WKVDQG’RJPDVRI%LRFRQWURO - Who we are | Horticulture … · ously published in a refereed journal arti-cle. Throughout this effort, ... evidence that these particular beliefs are

Plant Disease / April 2000 377

G. E. HarmanCornell University and BioWorks, Inc., Geneva, NY

0\WKV�DQG�'RJPDV�RI�%LRFRQWUROChanges in Perceptions

Derived from Research on

Trichoderma harzianum T-22

The biological control of plant diseaseshas long been an area of fruitful study forplant pathologists, and there are thousandsof publications on this topic. However, inspite of this extensive research effort onthe part of hundreds of research scientists,there are very few uses of biocontrol incommercial agriculture today.

This statement is correct if we considerbiological control to be the use of an addedmicrobial agent to an agricultural system tocontrol pests. It should be pointed out,however, that there are many biocontrolsystems in use in agriculture that rely onsome manipulation of an environment orecosystem to reduce diseases. For example,diseases of wheat may be ameliorated bymonoculture to induce suppressive soils,together with appropriate tillage, properfertilizer type and placement, and timelyapplication of herbicides (15,68). Croprotation with specific alternate crops canreduce nematode levels (77), and compostswill become suppressive to various plantpathogens due to the development of ap-propriate microbial communities if thecomposting process is properly managed(37,38). These and other cultural systemsare based on a sound understanding of the

microbiological processes involved andcan provide highly effective biologicalcontrol. However, this article will considerintroduced microbial agents or, in the par-lance of the Environmental ProtectionAgency (EPA), microbial pesticides.

I decided more than a decade ago that apriority for my research efforts would be todevelop biocontrol systems that are used incommercial agriculture. This effort hasincluded not only research but also othercritical issues such as patenting, registra-tions, and commercial production and de-velopment. As a component of this effort,two colleagues and I cofounded a com-pany, TGT Inc., now BioWorks, Inc., totranslate biocontrol research into biocon-trol reality as defined by sales of commer-cially useful products. This effort has beenlargely successful, and now products basedon a single strain of Trichoderma harzia-num, strain T-22 (a.k.a. 1295-22, KRL-AG2, or ATCC 20847), are sold in thegreenhouse, row crop, and turf industries.In 1999, retail sales of T-22 products to-taled around $3 million, and sales are ex-pected to grow substantially over the nextseveral years. This is one of only a veryfew biological control products for thecontrol of plant diseases to reach even thismodest level of commercial sales and ac-ceptance. In the process of this effort, Ihave gained an appreciation for the placeof biocontrol and its biological and com-mercial potential. However, I will soundsome warnings for those who expect thattheir research results will ever be used incommercial agriculture.

Nearly all of my research and develop-ment effort has focused on Trichodermaspp., and the lion’s share has dealt withstrain T-22. T-22 was produced using pro-toplast fusion in my lab (73) in an effort toobtain highly rhizosphere competentstrains that also possessed substantial abil-ity to compete with spermosphere bacteria.The term rhizosphere competence wasintroduced in the 1980s by the late TexBaker and his associates and is defined as“the ability of a microorganism to growand function in the developing rhizo-sphere” (1). Strains that were fused wereT-95 of T. harzianum, which was a rhizo-sphere competent mutant produced from astrain isolated from a Rhizoctonia-suppres-sive Colombian soil (1), and T. harzianumstrain T-12, which was more capable ofcompeting with spermosphere bacteria thanT-95 under iron-limiting conditions (29);both were strong biocontrol agents. Weobserved great diversity in progeny strains(73). Strains were selected that were morestrongly rhizosphere competent than eitherparental strain (34,69), that were stronglycompetitive in the spermosphere environ-ment, and that were broadly effective bio-control agents (34). Strain T-22 was themost effective of the strains produced(34,69), and research since then has beendirected primarily to development, pro-duction, and use of this single strain. Overthe past few years, this quest has beenaided immeasurably by access to data fromhundreds of field trials done by commer-cial and academic cooperators withBioWorks. None of the figures or the table

Dr. Harman’s address is: Departments of Horti-cultural Sciences and Plant Pathology, CornellUniversity, and BioWorks, Inc., Geneva, NY14456; E-mail: [email protected]

Publication no. D-2000-0208-01F© 2000 The American Phytopathological Society

378 Plant Disease / Vol. 84 No. 4

presented in this paper have been previ-ously published in a refereed journal arti-cle.

Throughout this effort, I have gainedsome insights that differ from those ofmany of my colleagues in biocontrol R&D.This article is written to provide the per-spective of biocontrol that has developedfrom immersion in both academic andcommercial aspects of biocontrol. It willbe colored and exemplified primarily byexperiences with Trichoderma, but I hopethat its underlying message will be usefulto those working with other microbes. Thepaper will deal not only with scientifictopics but also with the critical world ofcommercialization. If biocontrol is ever tolive up to expectations, real-world successis essential. But it is by no means guaran-teed.

Dogmas and MythsAs I attend meetings, read papers, and

review proposals, I am struck frequently bydogmas that have been gaining acceptancewithin the biocontrol community. Many ofthese dogmas are not universally applica-ble:• Biological control agents (BCAs) are

necessarily less effective and reliablethan chemical pesticides.

• Single BCAs added to roots or soil can-not affect microbial communities orcontrol root pathogens for long periodsof time. As a corollary, biological con-trol is likely to be effective for seed and

seedling diseases but not against dis-eases of the mature crop.

• Single BCAs cannot be effective in di-verse environments, on different crops,or against a wide range of plant patho-gens. As a corollary, mixtures of BCAswill be required for successful long-termcontrol, since individual componentscolonize different crops, are adapted todifferent environments, or have differentfunctions.

• BCAs have simple mechanisms of ac-tion that are controlled by one or only afew genes and gene products.

• Registration of BCAs with the U.S. EPAis relatively fast, inexpensive, and sim-ple.None of these statements are true for the

systems and BCAs with which I work.However, if biocontrol researchers acceptthese concepts as largely or wholly true,these assumptions and concepts can im-pede biocontrol progress. In this article, Iwill present evidence that, in the case ofthe biocontrol systems and agents withwhich I work, these dogmas are in factfalse.

I quote certain papers and articles asevidence that these particular beliefs arewidely held. These quotes should not beconsidered as confrontational to theauthors. I quote them because they all havemade excellent contributions to biocontrolresearch and because they are highly re-spected by others in the field, includingmyself. In particular, I quote from the re-

cent Plant Disease feature article byMathre et al. (60), since they clearly have avery different perspective on many aspectsof biocontrol than I, and they eloquentlystate their position.

Dogma 1. Biological control agents arenecessarily less effective and reliablethan chemical pesticides.

We frequently hear that biologicals havedifficulty in acceptance because there is aparadigm of chemical control in the agri-cultural community. For biologicals, weneed to introduce new concepts and uses,i.e., to substitute a biological paradigm forthe existing chemical one. However, itfrequently is unclear what this means inactual practice.

In my own research, I initially sought todevelop biological seed treatments for seedprotection. These efforts were largely suc-cessful as academic projects. We devel-oped strains of biocontrol agents, devisedmethods and principles, and demonstratedthat the biological agents could provide ahigh level of seed protection, especiallywhen combined with solid matrix priming(also known as biopriming [60]) (32,33,63,74,75).

To my initial surprise, there was verylittle interest from companies in commer-cializing this technology. In hindsight, thereasons are very clear and include the fol-lowing: (i) there are many highly effectivechemical pesticides available for seed pro-tection; (ii) these chemicals frequently areless expensive than biologicals, especiallyif the BCA requires solid matrix or bio-priming; (iii) the shelf life of the chemicalson seeds is superior to biologicals; and (iv)the chemicals protect seeds under a widerrange of temperatures and other environ-mental conditions than biologicals.

In short, we were trying to use our bio-logical agents in a system where chemicalfungicides provide a better and more eco-nomical fit, i.e., we were trying to fit ourBCAs into a chemical paradigm.

However, there are instances where bi-ologicals may be highly attractive to com-mercial agriculture. Some niches wherebiocontrol may fit well are given below.• Replacement of chemical pesticides lost

to regulatory action or pest resistanceand for which there are no adequatechemical replacements.

• Replacement, or reduction of use, ofchemical pesticides in sensitive envi-ronments.

• Applications where biologicals accom-plish tasks not possible for chemicalpesticides.

• Organic applications.The first niche is perhaps the most obvi-

ous and the one most frequently cited as agood reason for use of biologicals and onewhere there is a shift from “hard” chemi-cals to much softer alternatives. As anexample, control of Botrytis cinerea andpowdery mildews has become more diffi-cult as fewer chemicals are available and

Fig. 1. Colonization of sweet corn root hairs following seed treatment with Tricho-derma harzianum strain T-22. Root samples from plants grown in greenhouse pottingsoil were removed and washed. The root surfaces were then oxidized in 1% periodicacid, rinsed, and counterstained for 20 s in Schiff’s Reagent. They were then rinsedand mounted in 0.1 µg/µl 4 ′,6-diamidino-2-phenylindole (DAPI) to stain DNA of inhab-iting microbes and observed using epifluorescent microscopy. Fibrillar network of T-22 and bacterial colonies are indicated on photograph. Roots of similar plants grownin absence of T-22 lacked this hyphal network. Photograph courtesy of ThomasBjörkman, Cornell University, Geneva, New York. Similar photographs (30) and similarresults on creeping bentgrass roots with transgenic mutants of T-22 expressing β-glucuronidase have been published (55).


as pest resistance has increased. A range ofbiological alternatives is now or is ex-pected to be in the marketplace shortly,including TopShield (strain T-22 of T.harzianum, BioWorks, Geneva, NY),AQ10 (Ampelomyces quisqualis, Ecogen,Langhorne, PA), and Trichodex (strain T39of T. harzianum, Makhteshim-Agan Chem-ical Co., Israel). In addition, naturalchemicals such as components of cinna-mon oil (Mycotech, Butte, MT) and bakingsoda derivatives (H and J Agritech, Ithaca,NY, and TOAGOSEI Co., Tokyo, Japan)are registered for this use and are just be-ginning to be offered for sale. This is aniche where biologicals and natural chemi-cals can establish a presence and providean alternative to other pest control strategies.

