Screening of antimicrobial activities in Trichoderma isolates

representing three Trichoderma sections

Juan A. VIZCAINO1, Luis SANZ1, Angela BASILIO2, Francisca VICENTE2, Santiago GUTIERREZ3,

M. Rosa HERMOSA1 and Enrique MONTE1*

1Spanish-Portuguese Center of Agricultural Research (CIALE), Department of Microbiology and Genetics, University ofSalamanca, Edificio Departamental lab 208, Plaza Doctores de la Reina s/n, ES-37007 Salamanca, Spain.2Center for Basic Research, Merck, Sharp & Dohme de Espana S. A., Calle Josefa Valcarcel 38, ES-28027 Madrid, Spain.3University of Leon, Campus of Ponferrada, Superior and Technical Universitary School of Agricultural Engineers, Area ofMicrobiology, Avda. Astorga s/n, ES 24400 Ponferrada, Spain.E-mail : emv@usal.es

Received 22 July 2004; accepted 20 July 2005.

Methanol extracts from 24 Trichoderma isolates, selected as biocontrol agents and representating different species and

genotypes from three of the four taxonomic sections of this genus (T. sect. Trichoderma, T. sect. Pachybasium andT. sect. Longibrachiatum) were screened for antibacterial, anti-yeast and antifungal activities against a panel of sevenbacteria, seven yeasts and six filamentous fungi previously used in similar studies. Two different growth media were

tested (potato dextrose broth and CYS80), and all isolates included in the antimicrobial tests showed at least oneinhibitory activity against one of the target microorganisms in one of the two culture media. No statistically significantdifferences were detected in the number of active strains between the two culture media, but the highest number of

inhibitory strains against bacteria and fungi were found in strains from Trichoderma sect. Pachybasium, whereas strainsfrom T. sect. Longibrachiatum showed the highest anti-yeast values. In all cases, a correlation was found between thestrains that were active against yeasts and fungi. However, some degree of variability was detected for strains within the

same taxonomic section. In general terms, strains from T. asperellum (mainly in CYS80 medium), and T. longibrachiatumgave the best non-enzymatic antimicrobial profiles.

INTRODUCTION

Species of Trichoderma are commonly used in the bio-control of soilborne plant pathogens of several econ-omically important plant-pathogenic fungi (Howell1998). Although T. harzianum is the species of thisgenus most frequently used in biocontrol, molecularinformation from this genus has revealed thatT. asperellum, T. atroviride and T. koningii are alsoeffective in the biocontrol of plant pathogens (Hermosaet al. 2000, Sanz et al. 2004). The classical mechanismsof control have included antibiosis, mycoparasitism,and competition for nutrients. Trichoderma strains in-hibit or kill plant-pathogenic fungi through productionof antifungal antibiotics and/or hydrolytic enzymes.The ability to promote growth and induce resistancein plants are activities which have also been described

for members of this genus (Monte 2001, Harman et al.2004).

Antibiotic production by Trichoderma spp. was firstdescribed by Weindling (1934). However, the studiescarried out by Dennis & Webster (1971a, b) addressedthe role of antibiosis in the control of plant pathogens(Howell 1998). Since Dennis & Webster’s work, theinterest in the secondary metabolites produced byTrichoderma has been substantial, and the isolationand characterization of a considerable number ofmetabolites has been achieved. There are numerousreports of compounds derived from Trichodermaspecies with antibiotic activity : volatile (e.g. ethylene,hydrogen cyanide, alcohols, monoterpenes, aldehydesand ketones up to C4 chain-length), and non-volatile(e.g. peptaibols and diketopiperazine-like gliotoxinand gliovirin) compounds. For an extensive review seeSivasithamparam & Ghisalberti (1998).

The genus Trichoderma is subdivided into foursections: Trichoderma, Pachybasium, Longibrachiatum,* Corresponding author.

Mycol. Res. 109 (12): 1397–1406 (December 2005). f The British Mycological Society 1397

doi:10.1017/S0953756205003898 Printed in the United Kingdom.

and Hypocrearum. The first three sections includethe most important species and strains withhigh biotechnological value (Lieckfeldt, Kuhls &Muthumeenakshi 1998).

