesscreened. The first screening step on plants in sand was adaptedfrom that used by the Finnish research group (R. Tahvonen,pers. comm.). In Finland they screened more than 1700 fungalisolates and their best candidates also belonged to the genusClonostachys (Teperi et al. 1998).

Almost all of the commercialised BCAs have also beenselected using an in vivo screening approach and it is ourexperience that in vitro screening methods such as dual cultureson agar must be avoided. With such methods there will be ahigh risk of discarding potential antagonists, which do not showany effect in vitro but might perform well in the field or, effectsshown on agar, such as inhibition zones, might not be relevantin a natural situation. Evidence for this can be found in Knudsenet al. (1997), from work by the Finnish group (Teperi et al.1998) and also from the work by others (Whipps 1987). It isalso evident that mechanisms such as induced resistance andincreased plant growth response will not be selected for usingan in vitro screening approach. As discussed in Knudsen et al.(1997), dual cultures screening might, however, be relevant at alater stage to select the best candidate between isolates belongingto a specific group of organisms which already have been shownto be good candidates in field experiments and, where a specific

98 Australasian Plant Pathology D. F. Jensen et al.

mechanism such as antibiosis has been demonstrated to be animportant biocontrol mechanism for this group. Thus, selectingpotential antagonists in vitro as the first step in a hierarchicscreening procedure is less likely to be successful, although thisapproach is still used by many research groups.

Production, formulation and delivery

‘IK726’ can be produced using both liquid and solid statefermentation. Our experiences are restricted to small-scalefermentation but large-scale test production at a reasonable costof a strain of C. rosea has been carried out by the Germanfirm Prophyta. Thus, the general concern that upscaling is aproblematic step seems not to be the case with C. rosea.

The sand bioassay described above was developed furtherfor testing different formulations of ‘IK726’. Comparing theperformance of ‘IK726’ in the bioassay in sand with itsperformance in the field, showed a high correlation regarding thedisease index, although effects on plant emergence and plant dryweight seen in the field were not reflected in the sand test (Jensenet al. 2000). This was a unique situation to have a short bioassayreflecting the performance of ‘IK726’ under field conditions indifferent soils. Therefore, this has been a very useful tool fordeveloping the BCA further.

A main focus has been to optimise delivery of ‘IK726’withthe seed (Jensen et al. 2000, 2002). Testing dose response onbarley by coating seed with freshly harvested conidia of ‘IK726’showed a high efficacy in the biocontrol of both F. culmorum andB. sorokiniana using dosages above 104 cfu per seed (Jensenet al. 2000). Formulated products of ‘IK726’ either as a clayor as a peat bran formulation have been developed (Jensenet al. 2000; Møller et al. 2003). Solid fermentation resultedin conidia with better survival than those produced in liquidfermentation (Jensen et al. 2002). This result is in agreementwith results from solid fermentation of Trichoderma harzianum,which also showed that these conidia survive better at roomtemperature compared with conidia from liquid fermentation(Munoz et al. 1995). The effects of production length and storageconditions of ‘IK726’ on shelf life were also studied. Thus, thebest shelf life of the ‘IK726’ product was obtained by usingsolid fermentation production over 20 days followed by rapiddrying of the conidia (Jensen et al. 2002). A shelf life of morethan a year has been obtained following this procedure when thedry product is stored at 20◦C or below in sealed bags togetherwith a desiccant such as blue silica gel (B. Jensen et al., unpubl.data). Studies have shown that dried conidia germinate moreslowly than freshly harvested conidia (Jensen et al. 1996). Thelowest effective dose of the formulated dry and stored producton barley is, however, also ∼104 cfu per seed indicating thatthe conidia that survive retain their biocontrol efficacy. Survivalof ‘IK726’ and biocontrol efficacy after storage of coated seedwas, to begin with, poor but this has also been improved bystoring coated seed under dry conditions in sealed bags withsilica gel at or below 20◦C (Jensen et al. 2002). ‘IK726’-coatedseeds can in this way be stored for ∼6 months before sowing(Jensen et al. 2002).