Different agricultural markets differ inthe sensitivities of growers or other pur-chasers of products to the perceived andactual risks of chemical pesticides. Mostrow crop farmers, unless they cater to theorganic trade, use those legal chemicalpesticides that provide the most economiccontrol of pests. On the other hand, green-house operators are quite sensitive tochemical use issues. EPA-mandated reen-try times cause disruptions in their sched-ules, and they are concerned about workersafety issues. A biological with 0-h reentryand no known health risk to humans, asindicated by an exemption from residuetolerances on foods, is a big advantage tothese growers. Consequently, biologicalsmay have an advantage over chemicals forthe same purpose if they are similarlypriced and possess similar efficacy in en-closed environments. Other exampleswhere sensitivities of end-users to chemi-cals provide an advantage to biologicalsinclude high public use and urban areassuch as golf courses, homeowner proper-ties, sports fields, and parks.

The third niche for biologicals is per-haps the most solid. Biologicals can pro-vide advantages over chemical pesticides.Perhaps the best examples of this arestrains that are rhizosphere competent andcan provide advantages to the subterraneanportions of the plant throughout the life ofat least annual crops. Commercial biologi-cal control agents with this ability areavailable and will be discussed in the nextsection. Further, biologicals can be pro-duced on-site, for example using the Bio-Ject systems manufactured by EcoSoil-Systems, and this may confer a perceivedadvantage over chemicals that must bepurchased from a vendor and then applied.

The last niche, use of biologicals by or-ganic farmers and homeowners, has longbeen a prime target of biocontrol develop-ers. Biologicals may be acceptable to or-ganic certification agencies (T-22 is listedby the Organic Materials Review Institute)and so provide pest protection within anorganic framework. It should be noted thatmicrobial pesticides do not automaticallyqualify for organic agriculture; approval by

a certification agency likely will be re-quired for each new agent/product.

Thus, it is important to choose an appro-priate system for development of biologi-cals if commercialization is a goal of theresearch. Direct competition in a nichewhere chemicals are readily available andrelatively inexpensive, and where there isno perceived premium for use of biologi-cals in the mind of the user is likely to beunsuccessful.

Dogma 2. Single BCAs added to rootsor soil cannot affect microbial communi-ties or control root pathogens for longperiods of time. As a corollary, biolog-ical control is likely to be effective forseed and seedling diseases but notagainst diseases of the mature crop.

This perception is widely published; forexample, Deacon (20) stated that biologi-cal control agents “achieve only transitorylocalised dominance of the rhizosphere,and in only some soils and seasons…”Similarly, Mathre et al. (60) indicate thatnearly all commercialized microorganismsrely upon application of the antagonist“directly and precisely to the infectioncourt when and where needed.” Seed pro-tection, therefore, is a logical target, and asa corollary, the most that should be ex-pected from a seed treatment is seed pro-tection and perhaps increased vigor fromplanted seeds. However, this places thebiological squarely into the chemical para-digm where commercial success is difficultto achieve.

This need not be the case. At least twomechanisms, rhizosphere competence andinduced systemic acquired resistance(SAR), give long-term protection at a sub-stantial distance from the infection court.

Either fungal or bacterial agents may berhizosphere competent; for example, Pseu-

domonas species control root diseases ofwheat after colonizing roots (60), and Ba-cillus subtilis is sold as Kodiak (Gustafson,Inc., Dallas, TX) for the purpose of in-creasing plant yields through root coloni-zation.

SAR from bacterial colonization hasbeen well described, for example, byKloepper and his associates (47). Fungialso can induce SAR, and this will be de-scribed in the discussion of Dogma 4.When SAR functions, application of abiocontrol agent to one portion of the plantprovides protection to a wide range ofpathogens on another part of the plant(51,52); for example, an organism provid-ing SAR applied to roots may cause theplant to be resistant to pathogens on itsleaves.

An important point regarding any studyof rhizosphere competence is the physio-logical state of the colonizing microbe. Atleast in young seedlings, root hairs of cornare colonized by hyphae of T-22 and not byspores (Fig. 1). This is a critical distinc-tion, since spores are quiescent and inac-tive in biocontrol (49). Unlike many otherTrichoderma strains (49), T-22 can beadded as spores to the soil volume, forexample, as in-furrow or greenhouse soildrenches. The strain will then colonizeroots, which indicates that the spores ger-minate and grow in contact with the roots(Fig. 1).

The concept of rhizosphere competenceby strain T-22 has been extensively testedon a variety of crops. We have done a sub-stantial amount of root plating experimentsand usually find that the entire root iscolonized (30,53). BioWorks also con-ducted root assays; if customers wished toknow if T-22 “worked,” i.e., colonizedroots, they could send samples to the com-

Fig. 2. Standard assay performed by BioWorks in which samples were sectioned into2.2-cm segments from the upper, central, distal, and lateral portions of entire rootsystem. Root segments were plated onto potato dextrose agar amended with 0.1%Igepal Co-630 as a colony restrictor (71) and chlorotetracycline (50 mg/liter). Numer-ous fungal types and species grow on this medium, and Trichoderma spp. can readilybe detected. Typically, 80 to 100% of root segments were colonized by Trichodermaspp. over the entire 2.2 cm root length. In some cases, as in this example with poin-settia, T-22 was the primary fungus colonizing roots, while in its absence numerousother fungi were dominant.


pany lab with or without coded controls.Thousands of assays were done on cropsranging from ferns to beans to corn to or-namental flowering plants, and almostwithout exception, T-22 was found to colo-nize all parts of root systems. The basicassay technique is shown in Figure 2.These results frequently demonstrated notonly that T-22 colonized roots but also thatother microflora were displaced, therebychanging the root microfloral composition.Moreover, there was almost no effect ofsoil type or geographical location on thisability. Probably the only exception to thisgeneralization occurred on cotton roots atthe height of the summer from fields out-side Phoenix, Arizona. T-22 was presentonly sporadically on roots obtained from

these hot, dry soils. In other studies, T-22has provided advantages to cotton. Impor-tantly, T-22 has equal ability to colonizeroots in both alkaline and acidic soils (53)and across soil types ranging from sandy toheavy and with a wide variation in organiccontent (30). Further, the method of appli-cation was not important. Colonization wasobtained over the entire root surface whenT-22 was added as a seed treatment, asbroadcast granules on the surface ofplanted soil, as an in-furrow granule ordrench, as incorporated granules in green-house planting mixes, or as a conidial sus-pension in greenhouse potting mixes. T-22grew onto all newly formed root surfaces.

T-22 is not unique in this capability. Weobtained strain 41 of T. virens from roots of

pea plants in an Aphanomyces-suppressivesoil and discovered it was capable of con-trolling Phytophthora spp. and otherpathogenic fungi (71). We conducted a 3-year field study on the control of Phy-tophthora on raspberry. We evaluatedraised beds, metalaxyl treatments, meta-laxyl + T. virens, and T. virens added twicea year or once a month during the growingseason. We assayed the root and soil fungalpopulations at monthly intervals over thecourse of the season. In the absence ofapplication of strain 41, T. virens made uponly a minor part of the total fungalpopulation. However, when the biocontrolagent was added in any of the regimesnoted above (Trichoderma spp. generallyare resistant to metalaxyl), the T. virenspopulations became established and per-sisted as the dominant fungus colonizingroots and soil over the entire three growingseasons of the experiment (Fig. 3). Thefungus was present in similar numbersover the entire growing season. Clearly, itbecame the dominant culturable fungus inthe soil and persisted extremely well. Thebehavior of strain 41 differs from that of T-22 in one very important respect: T-22 doesnot become dominant in the soil but onlyon the roots. Conversely, strain 41 estab-lishes dominance in both habitats.

In the case of the raspberry study notedabove, one of the organisms that T. virenswas displacing was T. harzianum. On rootsof turfgrass, however, T. virens is the mostcommonly isolated native microfloralcomponent and makes up the majority ofthe isolates obtained. If, however, T-22granules were applied either at high ratesor repeatedly, T-22 displaced T. virens andother fungi and became the most numerousorganism, frequently making up more than50% of the total fungi isolated from theroot zone (K. Ondik and G. E. Harman,unpublished).

The consequences of root colonizationby Trichoderma spp. can be profound in

Fig. 4. Enhanced root development in field crops induced by Trichoderma harzianum strain T-22. (A) Roots of sweet corn grownfrom seeds either treated with T-22 Planter Box or not treated. Although roots colonized by T-22 were more abundant, in this par-ticular trial yields were similar regardless of treatment; conditions of growth were good, and no yield advantage was provided bythe improved root system. (B) Soybean plants with roots grown from seeds either treated with T-22 or not. A 123% increase in yieldwas obtained in this trial as a consequence of treatment with T-22.