The ability to produce antibiotics is dependent on themicroorganism, environment (pH and temperature)and substrate. A given Trichoderma species can pro-duce several antibiotic compounds and, in a similarway, a given antibiotic can be produced by differentTrichoderma species (Sivasithamparam & Ghisalberti1998). Moreover, different isolates of the same speciescan produce different compounds (Luckner 1990).The variability among strains of T. harzianum in theirability to reduce wheat take-all has been related to theproduction of antibiotic compounds (Ghisalberti et al.1990). It has been also demonstrated that antibioticsplay an important role in the antifungal activity ofT. virens (Howell & Stipanovic 1983). However, thespecies concepts for T. harzianum and T. virens havechanged (Gams & Bissett 1998, Gams & Meyer 1998).Molecular data derived from enzymes and DNA havebeen useful to characterize species more objectivelythan traditional methods (Lieckfeldt et al. 1998) andhave served to describe the biodiversity of biocontrolstrains within the genus (Grondona et al. 1997,Hermosa et al. 2000, 2004).

An increasing number of metabolites from naturalsources have been discovered by the application ofbiochemical assays that are used to identify metabolitesin an extract exhibiting a particular biochemicalactivity. In this way, biological activities other thanantibiotic properties have been recognized for manycompounds (Sivasithamparam & Ghisalberti 1998).

In this study we used two different media to assay theantibacterial, anti-yeast and antifungal activities, otherthan cell-wall-degrading enzymes, of 24 Trichodermastrains, representative of accepted species and geno-types, that were molecularly and biochemically charac-terized previously (Hermosa et al. 2004, Sanz et al.2004), and selected as biocontrol agents against suchplant-pathogenic fungi as Phoma betae, Rosellinianecatrix,Botrytis cinerea and Fusarium oxysporum f. sp.dianthi. The correlation between non-enzymatic bio-logical activities and their distribution in Trichodermais analyzed.

MATERIALS AND METHODS

Fungal isolates, maintenance and growth conditions

Trichoderma biocontrol strains investigated in thisstudy are listed in Table 1. Strains were stored atx80 xC in 50% glycerol. Trichoderma strains weregrown in potato dextrose agar (PDA, Difco, Detroit,MI) at 25 x for 4–7 d. A spore suspension of each strainwas obtained and an equal number of spores (106) wasused to inoculate 25 ml flasks containing 10 ml ofeither potato dextrose broth (PDB, Difco) or CYS80(sucrose 80 g lx1, yellow corn meal 50 g lx1, yeast

extract 1 g lx1 ; Suay et al. 2000). Each strain wascultured at 25 x and 220 rpm in constant dark for 14 d.

Target microorganisms

In vitro antimicrobial susceptibility tests were per-formed using a panel of 20 microorganisms thatincluded both human clinical pathogens and labora-tory control strains : Three Gram-negative bacteria(Pseudomonas aeruginosa MB979, Proteus vulgarisMB838 and Escherichia coli MB4926), three Gram-positive bacteria (Bacillus subtilisMB964, Enterococcusfaecium MB5571 and Staphylococcus aureus MB5393),one mycobacterium (Mycobacterium smegmatisMB2233), seven yeasts (Candida albicans MY1055,C. glabrata MY992, C. tropicalis MY1012, C. rugosaMY1022, Cryptococcus neoformans MY2062,Saccharomyces cerevisiae MY34 and S. cerevisiaeMY2141), and six filamentous fungi (Aspergillusfumigatus MF5668, A. niger MF442, Alternaria solaniMF3550, F. oxysporum MF4014, B. cinerea B05.10and Colletotrichum acutatum IMI 364856). Except forC. acutatum deposited by us in the Fungal GeneticResources Collection of CABI Bioscience (IMI,Egham) and B. cinerea, originally isolated from Vitisvinifera (Faretra 1988), all other test microorganismsbelonged to the Merck Sharp & Dohme (MSD)antimicrobial testing panel used for the screening ofnatural compounds with potential antibiotic activities.

Some bacteria included in this test were selected bytheir special resistant attributes previously described:S. aureus MB5393 was methicillin-resistant, E. faeciumMB5571 was resistant to vancomycin and b-lactamantibiotics, M. smegmatis MB2233 was resistantto aminoglycosides, macrolides and penicillin andP. aeruginosa MB979 was resistant to cephalosporins(up to the third generation), macrolides, penicillin andimipenem (Zak 1980, Al-Obeid, Collatz & Gutmann1990, Davis & Stone 1986, Sunderam et al. 1986).