It is of considerable interest that biological control measuresare compatible with existing technologies. ‘IK726’ has beenshown to be compatible with several insecticides, and otherchemical compounds in concentrations used in seed technologies

such as seed coating and pelleting (Danisco Seed, unpubl. data)or it has been found to be compatible with several fungicides(J. Larsen, pers. comm.). Biocontrol should, in general, beplaying an important role in integrated pest and diseasemanagement where several control measures are combined asoutlined in Jensen and Lumsden (2000).

Some vegetable seeds are primed at high moisture content fora defined period to trigger seed germination processes and thensubsequently dried to retain moisture content and stored untilsowing. The seed will then germinate ∼4–5 days sooner whenplanted in the field – especially when planted early in the seasonat low temperatures. Seedborne pathogens and saprotrophicfungi can be troublesome during priming, causing poor seedquality with lowered field emergence. By introducing ‘IK726’at the start of carrot seed priming (called biopriming), wedemonstrated that seedborne Alternaria spp. were controlledduring priming by the biocontrol agent (Jensen et al. 2004) andthat the field emergence was improved significantly followingbiopriming with ‘IK726’ (Fig. 2).

Field experiments with ‘IK726’ have also been conductedby others in the EU-project ‘STOVE’ (http://www.stove-project.net/index2.html, verified 7 February 2007). High fieldefficacy of ‘IK726’ against Alternaria spp. in carrot was reportedin this project in 2006 from Germany, Italy, the UK and Sweden.Combinations of BCA treatment and physical seed treatmentswere also tested in the field. A significant additional effect wasseen in plots where a hot water seed treatment was combinedwith an application of ‘IK726’ to carrot seed. Effects againstAscochyta pisi in pea and Colletotrichum lindemuthianum inbean were reported from greenhouse trials in the EU project,but the efficacy of ‘IK726’ against these diseases was not testedin the field.

‘IK726’ has also been shown to efficiently control seedbornepathogens as well as saprotrophic fungi on acorn (Quercus robur)during cold storage at high humidity, as acorns, being recalcitrantseeds, must be stored moist (Knudsen et al. 2001). Also,broadcast application of a sprayable ‘IK726’ clay formulation toChinese cabbage plants controlled bottom rot caused by Pythiumtracheiphilum (Møller et al. 2003).

‘IK726’ can also be incorporated in soil or in greenhousesubstrates such as sphagnum peat or composted plant materialas a wheat bran formulation and control soilborne pathogens(D. F. Jensen, unpubl. data). Thus, the choice of appropriateapplication methods ensures that the BCA can be delivered atthe right place and at the right time for controlling a range ofseed and soilborne pathogens.

Ecology and biocontrol interactions

Basic information on ecological and biocontrol interactionshas been important for optimising the use of BCAs in cropproduction. ‘IK726’ transformed with constitutive expressedreporters [β-glucuronidase (GUS), green fluorescent protein(GFP), red fluorescent protein (DsRed)] are used for in situstudies (Lubeck et al. 2002; Jensen and Schulz 2003; Mikkelsenet al. 2003; Jensen et al. 2004). Conidial germination, hyphalgrowth, colonisation and conidiogenesis have all been studiedboth in soil, in greenhouse mix and in the rhizosphere as wellas in the spermosphere and on leaves, by exploiting fluorescentreporter technology (Lubeck et al. 2002; Jensen et al. 2004).

Development of a biocontrol agent for plant disease Australasian Plant Pathology 99

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Fig. 2. The effect of ‘IK726’ coating and ‘IK726’ biopriming on seedlingemergence of carrots in the field (a) early and (b) late in the season.Seeds were primed and bioprimed at 15◦C for 14 days. Thiram was usedas a standard fungicide seed treatment. Columns with different letters aresignificantly different (P < 0.05).