Fig. 3. Replacement of endogenous fungi by application of Trichoderma virens strain41; percentage of total culturable fungi obtained from (A) roots and (B) soil. Strain 41was applied as a granular application (62 kg/ha) in a strip 1 m wide on rows of rasp-berries once each in spring and fall. Initial application was at planting, and thereaftergranules were banded on soil surface. Two soil cores were taken from each plot anddilution plated onto acidified potato dextrose agar (PDA) and amended with Igepal Co-630 as a colony restrictor (71). Fungi other than Trichoderma were easily distin-guished on basis of morphology, and T. virens could easily be separated from otherTrichoderma strains on the acid PDA medium. T. virens has a pure white coloration onits lower surface, while native Trichoderma spp. were tan to brown. Data shown werefrom plots of cv. Newburgh raspberries on raised beds in a field soil heavily infestedwith Phytophthora fragariae var. rubri. Arrows indicate application times. Each treat-ment was replicated four times, and error bars represent standard deviations. Data arefrom a larger trial conducted by W. Wilcox, G. Harman, P. Nielsen, and K. Ondik nearGeneva, New York.


terms of plant disease control and plantgrowth and productivity (6,13). Not onlycan colonized roots be protected againstdisease by T-22, but they frequently arelarger and more robust (Fig. 4). This in-creased root development may occur as aconsequence of control of clinical or sub-clinical pathogens, as suggested severalyears ago for Pseudomonas spp. (10).However, T-22 probably also has directeffects upon plant metabolism. In limitedstudies, we observed that T-22 was as ef-fective as a commercial rooting hormonein inducing rooting of tomato cuttings,although callus tissue was not formed onthe base of the cuttings as occurs withcommercial hormone preparations (Table1). Further, T95, which was one of theparental strains used in the fusion that gaverise to T-22, increased plant growth evenunder axenic conditions (79). Also, cu-cumber plants grown in axenic hydroponicconditions were larger in the presence of T.harzianum strain T-203 than in its absence(81). Therefore, T-22, as well as othersimilar Trichoderma strains, probably en-hances root growth and plant developmentboth by displacement and control of delete-rious root microflora and by direct effectson plants by as yet unidentified biochemi-cals.

The length of time that T. harzianumstrain T-22 can persist and proliferate onroots is rather remarkable, as was alreadydemonstrated with strain 41 of T. virens. Inan early field trial, we applied broadcastgranules of T-22 in October 1993 to a fieldthat had just been seeded to a rye graincover crop; control plots were seeded tothe rye cover crop without T-22. Early nextspring, we sampled the roots and found ahigh population of T-22 (around 105 CFU/gdry weight of roots) on the roots where T-22 was broadcast but not on the controlareas. We then killed the small rye grainplants with glyphosate and planted sweetcorn into the untilled plots. Differences ingrowth of the corn between the treated andnontreated plots were evident over theentire season and were different at the endof the season (Fig. 5). As would be ex-pected from the size of the plants, the yieldof corn was also different, with 1.7 timeshigher weight of ears being harvested fromthe T-22–treated plots than from thoseoriginally planted to the rye cover cropswithout T-22.

Cover cropping also was investigatedwith a quite different system. In the fall of1997, plots were planted to T-22 treated ornontreated seeds of a Sudan-sorghum(Sudex) hybrid on a highly organic (muck)soil near Oswego, New York. The roots ofthe Sudex were sampled after the cropbecame established, and there was a oneorder of magnitude higher population ofTrichoderma spp. on Sudex grown fromtreated seeds than from nontreated seeds.The next spring, onions were planted onthe Sudex + T-22 and the Sudex without T-

22 plots. Yields were greater in the pres-ence of T-22 than in its absence. The bestyields occurred in rows that also received amaneb + metalaxyl (Ridomil) drench tocontrol early-season diseases. With theintegrated T-22 + fungicide program,yields were increased 10% relative to thesame program without T-22, which washighly significant both economically andstatistically.

All of these examples demonstrate that,with T-22 and T. virens strain 41 at least, itis possible to achieve much more than“transitory localised dominance of therhizosphere, and in only some soils andseasons” (20). They indicate that rhizo-sphere competence is real and can result inlong-term root colonization. If so, it shouldprovide quantifiable improvements in plantperformance. The following section indi-cates that substantial and unexpected ad-vantages to plant growth and productivitycan be conferred by strongly rhizospherecompetent BCAs.

An important question regards the depthand degree of root enhancement with T-22.In 1999, we conducted trials with field

corn in a sandy loam grower field. Thefield was planted to corn either treated ornot treated with the commercial formula-tion of T-22 in alternating bands six rowswide throughout the field. At about thetime of tasseling (about 60 days afterplanting), we dug trenches about 3.2 mdeep with a back hoe about 15 cm in frontof rows of corn about 2 m tall. The ex-posed soil profile was washed with apower washer to reveal the roots. We thenestablished 25 × 25 cm grids across anddown the soil profile. Each root interceptwas marked with a map pin with a differ-ently colored head (different colors foreach adjacent grid), the grids were indi-vidually photographed, and the pins ineach grid were removed, placed in separateboxes, and counted. Numbers of root inter-cepts were similar in corn with and withoutT-22 in the upper 25 cm

of soil. However,

roughly twice as many intercepts werefound in the second and third grids whenT-22 was present than when it was absent(Fig. 6A and B). This greater deep rootdensity may confer substantial benefits tocorn and other crops, especially in dry

Fig. 5. Growth of sweet corn without Trichoderma harzianum strain T-22 (left) andcorn with roots colonized by T-22 (right). A granular formulation of T-22 was appliedby broadcast (about 10 kg/ha) at the same time (October 1993) that a rye grain covercrop was seeded. In spring 1994, roots of rye were found to be colonized by T-22, andthe rye was killed by spraying with glyphosate. Sweet corn (cv. Jubilee Supersweetwith standard fungicide seed treatments) was planted into the field without tillage.Data are from G. Harman, H. Price, and P. Nielsen (pictured), Cornell University.

Table 1. Enhancement of rooting and root elongation of cuttings of a Solanum × Lycopersi-con hybrid as a consequence of dipping of stem cuttings in Rootone powder (1% indolebutryic acid) or in T-22 in the form of RootShield drench

Treatment Numbers of rootsa Length of roots (mm)

None 1.2 ± 0.8 1.8 ± 0.6Rootone 1.1 ± 0.3 1.6 ± 0.6T-22 2.5 ± 1.0 2.4 ± 0.6

a Thirteen days after treatment just as roots were being initiated.


growing seasons when root colonization byT-22 can reduce sensitivity of crops todrought stress (Fig. 6C).

Further, we sometimes noted that cornplants that grew from seeds treated with T-22 were greener than plants without T-22.Increased greenness in corn frequently isassociated with higher levels of nitrogenuptake (66). We began to consider the pos-sibility that one of the effects of T-22 oncolonized roots was increased efficacy ofuse of applied fertilizers, especially nitro-gen. In 1998, we established a trial withfield corn in a commercial grower’s fieldon a sandy loam. The entire field wasplanted in bands six rows wide with seedtreated with T-22 Planter Box alternatingwith six rows without the biocontrol agent.We expected that the initial nitrate levelwould be low, since the field had not re-ceived recent manure applications andbecause corn had been grown the previousseason. Pre-sidedress nitrate soil tests(PSNT) verified that the endogenous nitro-gen was indeed low, about 20 kg/ha. Nitro-gen, in the form of ammonium nitrate, wasbanded and incorporated beside rows ofcorn at about the four-leaf stage to givetotals, including the 20 kg/ha of residualnitrogen, of 20, 40, 80, 160, and 240 kg oftotal nitrogen per ha.

Differences were observed almost im-mediately. Heights of corn were measured2 weeks after side-dressing, and those withT-22 responded more rapidly and remained

larger for most of the growing season thancontrol plots (Fig. 7B and C). At 2 and 4weeks after nitrogen application, there wasno difference in corn greenness as deter-mined by readings with a SPAD meter(66). However, later, at the time of tassel-ing, the T-22–treated plants were greenerin a nitrogen dose–dependent manner (Fig.7D). At maturity, there was a difference instem diameter and grain and silage yields(Fig. 7E to G).

Perhaps the most important yield com-parisons were the points at which nitrogenno longer gave a yield increase, presuma-bly because the plants had as much nitro-gen as they could utilize. The unused ni-trogen would not be expected to provideany yield benefit and probably would bevolatilized or contaminate groundwatersupplies (25). No increase in yield of eithersilage or grain occurred above 150 kg of Nper ha in the presence of T-22. In the ab-sence of T-22, however, the full rate of 240kg was required for maximum yields (Fig.7F and G), i.e., maximum yields were ob-tained with 38% less nitrogen in the pres-ence than in the absence of T-22. The EPAis requiring a plan to reduce hypoxia in theGulf of Mexico and may mandate a reduc-tion in nitrogen fertilizer use in the Missis-sippi River Basin (see EPA web site:http://www.epa.gov/msbasin/legis98.htmland American Farm Bureau Federationweb site, news from 2000 annual meeting:http://www.fb.com/2000annual/amnews/hy

poxia.html [Note: web addresses oftenchange]). A microbial agent that increasesnitrogen use efficiency by crop plantswould appear useful in this regard.

A similar trial was established in thespring of 1999. Soil types and plantingconditions were quite similar, but therewas a high level of residual nitrogen in thefield. PSNT values ranged from 40 to 90ppm; corn usually does not respond tonitrogen fertilizer if values are above 20 to30 ppm (35). Under these conditions, therewas no measurable response of the corn toapplication of T-22. However, roots werecolonized by the fungus, and deep rootgrowth was enhanced; the data in Figure 6are from this plot. Even in the presence ofoptimal or supraoptimal levels of N, T-22apparently enhances corn root develop-ment, but unless N is limiting during somephase of the growth cycle of the corn, thisenhanced root development does not affectplant growth or yield in the absence ofother stress factors such as drought.

More than 70 separate T-22 trials havebeen done on field corn alone, more than30 on soybeans, and numerous trials havebeen done on crops such as wheat, peas,sugar beets, and potatoes. Specific advan-tages and uses of T-22 on these crops arenumerous and diverse. When T-22 is ap-plied as a seed treatment on potatoes, bothsize (increased grade) and yields frequentlyare increased. When T-22 or T. virensstrain 41 was applied in Brazil to wheat

Fig. 6. Enhanced deep rooting in field corn induced by Trichoderma harzianum strain T-22. (A) Numbers of roots at different depthsof mature (2 m tall) field corn (E238) in field trials near Geneva, New York, in 1999 with and without root colonization by T-22.Separate trenches (about 3.2 m deep × 5 m long) were dug with a backhoe about 15 cm in front of rows of corn. The resulting soi lfaces were washed with a power washer to expose corn roots, and grids of string were placed over the soil profile. Root interce ptsin each square were marked with map pins, each square was photographed, and the number of pins in each square was counted.Data were modeled by logistic curves (72) of the form: Numbers of roots = a + c/1 – b(depth – m), where a = lower asymptote, a + c =upper asymptote, m = point of inflection and b = slope parameter. Lines are significantly different at P = 0.006. Data for the lowerfour regions of soil were fitted to linear regressions; there was no significant difference in slopes, but elevations were diff erent at P= 0.001 (graph not shown). (B) Appearance of root intercepts in the same trial as (A) marked by map pins in single 25 × 25 cmsquares 25 to 50 cm (top) or 50 to 75 cm (bottom) below the ground level. Squares shown were chosen because they approximatethe average values for each treatment. (C) Drought tolerance in corn induced by T-22 from trials in 1999 in Ohio which probably wasa consequence of enhanced deep rooting. Corn without T-22 (right) exhibited the typical leaf curl to reduce transpiration sympt o-matic of drought stress, while leaves of T-22–treated corn (left) did not. In both New York and Ohio, T-22 was applied as a dus t tothe seed according to labeled directions for T-22 Planter Box. Data in (A) and (B) are from field trials conducted by R. Petzol dt andG. E. Harman, Cornell University, while (C) is a photo from Ed Winckle, Hymark Consulting, Blanchester, Ohio.


seeds infected with Pyrenophora tritici-repentis (17), both stands and yields wereincreased significantly and substantially(yields increased from 1,666 to 2,166 kg/hafor strain T-22 and to 1,953 kg/ha for strain

41). The same authors obtained similarresults over 2 years with corn seeds in-fected primarily with Fusarium gramin-earum and F. moniliforme (W. C. da Luzand G. C. Bergstrom, unpublished). Addi-

tionally, in 1999, trials were conducted oncontrol of take-all in wheat. Seed treatmentwith T-22 enhanced spring green-up andsignificantly reduced white heads causedby Gaeumannomyces graminis var. tritici

Fig. 7. Interaction of Trichoderma harzianum strain T-22 and nitrogen fertilizer on corn growth and yield. (A) Appearance of cornfrom a grower trial in 1996 produced from T-22–treated plants and from nontreated plants. (B-G) Results from trials in a grower fieldon deep sandy loam soil at various levels of N fertility. Such treatments have been used for other studies in order to determin e ni-trogen use efficiency as imposed by various treatments using negative exponential models (46). (B) Corn growth response to dif-ferent levels of N from T-22–treated and nontreated plots 2 weeks after side-dressing. Plants whose roots were colonized with T -22responded to N, while those without T-22 did not. Both position and slope were significantly different based on regression anal ysis(P = 0.001 and 0.04, respectively). (C) Corn growth response to N 4 weeks after application of various levels of N. Slopes were s imi-lar, but intercept of curve was significantly different ( P = 0.001). (D) Leaf greenness of corn at tasseling as a function of treatmentwith T-22 and nitrogen application level as measured with a Minolta SPAD 502 meter. All plants were of similar height, but leafgreenness was significantly different (slope parameters were not significantly different but position was significantly differe ntbased on regression analyses at P = 0.002). (E) Stalk diameter of corn at harvest as influenced by treatment with T-22 and differentlevels of N. Slope parameters were similar, but positions of lines were significantly different at P < 0.001. (F) Yield of grain as influ-enced by treatment with T-22 and different levels of N. Analysis of shape of curves gave no evidence for any other than a linea rrelationship between N level and yield in the absence of T-22, but in its presence a curvilinear relationship was a better fit. Regres-sion analysis indicated no significant difference in shapes or positions of lines generated for T-22 and nontreated corn, but a naly-ses of means using a pooled standard error and a Bonferroni-adjusted 5% least significant difference (62) indicated that there is asignificant difference in yields of corn grain between T-22 and nontreated corn at 80 and 160 kg/ha of N. (G) Yield of silage a s influ-enced by presence or absence of T-22 and level of nitrogen fertilizer. Analysis of shape of curves gave no evidence for any oth erthan a linear relationship between N level and yield in the absence of T-22, but in its presence a curvilinear relationship was a betterfit. Comparison of shape and position of curves indicated that position of curve generated by T-22–treated corn was significant lyhigher than that from untreated corn ( P = 0.055). Photograph in (A) is from BioWorks and shows Christopher Hayes. All other dataare from G. E. Harman and R. Petzoldt, Cornell University.


from 32 to 21% (D. Huber, Purdue Univer-sity, unpublished). However, in spite of theresults just cited, in other trials no yieldincreases have been noted. I will provide arationale for this difference and a descrip-tion of limitations of T-22 at the end of thediscussion of Dogma 3.

In nearly all cases, the best results in di-rect-seeded field crops are obtained withan integration of chemical seed treatmentsand T-22. Extensive testing has revealedalmost no chemical seed treatments thatprevent subsequent root colonization by T-22 (full rates of thiram and tebuconazoleare exceptions). This resistance permits theuse of both the biological root colonizingagent and chemical seed protectants.

Granules of T-22 frequently are incorpo-rated into potting mixes in greenhousecrops for production of vegetable andflower transplants and also for pot cropssuch as chrysanthemums and poinsettias.

For example, tomatoes were grown in apotting mix containing the granular for-mulation of T-22, which permitted roots tobecome colonized, then transplanted to thefield. Fusarium crown and root rot at har-vest of mature fruit was reduced (18,64).T-22 was not the only effective organism—the rhizosphere competent Bacillus subtilis(the active ingredient in Kodiak) fromGustafson was also effective. The combi-nation of T-22 and the mycorrhizal fungusGlomus intraradices was more effectivethan either organism alone (18). Thus,application of a very small amount of anyof several biocontrol organisms at the timeof seeding of transplants provided a sea-son-long benefit to tomato health andyield. Again, this is hardly a localized ortransitory effect or one confined to seeds,seedlings, or the infection court to which

the biocontrol agent was originally applied.There are similar studies on other trans-

plant vegetables. Pepper seedlings wereproduced in the greenhouse with or withoutthe addition of RootShield, the greenhouseproduct containing T-22. The peppers weretransplanted into the field under less thanideal conditions. When their roots lackedT-22, fewer pepper plants survived trans-planting and early yields were lower (Fig.8).

A number of trials have focused on or-namentals; generally, T-22 either applied asa drench or incorporated as granules givesadequate disease control over an extendedperiod of time (several months). Further,root development frequently is better inplants where disease is controlled by T-22rather than by fungicides; many fungicidesinhibit root growth to some extent, whereasT-22 enhances root development, as shownfor poinsettias (Fig. 9). There have beennumerous other demonstrations of similareffects of T-22 on greenhouse ornamentals.

These data clearly demonstrate that T-22, as well as other rhizosphere competentbiocontrol agents, can provide long-termprotection or other advantages to plantsfrom a single application at the beginningof the season. These biocontrol agents canestablish themselves on roots, grow withthe developing root system, and remainfunctional for at least the life of an annualcrop.

Thus, biologicals can be more effectivethan chemical pesticides for root protectionand plant growth enhancement. However,the assessment of efficacy is dependentupon the particular parameter being meas-ured.

Dogma 3. Single BCAs cannot be ef-fective in diverse environments, on dif-

ferent crops, or against a wide range ofplant pathogens. As a corollary, mix-tures of BCAs will be required for suc-cessful long-term control since individ-ual components colonize different crops,are adapted to different environments,or have different functions.

The dogma that single biocontrol agentscannot be widely effective was very wellstated in the recent article by Mathre et al.(60), “One rather daunting principle thatapplies across all biological methods fordisease and pest control with introducedagents…is that, almost invariably, a differ-ent agent…is needed for each disease orpest.” Similarly, Cook (16) stated that“biological control is widely recognizedboth scientifically and based on empiricalexperience as highly pest- or disease-spe-cific.” Further, he advocates an approach tobiological control that uses mixtures ofnumerous agents for each pest or disease.This view is inconsistent with our experi-ences and others, at least with Trichodermaspp. A large number of papers indicate thatsingle strains of Trichoderma spp. are ca-pable of controlling diverse pathogens, andChet (14) has summarized some of these.