Moreover, in situ interactions with pathogens have also beenstudied (Lubeck et al. 2002). Fig. 3 is a confocal laserscanning image which shows spores and germlings of ‘IK726’in sphagnum peat illustrating the use of the GFP reportertransformed into ‘IK726’ for ecological studies. It is plannedthat environmental factors such as effects of pH, water potentialand conductivity will be studied in more detail using fluorescent

Fig. 3. Confocal laser scanning image of Clonostachys rosea strain‘IK726’ following its inoculation to Sphagnum peat moss. Germinatedconidia can be observed in green colours. ‘IK726’ has been transformedwith the green fluorescent protein reporter gene. Photograph supplied byDan Funck Jensen and Michael Hansen.

reporters. From work using both GFP and GUS reporters,it is clear that the antagonist can germinate and start togrow but needs organic substrates for extensive growth insoil, compost and greenhouse mixes such as sphagnum peat(D. F. Jensen, unpubl. data). It will also germinate on leafsurfaces and seed coats and start growing without the additionof nutrients (Lubeck et al. 2002; Jensen et al. 2004). Workfocusing on enzymatic activities and gene expression in resourcecapture and in biocontrol interactions is in progress. Increasedchitinase activity has been shown in the rhizosphere of barleycolonised by ‘IK726’ (Johansen et al. 2005). Chitinase genesencoding both exo- and endochitinases from ‘IK726’ havebeen cloned (Fig. 4) (Mamarabadi 2007) and methods basedon fluorescent reporter technology for following their in situexpression will be developed. Chitinase gene expression in‘IK726’ in interactions with Botrytis cinerea on strawberry hasalso been studied using real time reverse transcription PCR(Mamarabadi 2007).

Genes encoding proteins secreted from ‘IK726’ inmycoparasitic interactions with Pythium ultimum are beingrevealed in an ongoing research program by using transposonassisted signal trapping or TAST (Becker et al. 2004), which isa new approach to secretome analysis. This might lead to thediscovery of some, as yet unrecognised, genes involved in thecomplex interactions of ‘IK726’ with plant pathogens.

Non-target effects of IK726

‘IK726’ has never shown any direct deleterious effects on plantgrowth – on the contrary, a direct plant growth promotioneffect has sometimes been observed (Johansen et al. 2005;

100 Australasian Plant Pathology D. F. Jensen et al.

Cr-ech37 Single exon gene

3 exons & 2 introns

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Fig. 4. Three endochitinase genes and one exochitinase gene were clonedfrom Clonostachys rosea ‘IK726’. The genes for the endochitinases werenamed according to their predicted molecular mass (Mamarabadi 2007).

Ravnskov et al. 2006). Non-target effects of ‘IK726’ on theindigenous microbiota were studied in soil systems. In a barleyand sugar beet system, ‘IK726’ generally had a stimulatingaffect on soil enzyme activity and on the soil microbiota,especially culturable pseudomonads and Gram-positive bacteriain comparison with the non-inoculated control treatment(Johansen et al. 2005). In addition, ‘IK726’ had a direct plantgrowth promoting effect on barley (Johansen et al. 2005).In a comparable low nutrient system including tomato andGlomus intraradices symbiosis, the same increasing trend formost groups of bacteria was observed. The two inoculants,‘IK726’ and G. intraradices were, however, mutually inhibitory(Ravnskov et al. 2006). Nevertheless, dual inoculation resultedin a significantly increased plant dry weight of tomato and thiswas also the case when soil was inoculated with ‘IK726’ alone(Ravnskov et al. 2006).

Conclusion

Most of the aspects listed in Table 2 have been addressed in theattempt to develop C. rosea ‘IK726’ as a biological control agent.Approaches for isolation of potential antagonists and the use ofa hierarchic in planta screening procedure successfully resultedin the selection of a superior antagonist – strain ‘IK726’ – outof more than 400 fungal isolates. ‘IK726’ has been shown tobe efficient in the biocontrol of several plant diseases. Methodsfor production, formulation and delivery have been investigatedand prototypes of BCAs with a good shelf life have beendeveloped, which can be used for either delivery with the seed,soil incorporation, or furrow or broadcast applications. The basicecology of the antagonist and its interaction with pathogens havebeen studied using molecular methods and advanced microscopetechniques. Future work will focus on gene expression inbiocontrol interactions with special emphasis on the role ofcell wall-degrading enzymes. Gaining more knowledge on thebasic biology will help optimising strategies for efficient diseasecontrol. Risk assessment and registration of the BCA stillneed to be addressed although relevant information has beengathered including information mentioned above. Toxicologicaland ecotoxicological data must be obtained and, demonstrationof efficacy based on good experimental practices is a requirementfor EU approval. Thus, future work on commercialisation of‘IK726’ must be done in close collaboration with industry.

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Received 12 January 2006, accepted 15 January 2007

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