This is particularly important in light ofeconomic realities. In my view, as I willdiscuss in the last section of this paper, itprobably will be economically impossibleto commercialize mixtures of strains, atleast until there are significant economicsuccesses in biocontrol. In other words, notonly is one strain all that you may need,but perhaps more importantly, one strain isvery likely all that you can afford! If thisview is even remotely true, then it is im-portant to determine what can be accom-plished with a single strain, in this case T-22.

Fig. 8. Appearance of pepper plants in a field in New York a few weeks after transplanting from plugs grown in absence (A) andpresence (B) of Trichoderma harzianum strain T-22 in the form of the commercial product RootShield. (C) Yield data were taken onthree cultivars for first and third pickings. Yields for first picking for transplants produced in absence of RootShield were 5 .9, 3.6,and 5.4 kg per plot for Merlin Belle, Vanguard Belle, and Bonanza peppers, respectively, while for plants grown in the presence of T-22, yields were 10.2, 8.6, and 9.6 kg per plot. In third picking, yields were similar: in the absence of RootShield, 21.4, 11.7, and 18.1kg per plot were harvested from the same three cultivars, while in presence of RootShield yields were 24.1, 11.4, and 23.6 kg. Pho-tos and data are from Russell Wallace, BioWorks.


In our first article on T-22 (34), we re-ported that it controlled F. graminearum,R. solani, Pythium ultimum, and Sclero-tium rolfsii in laboratory tests. This list hasbeen expanded to include other pathogensof both above- and belowground plantparts in real-world research evaluations.

Control of R. solani has been welldocumented in the greenhouse on a numberof different crops (Fig. 10A to C). In manycases, T-22, as its commercial productRootShield, substantially enhances plantquality relative to fungicides. Similar re-sults have been obtained for control ofPythium (Figs. 11A to C). In many casesfor both pathogens, RootShield is as effec-tive as the competitive chemical fungi-cides, even when the biocontrol product isapplied infrequently and the chemicals are

applied more often, as per their label rec-ommendations.

However, other trials have indicated thatproducts based on T-22 are less effective incontrolling Pythium and R. solani than arechemical fungicides. This apparent para-dox will be addressed in a subsequent sec-tion, but at least two important factorsshould be mentioned here. Like other bio-control agents, T-22 can be overwhelmedby heavy disease pressure. Therefore, T-22must be used strictly as a preventativemeasure; it cannot cure existing infections.The biocontrol agent must be used withinits limits and as part of a total managementstrategy.

In general, we have not focused on theability of T-22 to control these pathogensin the field except on golf course turfgrass

(53–55), where T-22 reduces disease lev-els. Pythium and R. solani on field cropscause seed or seedling disease, and werecommend that these diseases be con-trolled by appropriate chemical fungicides,for the reasons noted earlier.

Diseases caused by Fusarium spp., es-pecially root and crown rot, also are con-trolled. Earlier in this paper I summarizedresearch (18,64) that demonstrated that asingle application of T-22 as RootShieldgranules in the greenhouse provided pro-tection of the tomato crop against Fusariumcrown and root rot of the mature crop.Similarly, a single application of T-22 tored onions at the time of planting resultedin significantly reduced Fusarium basal rotat harvest. An in-furrow drench was moreeffective in both root colonization anddisease control than a seed treatment (Fig.12A and B).

In the greenhouse, other pathogens suchas Cylindrocladium and Myrothecium, bothon Spathiphyllum, also were effectivelycontrolled (Fig. 13A and B). However,weekly applications of RootShield wererequired, as opposed to less frequent appli-cations.

A great need in agriculture, particularlyfor crops such as vegetables and strawber-ries in California and Florida, is an effec-tive replacement for methyl bromide. Con-ceptually, a broadly effective and rhizo-sphere competent biocontrol agent shouldbe useful. However, there are such diverseand numerous pathogens that a stand-alonebiocontrol agent probably cannot be totallyeffective. Recently, a combination of ozonefumigation followed by T-22 has shownpromise on strawberries in California. T-22alone, ozone alone, and ozone fumigationfollowed by T-22 were compared in onetrial; in another part of the same field, thestandard methyl bromide fumigation andno treatment were compared. Yields with

Fig. 10. Control of Rhizoctonia solani by Trichoderma harzianum strain T-22 added as commercial product RootShield. (A) Freshweight of Catharanthus cv. Peppermint Cooler in the greenhouse in the presence of the pathogen (AG-4) as affected by treatmentwith Ban-rot (etridiazole and thiophanate methyl) or RootShield. Plants were grown in Earthgro composted soil (George Elliot, Uni-versity of Connecticut, Storrs, CT, and Wade Elmer, Connecticut Agricultural Experiment Station, New Haven, CT, unpublisheddata). (B) Comparison of effects of a single application of RootShield drench and Cleary’s 3336 (thiophanate methyl) on top grade ofpoinsettias. Pathogen was added to pots with all treatments except untreated control (BioWorks data from A. R. Chase, Chase Re-search Gardens, Mt. Aukum, CA). (C) Comparison of effects on plant height of zonal geraniums of a single addition of RootShieldgranules 7 days before inoculation with the pathogen and 10 days before transplanting with Cleary’s 3336, applied according tolabel directions. Data generated by Jean Williams-Woodward, Department of Plant Pathology, University of Georgia, in cooperatio nwith BioWorks. In all cases, asterisks above bars indicate significant differences ( P = 0.05).

Fig. 9. Appearance of poinsettias grown in a commercial greenhouse in a trial estab-lished by Mark Arena, Clemson University, in cooperation with BioWorks. First plant(far left) received no soil fungicide treatment, second received a single treatment ofBan-rot (etridiazole and thiophanate methyl), third plant received a single treatment ofBan-rot plus monthly applications of Subdue (metalaxyl) and Cleary’s 3336(thiophanate methyl), and plant on right received a single early application of Root-Shield, all according to label directions. This photograph is the property of BioWorksand has been published in GM Pro magazine.


the ozone + T-22 (RootShield granule)treatment or the standard methyl bromidetreatment were similar, and the combina-tion was significantly better than either T-22 alone or ozone alone (Fig. 14).

All results discussed above have beendirected toward control of root and soilpathogens. However, T-22 also is effectivein the control of fruit and foliar diseaseswhen applied as a spray to these plantparts. Diseases controlled include powderymildews on Catharanthus and pumpkins(Fig. 15A and B). Similar formulations areactive against B. cinerea on greenhousecrops and strawberry (Fig. 16C and D) andagainst downy mildews (Fig. 16E). T-22has also demonstrated activity against B.cinerea on grape, although results were notalways consistent (31), and against turf-grass pathogens such as R. solani (brown

patch), Pythium spp., and S. homoeocarpa(dollar spot) (54,55). For these appli-cations, T-22 must be applied frequently, atleast once every 10 days when diseasepressure is high, since it cannot extensivelygrow on and colonize newly formed leaftissues. However, on flowers or fruits, it ishighly persistent; an application to eithergrape or strawberry flowers results in ahigh percentage of colonization of theimmature fruits (unpublished). Spores of T-22 germinate on strawberry flowers (G. E.Harman and R. Petzoldt, unpublished) andturf leaves (55). However, they may notgerminate well on strawberry leaves. Themethod and adjuvants required foreffective control are very important, butthis topic is beyond the scope of thisarticle. T-22 is not unique in its ability tocontrol powdery mildews and B. cinerea.

T. harzianum strain T-39, the activeingredient of Trichodex, also controls thesepathogens (24).

Further, we have obtained encouragingresults with bee delivery of T-22, a conceptpioneered by John Sutton and his col-leagues at Guelph (65). Conidia of T.harzianum are smaller than pollen grainsand can adhere to the body of a bee in asimilar manner. Bees exiting the hive passthrough a device that requires them tocome into contact with products containingthese spores. They subsequently deliversubstantial amounts of T-22 or similarfungi to the strawberry or other flowers. Inour hands, this method of delivery is moreeffective than spray applications for con-trol of B. cinerea and has proven as effec-tive, over several years and trials, as stan-dard chemical applications (Fig. 16E) (48).

The last two sections have provided nu-merous examples of the uses of a singlebiocontrol agent, T. harzianum strain T-22.However, T-22 and the products basedupon it are management tools for growers.It is not magical nor is it a “silver bullet”that solves all problems. A summary oflimitations follows:• T-22 is strictly preventative. It cannot

control existing diseases, and so a goodsystemic fungicide must be used if dis-eases already exist.

• It is less effective against systemic dis-eases than against more superficial ones.Therefore, it is more effective, for ex-ample, against Fusarium crown and rootrot than against Fusarium wilts (18,36).

• In conditions of high or very high dis-ease pressure, T-22 should be used aspart of an integrated chemical–biologi-cal system. For example, for control ofB. cinerea on strawberries in Florida, itsbest use is probably as a tank mix or asan alternating spray to reduce, but noteliminate, the chemical fungicide appli-cation.

• In other cases, maximum benefit to thecrop requires use of both biological and

Fig. 11. Control of Pythium spp. by Trichoderma harzianum strain T-22 added in the form of commercial product RootShield. (A)Comparison of Subdue (metalaxyl) and RootShield drench in control of root rot of geraniums caused by P. ultimum. Data generatedby Gary Moorman of Pennsylvania State University in cooperation with BioWorks. (B) Comparison of Subdue and RootShield gran-ules in control of Pythium root rot as measured by foliage dry weight in greenhouse impatiens and (C) in petunias. Data generatedby Jean Williams-Woodward, University of Georgia, in cooperation with BioWorks. Asterisks placed above bars indicate significantdifferences (P ≥ 0.05).

Fig. 12. Incidence of Fusarium basal rot (solid bars) of red onions at end of seasonand yield (hatched bars) as affected by T-22 PlanterBox seed treatment or an in-furrowdrench (1 kg/ha as RootShield drench) of T-22. Onion plants were direct seeded in amuck soil near Oswego, New York, in a randomized complete block design with fourreplications per treatment. Data generated by J. van der Heide, R. Petzoldt, and G.Harman, Cornell University.


chemical agents. For example, the com-bination of chemical seed treatment formaximum seed and seedling protectiontogether with the long-term root protec-tion and enhancement by T-22 is highlyeffective.

• While T-22 is extremely persistent onroot surfaces, it does not persist at bio-logically significant levels in the ab-sence of roots. Perhaps the only excep-tion to this generalization is in highlyorganic soils such as those in which on-ions are grown in upstate New York (seediscussion regarding onions and a Sudexcover crop).

• In spite of the pictures presented in thefigures, T-22 does not always, or per-haps not even in the majority of situa-tions, give obvious visual enhancementof plant growth or yield. T-22 providestolerance to a variety of biological andedaphic stresses. If no stresses occur andplants are always growing at near-opti-mal conditions, T-22 can provide littlevisual or yield improvement.Thus, T-22 is a highly versatile biocon-

trol and plant growth–enhancing agent. Ithas been the subject of intense public andprivate sector development for about 15years. A critical consideration in all ofthese studies is the mechanisms wherebyT-22 and other BCAs exert their beneficialeffects.

Dogma 4. Biocontrol agents have sim-ple mechanisms of action that are con-trolled by one or only a few genes andgene products.

Our expectations of the abilities of bio-control agents are partially conditioned byour expectations of their mechanisms ofaction. If we assume that a specific agent(or group of agents such as Trichodermaspp.) has a single or a very limited numberof mechanisms of action, we could con-clude that its activity might be specific toparticular crops or pathogens. However, ifwe determine that there are many differentbiochemicals, genes, and even generalmodes of action for a specific BCA, then itis much more reasonable that the particularorganism might have manifold and diverseadvantages.

Further, the general conceptual frame-work in which we view the particular or-ganism also affects our thinking about itspotential uses. In my view, Trichodermaspp. frequently are very numerous andeven dominant in agricultural soils becausethey persist and multiply in the presence ofhealthy plant roots. In the absence ofhealthy roots, their numbers are likely todecline. Certainly this is true of T-22. If itis generally true of T. harzianum, we needto consider these fungi as opportunisticplant root colonists or even symbionts. Ifso, then it is reasonable to assume that theywill have developed numerous mechanismsto promote their ecological niche, i.e.,abundant and healthy plant roots.

The following are the mechanisms bywhich strains of T. harzianum function.The first three are the ones by which thesefungi have always been considered tofunction; those added in the last 1 to 3years are in italics. In addition, it is ex-tremely likely that other mechanisms existbut have not yet been discovered.• Mycoparasitism• Antibiosis• Competition for nutrients or space• Tolerance to stress through enhanced

root and plant development• Induced resistance• Solubilization and sequestration of inor-

ganic nutrients• Inactivation of the pathogen’s enzymes

Mycoparasitism. Mycoparasitism haslong been considered an important mecha-nism of action of biocontrol by Tricho-derma spp. This is a complex process thatinvolves tropic growth of the biocontrolagent toward target fungi, lectin-mediatedcoiling of attachment of Trichoderma hy-phae to the pathogen, and finally attack anddissolution of the target fungus’ cell wallby the activity of enzymes, which may beassociated with physical penetration of thecell wall (14). Probably the killing of targethyphae a short distance away from Tricho-derma hyphae (50,55) can be consideredsimilar to mycoparasitism. Numerous sepa-rate genes and gene products have beenproposed to be involved in mycoparasitic

Fig. 14. Cumulative yield of strawberries from a trial in 1997-98 at Monterey Bay Acad-emy, Watsonville, CA, following application of standard rates of methyl bromide: chlo-ropicrin (67:33 at 325 kg/ha), Trichoderma harzianum strain T-22 applied as Root-Shield granules incorporated into the soil at the rate of 135 kg/ha prior to planting,ozone applied at the rate of about 400 kg/ha and combination of preplant applicationof ozone followed by T-22. Bars with dissimilar letters are significantly different (P =0.05). Methyl bromide + chloropicrin and untreated control data were from other partsof the same field but were not part of the replicated trial that included T-22, ozone, andthe combination. Pathogens present in soil included Verticillium and Phytophthoraspp. Data courtesy of John Duniway, University of California, Davis. Other data by J.Duniway indicate significant responses to combinations of T-22 and ozone in othersoils and experiments.

Fig. 13. Comparison of weekly doses of RootShield and the fungicide Medallion oncontrol of (A) Cylindrocladium and (B) Myrothecium petiole rots on Spathiphyllum. Allexperiments were done in randomized complete block designs, and asterisks indicatesignificant differences (P = 0.05). Data from A. R. Chase, Chase Research Gardens,Inc., Mt. Aukum, CA, in cooperation with BioWorks.


interactions. As recently as 1990, we na-ively expected to test the involvement of“ the Trichoderma chitinase and β-1,3-glu-canase” in biocontrol by inactivating thegenes encoding these two enzymes. Wenow know that for each of these two func-tional groups, there are multiple classes ofenzymes, and within each class, there aredistinctly different enzymes. In 1998, Lo-rito listed 10 separate chitinolytic enzymesalone (57). Similar levels of diversity existwith β-1,3-glucanases (8). In addition, β-1,6-glucanases (56) and proteases (26)likely also are involved. For mycoparasit-ism of Pythiaceous fungi, β-1,4-glucanasesmay also be important (76). To add evenmore complexity, peptaibol antibiotics arespecifically produced by T. harzianum inthe presence of fungal cell walls, so theyprobably also should be considered as partof the mycoparasitic complex (67). Addingstill further to the complexity is the factthat the regulation of individual genes pre-sumably involved in mycoparasitism dif-fers; for example, the 42-kDa endo-chitinase is induced before T. harzianumcomes into contact with B. cinerea, whilethe 72-kDa N-acetylhexosaminidase isinduced only after the two fungi are indirect contact (82). Therefore, a single stepin the mycoparasitic process of T. harzia-num may involve more than 20 separategenes and gene products under complexregulatory control. Further, most of thesegene products are synergistic with oneanother (see 57 for a summary). Given thisentire arsenal of synergistic gene productsthat are part of only one mechanism bywhich Trichoderma species attack and gainnutrition from other fungi, it is not sur-prising that this genus has been reported tobe pathogenic against, and provide controlof, very diverse groups of fungi (14).

It should also not be surprising, in viewof this complexity, that studies with thisorganism involving knock-out or overpro-ducing mutants for single genes providecontradictory results. In one recent study, a

strain of T. harzianum deficient in the abil-ity to produce endochitinase had reducedability to control B. cinerea but increasedability to control R. solani (80). In anotherstudy, activity of the same enzyme eitherwas deleted or increased in T. harzianum,but these changes had no effect on biocon-trol ability against R. solani or S. rolfsii(12). In a third study (4), this same enzymewas disrupted or overproduced in T. virens,and the resulting strains were found tohave decreased or increased biocontrolability, respectively, toward R. solani rela-tive to the wild strain. Thus, in three stud-ies, almost every conceivable result(ranging from decrease to no effect to in-crease of biocontrol ability) was obtainedby deletion of a presumed biocontrol gene.Finally, transformed plants expressing thissame endochitinase have much greaterresistance to several plant pathogens thando nonexpressing plants (9,59). This, to-gether with the strong antifungal activity ofthis enzyme against a variety of plant-pathogenic fungi (57), suggests that thisenzyme probably is involved in biocontrol.However, since there are so many differentgene products that may also be involved,nonexpression or overexpression of anyone product may provide ambiguous re-sults.

Antibiotics. Antibiotics have long beensuggested to be involved in biocontrol byTrichoderma (78). Sivasithamparam andGhisalberti (70) list 43 substances pro-duced by Trichoderma spp. that have anti-biotic activity (enzymes are not included).Of these, alkyl pyrones, isonitriles,polyketides, peptaibols, diketopiperazines,sesquiterpenes, and steroids have fre-quently been associated with biocontrolactivity of some species and strains ofTrichoderma (40). A substantial number ofreports show that mutation to eliminateproduction of specific antibiotics is associ-ated, in some strains, with a loss of activityagainst particular pathogens (40). How-ever, particular antibiotics that may be

important in activity of one strain may noteven be produced by another highly effec-tive biocontrol strain of Trichoderma. Forexample, gliotoxin appears to be necessaryfor activity of the T. virens strain that is theactive ingredient of SoilGard, but it is notproduced by T-22. Again, to add to thecomplexity, many of these antibiotics aresynergistic with cell wall degrading en-zymes (21,58).

Further, as noted regarding the study byWoo et al. (80), some mutants preparedwithout activity of a specific componentexpected to be involved in biocontrol mayactually be more active than the parentalstrain. For example, Graeme-Cook andFaull (27) prepared UV mutants of a strainof T. harzianum that produced the antibi-otic 6-n-pentyl pyrone but lacked ability toproduce isonitrile antibiotics. Mutantswere obtained that possessed unexpectedproperties, including a newfound ability toproduce isonitrile antibiotics. These twoclasses of antibiotics are different chemi-cally and probably arise from distinctlydifferent pathways (70), so it is unlikelythat the UV mutation simply gave rise to abranch in a biosynthetic pathway to pro-duce an alternative product. Instead, asGraeme-Cook and Faull (27) stated,“There are several silent pathways forantibiotic production whose expression hasbeen lost.” By this reasoning, the mutationwould have induced changes in regulatorycontrols that activated the “lost” pathway.These results strongly suggest that theparental strain possessed cryptic genes thatwere not expressed until their activitieswere in some way released through themutation process. This alteration of ex-pression indicates that biocontrol strains ofTrichoderma (and no doubt other mi-crobes) have genes that may only be ex-pressed if some change in regulatory proc-esses occurs. Therefore, studies withknock-out mutants, no matter how care-fully the study is done to avoid changes toother than the gene of interest, may iden-tify phenomena that act, by analogy, like“snake’s teeth.” If a snake’s fangs are lostor removed, others take their place. If ac-tivity of one gene is deleted and a mecha-nism of biocontrol is lost, another may takeits place.

Competition. Competition for space ornutrients has long been considered one ofthe “classical” mechanisms of biocontrolby Trichoderma spp. (14). However, inmany cases, this mechanism was surmisedto occur because no evidence for myco-parasitism or antibiosis could be discov-ered in a particular interaction. Competi-tion very likely is an important mechanismof biocontrol, but it is extremely difficultto prove. Is the displacement of other or-ganisms by either T. virens or T-22 a con-sequence of competition or some othermechanism? Elad et al. (22) presentedinformation regarding biocontrol of B.cinerea by T. harzianum strain T39. B.

Fig. 15. Appearance of Catharanthus cv. Parasol plants grown in planting mix at aboutpH 7 in the absence and presence of Trichoderma harzianum strain T-22 applied to thesoil mix as RootShield. Experiment conducted by George Elliot, Department of PlantScience, University of Connecticut, Storrs. Photograph by Russell Wallace, BioWorks.


cinerea conidia require external nutrientsfor germination and infection. When con-idia of T-39 were applied to leaves, germi-nation of conidia of the pathogen wasslowed, an effect attributed in part to com-petition (22).

Tolerance to biotic and abiotic stressesthrough enhanced root development.Another possible mechanism recentlygaining credence is tolerance to stressthrough enhanced root and plant develop-ment. The drought tolerance and enhancednitrogen utilization indicated in Figures 6and 7 are examples of this mechanism. Theenhanced rooting by T-22 probably alsoinduces tolerance to pests that it does notdirectly control. For example, we do notbelieve that T-22 has the ability to controlPhytophthora spp. because it has nomechanism to intercept or attack zoo-spores. Indeed, in several studies, it wasfound to have no effect on this pathogen(e.g., 71). However, growers have indi-cated that Phytophthora-attacked plantswere larger in the presence of T-22 than inits absence. One possible explanation forthis result is that the larger root systems ofplants colonized by T-22 were better ableto withstand the damaging effects of thepathogen.

Solubilization and sequestration ofinorganic plant nutrients. In soil, variousplant nutrients undergo complex transitionsfrom soluble to insoluble forms thatstrongly influence their accessibility andabsorption by roots. Microorganisms maystrongly influence these transitions (2).Iron and manganese in particular have beeninvestigated with regard to both microbialsolubilization of oxidized forms of theseelements and their influence on plant dis-ease (28). Pseudomonas spp. produce com-pounds (siderophores) with very highaffinities for iron. Iron chelated with thesesiderophores is unavailable to plant patho-gens, so their activity is thereby reduced,but plant roots can take up iron in this formeither directly or after reduction of Fe3+ toFe2+ (7). Manganese is a microelementessential for diverse physiological func-tions in plants, including both plant growthand disease resistance (28). Only the Mn2+

form of this element is soluble; the morehighly oxidized forms are insoluble. Sev-eral pathogens, including Streptomycesscabies and G. graminis var. tritici, canoxidize manganese and thereby inactivate amajor part of the plant defense mecha-nisms (43,44).

Earlier, we documented the abilities ofstrain T-22 to enhance nitrogen use effi-ciency in corn. However, this strain canalso solubilize a number of poorly solublenutrients. In in vitro studies, T-22 has beenshown to solubilize rock phosphate, Znmetal, Mn4+, Fe3+, and Cu2+; these activi-ties are not due to medium acidification orproduction of organic acids (2). There is nocommon biochemical mechanism for theseabilities. Strain T-22 produces separate

substances that reduce and chelate Fe3+,while for Mn4+ only reductive materials areproduced. The substances that reduce Cu2+

or Fe3+ are separate biochemical entities(2). Therefore, this strain of T. harzianumproduces a large number of chemicals in-volved in nutrient solubilization.

A direct role for the nutrient solubiliza-tion and chelating abilities of Trichodermametabolites has not yet been demonstrated,but circumstantial evidence of its ability tosolubilize iron and make it usable to plantsis available. Catharanthus is intolerant ofalkaline potting media because of suscepti-

Fig. 16. Control of various foliar and fruit diseases by spray applications of Tricho-derma harzianum strain T-22 as the prototype commercial formulation TopShield. (A)Comparison of Cleary’s 3336 (thiophanate methyl) and T-22, both applied with LatronB-1956 added as an adjuvant in control of powdery mildew on New Guinea impatiensin the greenhouse. Data from M. Daughtrey, Long Island Horticultural Research Labo-ratory, Cornell University, in cooperation with BioWorks. (B) Control of powdery mil-dew (Sphaerotheca fuliginea) on upper surfaces of field-grown pumpkins with strobi-lurin fungicide Quadris applied on a 14-day schedule and T-22 applied on a 7-dayschedule. This information is part of a larger study (61). (C) Comparison of Chipco26019 (2.4 lb/liter) and T-22 (7 g/liter) for control of Botrytis cinerea on geraniums.Data from M. Daughtrey in cooperation with BioWorks. (D) Comparison of field appli-cations of the chemical fungicide Topsin M with T-22 either as a spray (TopShield) orby bee delivery. For spray applications, T-22 was applied at the rate of 3.2 kg/ha, whilefor bee delivery, a dry, powdered formulation of T-22 containing about 4 × 10 8 CFU/gwas placed at exit of hive so that bees had to pass through it as they exited (48). (E) Acomparison of Subdue Maxx (mefenoxam, 37 mg/liter) and TopShield (3 g/liter) appliedto the point of drip on a 7-day schedule for control of downy mildew ( Peronosporaantirrhini) on snapdragon. Infected plants were intermingled with healthy ones, andspores were distributed using a fan throughout the trial. Data are from A. R. Ch ase,Chase Research Gardens, Inc., Mt. Aukum, CA). In all cases, asterisks indicate thatdata are significantly different, with four asterisks different from two ( P = 0.05).


bility to iron deficiency at pH values aboveabout 6.5. Catharanthus cv. Parasol plantsgrown in a soilless potting mix with a pHof about 7 were highly chlorotic in theabsence of T-22 but less chlorotic whengrown in a medium containing RootShield(Fig. 15). This difference probably is aconsequence of enhanced iron uptake, sug-gesting that T-22 can increase iron avail-ability. Plant tissue culture analyses areunderway to test the hypothesis of en-hanced iron nutrition (G. Elliot, personalcommunication).

Induced resistance. Elicitation of re-sistance in plants by Trichoderma spp. hasbeen a subject of several papers and isbecoming a more researched topic. Studiesthrough 1997 have been reviewed by Bai-ley and Lumsden (5). Of particular note isthe ability of a xylanase or other elicitorsfrom Trichoderma spp. to induce resistance(3,5,11).

Some Trichoderma strains clearly arepotent inducers of SAR-like responses.Strain T39 of T. harzianum, which is theactive ingredient in Trichodex, can be in-oculated onto roots or onto leaves and pro-vide control of disease caused by B. cine-rea on leaves spatially separated from thesite of application of the biocontrol agent(19). Analyses of protected leaves to whichT39 was not applied demonstrated that thebiocontrol agent was not present. The levelof protection afforded by T39 applied toroots was similar to the known bacterialSAR-inducing organism Pseudomonasaeruginosa KMPCH (19). Elad and hiscolleagues (22) consider induced resistanceto be the primary method whereby T39controls powdery mildews. By extension,we would presume that T-22 has a similarmode of action against this pathogen.

In another recent study (81), cucumberswere grown in the presence or absence ofstrain T-203 in an axenic hydroponic sys-tem. Plants were always larger in the pres-ence of T-203 than in its absence and, un-like T-22, this strain penetrated rootcortical tissue. Cell walls of the roots werestrengthened in the area of penetration.Both chitinase and peroxidase activitieswere increased in both root and leaf tissuesof treated seedlings, which is an indicationof SAR (81).

Finally, Howell and his colleagues (41)also have demonstrated the presence ofSAR induced by T. virens. Effective nativestrains of T. virens usually producegliotoxin or gliovirin. Mutants were pre-pared that lacked both mycoparasitic abil-ity and the capacity to produce these orother detectable antibiotics. Surprisingly,many of the mutants were as effective ormore effective than the parental strains inbiocontrol of R. solani (39,42). This highlevel of protection was associated withsignificantly enhanced levels of the variousterpenoid phytoalexins known to be in-volved in disease resistance in cotton (41).Levels of phytoalexin production induced

by the mutants were significantly greaterthan the parental antibiotic-producingstrains. Further, as a seed protectant, it issuperior to our strain T-22, which has noknown ability to enhance phytoalexin pro-duction in cotton. Thus, at least a portionof the mechanism of action of the mutantstrains of T. virens is probably SAR (41).

Inactivation of the pathogen’s en-zymes. Yet another mechanism of biocon-trol by Trichoderma spp. has been at leasttentatively identified. B. cinerea dependsupon production of pectolytic, cutinolytic,and cellulolytic enzymes to infect livingplants (45). However, conidia of twostrains of T. harzianum (T39 andNCIM1185), when applied to the leaves,produce a serine protease that is capable ofdegrading the pathogen’s plant cell walldegrading enzymes and thereby reducingthe ability of the pathogen to infect theplant (23). The biocontrol activity of T39could be enhanced by adding additionalquantities of its protease; further, severalprotease inhibitors reduced the biocontrolactivity of T39. Interestingly, while theproteases of T39 could be detected on leafsurfaces, no activity of chitinolytic or β-1,3-glucanolytic enzymes, which are pre-sumed to be involved in mycoparasitism,could be detected. Thus, there is a solidcase for the role of proteases from T.harzianum in inactivation of the pathogen’sprimary modes of ingress into plants. Theauthors suggest that the proteases may bedirectly toxic to germination of the patho-gen and also may inactivate its enzymes(23).

In conclusion, there is an almost bewil-dering array of mechanisms by whichTrichoderma spp. exert biocontrol. Thus, itis not surprising that different strains havevery different biocontrol capabilities; evenmutants of the same original strain mayexhibit very different biocontrol mecha-nisms. Further, it is not surprising that, inthe case of Trichoderma, both dogmas oneand two are false. With this array of bio-logical weapons, these fungi should behighly versatile and have very little speci-ficity as to the pathogens they control.Definitive studies on single mechanisms ofbiocontrol are rare because there are somany genes and gene products involved inbiocontrol and because these strains canadapt to the loss of one mechanism byturning on another.

Dogma 5. Registration of BCAs withregulatory agencies is relatively fast,inexpensive, and simple. This dogma is asubset of the concept that biocontrolagents of plant pathogens can becomeboth profitable and useful.

Commercialization of biocontrol agentsrequires several steps, beginning with ini-tial discovery and then proceeding throughtesting of efficacy, prototype and thencommercial production, extensive large-scale field testing, toxicology and envi-ronmental tests, registration, and market-

ing. Nonprofit research institutions arewell suited only to the first two steps, andcommercial companies are required for thelast several steps. For agents used in con-trol of plant disease, these steps have beentaken by small to medium-sized companieswhere the required levels of innovation andtechnology development have been verysubstantial. As a consequence, the proc-esses, procedures, and equipment requiredfor economical production of biocontrolagents are jealously guarded and highlyproprietary. Together with patents andregistrations, this knowledge and capabilityare among the most valuable componentsof most of the companies that currentlyproduce biocontrol agents. As a result,these processes are unavailable to aca-demic researchers except in collaborationwith the biocontrol companies.

Costs just to reach and validate com-mercialization steps are expensive (severalmillion dollars) and require a substantialamount of time (two or more years) toaccomplish. Funding for commercial bio-control projects usually comes from in-vestors who are looking for a relativelyhigh payoff of their investment in a rela-tively short period of time. Unfortunately,increase in sales of most biocontrol prod-ucts does not ramp up quickly regardless ofthe product. Success in selling biocontrolproducts requires that potential users anddistributors be educated and convincedabout the value of a biological product thatis probably more conceptually difficult touse than standard pesticides. Further, oneof their chief advantages, that they mayhave numerous uses in addition to killingpests, adds to the complexity of the bio-logical paradigm. Therefore, adoption ofbiocontrol technologies by growers pro-ceeds more slowly than if these technolo-gies had simpler bases.

Given the above, it is relatively easy tosee why biologicals have never gained asubstantial market share. Costs are rela-tively high, time of development is rela-tively long, and gaining market share is aslow process. BioWorks and a few othercompanies are working to change this, butit is extremely difficult.

Time and expenses of complying withEPA, state, or international regulations aresignificant factors in the development andsuccessful commercialization of biocontrolagents.

I frequently hear statements in meetings,or read in grant proposals and other docu-ments, that EPA registrations are relativelyeasy and take less than a year to obtain.This is incorrect—in BioWorks’ experience(and that of other companies as well), EPAregistrations for microbials, especially forbroad-scale uses on food crops, commonlytake 3 years or more to obtain even if thereare no negative environmental or healthissues of the microbial in question. Thereare exceptions, but generally this timerequirement is correct. The time required


for registration of microbial pesticides hasincreased significantly since the passage ofthe Food Quality Protection Act. In manyother countries, time to registration is atleast as long, or even longer.

This slow pace causes problems forcompanies that attempt to comply withEPA regulations. Business plans may as-sume that a registration may be obtained in1 year or less since this concept has beenwidely publicized and stated. However,when timelines stretch out, sales do notoccur, and companies face bankruptcy.This is a major factor in the lack of bio-logical alternatives to synthetic pesticidesin the marketplace.

Companies therefore have a choice.They can cease to exist or they can sellproducts in violation of EPA regulations,which state that any product that makespesticidal claims is a pesticide and must beregistered before sales can occur. Somecompanies faced with this dilemma chooseto sell their product. Frequently, productsare labeled as plant growth promoters, andthen the product literature gives pages ofdata on the biocontrol activity of the activeingredient. I am aware of another approachthat states that the product is “EPA regis-tration pending.” Either of these cases is inblatant violation of EPA regulations, thelatter even stating that it is selling a pesti-cide without registration! To date, EPA andstate regulators have chosen NOT to en-force EPA and state regulations, so thenumber of unregistered products, espe-cially disease-controlling biocontrol prod-ucts, is becoming very large; perhaps onlyabout 5% of such products offered for saleare EPA registered. This is a disturbingsituation for the biocontrol industry. First,companies that comply with regulationsare severely penalized by being unable tosell in markets where unregistered micro-bial pesticides already are gaining a foot-hold and by being encumbered by the highcosts of obtaining appropriate state andfederal registrations. More importantly, asthe number of unregulated products prolif-erates, sooner or later it is likely that therewill be placed for sale a microbial withhealth or environmental hazards. The en-suing furor is likely to be extremely dam-aging to the entire biocontrol industry. Thisis, in my view, a serious matter that thebiocontrol community should considercarefully.

In summary, it will require several yearsand several to many millions of dollars tobring a single biocontrol agent to marketand to become profitable. I frequently readproposals and other documents that decrythe “silver bullet” (single biocontrol agent)approach. Therefore, the use of and dis-covery of multiple agents and mixtures isfashionable. In my view, it will be eco-nomically impossible to bring such com-plex mixtures to market. For EPA registra-tion alone, the time and funds required canbe multiplied by the number of agents in

the proposed product (unless, of course,EPA continues to avoid enforcement of itsown laws).

Therefore, I view the development ofwidely adapted and broadly active agentsas the only economically feasible approachto the development of commercially usefulbiocontrol. Contrary to frequently stateddogmas of biocontrol, such agents do exist,and they can provide broad-spectrum ac-tivity with unique advantages to growersand other users.

AcknowledgmentsI thank Kristen Ondik for her very capable

editorial assistance and John Barnard for provid-ing statistical expertise and analyses. I also thankDon Mathre and R. J. Cook, Charles Howell, IlanChet, and Yigal Elad for providing access to un-published data and manuscripts. I thank theBioWorks employees and its university and pri-vate cooperators for use and review of data. Thereare also a number of other cooperators ofBioWorks and my program at Cornell who haveprovided useful insights and information. DonHuber is one of these and was the first to suggestto me that one mechanism of biocontrol/growthpromotion by Trichoderma spp. may be solubili-zation of sparingly soluble or insoluble plantnutrients. Key portions of this work were fundedat Cornell in part by the US-Israel BinationalAgricultural Research and Development (BARD)Fund, including IS-2880-97 and US-1224-86, bythe Cornell Center for Advanced Technology inBiotechnology, which is sponsored by the NewYork State Science and Technology Foundationand industrial partners, by the USDA IR-4program, and by grants from the NY State SweetCorn and Onion Grower’s Associations. Inaddition, research at BioWorks was funded in partby grants from the Small Business InnovationResearch grant programs of the United StatesDepartment of Agriculture and the EnvironmentalProtection Agency.

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Dr. Harman is a professor in the Departments of Horticultural Sciences andPlant Pathology of Cornell University, Geneva, New York. He also is acofounder, consultant, and vice president for technology development ofBioWorks, Inc., also in Geneva. He received his B.S. degree in botany fromColorado State University in 1966 and completed requirements for his Ph.D.from Oregon State University in 1969. He was a postdoctoral associate at NorthCarolina State University from 1969 to 1970. In 1970, he joined the faculty ofCornell University, where he has remained. He received the Award of Merit fromthe American Phytopathological Society’s northeastern division and is a Fellowof the Society. Recently, he coedited a two-volume book, Trichoderma andGliocladium, which is a comprehensive monograph of these fungi. His interestsand research are directed toward the biological control of plant pests andcommercial development of biocontrol technologies. Included in this range ofinterests are mechanisms and uses of microbial biocontrol agents and of theirgenes and gene products.

Gary E. Harman

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