Antimicrobial activity assays

The Bacillus subtilis MB964 assay plates were preparedby adding 1 ml of a spore suspension (Difco) directly to1 liter of cooled nutrient agar yeast extract medium(nutrient agar 23 g lx1, yeast extract 2 g lx1). Inoculafor the remaining bacterial strains were prepared byadding the contents of thawed cryovials to the relevantmedium and they were incubated overnight at 37 x.Enterococcus faecium MB5571, E. coli MB4926 andStaphylococcus aureus MB5393 were grown in shakencultures of brain heart infusion broth (BHI, Difco,37 g lx1) at 37 x for the two first or 28 x for the last one.Mycobacterium smegmatis MB2233 was grown innutrient broth supplemented with Tween 80 (nutrientbroth 8 g lx1, 0.2% Tween 80) at 37 x. Pseudomonasaeruginosa MB979 and P. vulgaris MB838 were grownin nutrient broth yeast extract (nutrient broth 8 g lx1,yeast extract 2 g lx1) at 37 x static or shaken,

Screening Trichoderma for antimicrobial activities 1398

respectively. The bacterial assay plates were preparedby inoculating the same cooled media plus agar(15 g lx1) for each microorganism with 3.3% of theinoculum adjusted to an optical density (OD) range of0.22–0.35 at 660 nm.

Thawed cryovials of the stock inocula of Candidaspecies and Cryptococcus neoformans were used to seedSauboraud dextrose broth (Difco) in culture inocu-lation flasks that were incubated overnight at 28 x.Stock cultures of the two strains of Saccharomycescerevisiae were used to inoculate yeast peptone adeninedextrose-KCl broth (peptone 20 g lx1, yeast extract10 g lx1, adenine 0.04 g lx1, glucose 20 g lx1, KCl45 g lx1). Suspensions were adjusted to 40% transmit-tance at OD 660 nm and were added to yeast nitrogenbase-dextrose agar (yeast nitrogen base 6.75 g lx1,dextrose 1 g lx1, agar 15 g lx1) in the proportion of10 ml lx1.

For target fungi, stock conidial suspensions weregrown on oatmeal agar (Difco), excepting Fusariumoxysporum MF4014, which was grown on PDA.A spore suspension was obtained from each strain anddiluted into yeast nitrogen base broth to 65% trans-mittance at OD 660 nm. Ten ml of this inoculum wereadded to 1 liter of yeast nitrogen base-dextrose agar.In all cases, 100-ml aliquots of the seeded agar mediawere poured into Nunc (Nalge Nunc International,Rochester, NJ) square plates (24r24 cm).

Trichoderma methanol extracts were prepared bymixing 2 ml of the production culture with 2 ml of100% methanol, shaking for 15 min and then cen-trifuged at 1500 g for 15 min. Two-ml aliquots of themethanol extracts were evaporated to half their volumein a nitrogen flow in order to increase the concentrationof the metabolites and reduce any toxic effect due tothe solvent. Ten ml of the Trichoderma extracts wereapplied into the surface of the seeded assay plateswith the target microorganims, which were incubatedat either 28 x (yeasts and filamentous fungi) or 37 x

(bacteria). Inhibition zones around the applicationpoints were measured after 24 h. A 1:1 solution ofDMSO and methanol was used as a negative control.

We considered that the antimicrobial activity waspositive when an inhibition zone around the appli-cation point was observed. For example, if for onesingle strain of Trichoderma, the inhibition zones werefound in 10 of the 20 target microorganisms, the totalantimicrobial activity was considered to be the 50%.

Statistical studies

Statistical analyses of the distribution of the anti-microbial activities into the three Trichoderma sectionswere carried out using the non-parametric Kruskall-Wallis test, and the distribution of antimicrobialactivities into the two culture media was done with the

Table 1. Trichoderma isolates used in this study. The strain code is the same used in a previous study (Hermosa et al. 2004).

Strain code SpeciesaGenetic source

collectionb Geographical origin, source

Trichoderma sect. Pachybasium

8 T. harzianum NBT 8 Argentina, soil

18 T. harzianum NBT 18 Argentina, soil

24 T. harzianum IMI 352940 Spain, soil

36 Trichoderma sp. IMI 293162 India, soil

37 Trichoderma sp. IMI 296235 Colombia, soil

39 T. harzianum IMI 298372 UK, soil

40 T. harzianum IMI 298373 UK, soil

41 T. harzianum IMI 298374 UK, soil

42 T. harzianum IMI 304056 India, soil

43 T. harzianum IMI 304057 India, Lycopersicon esculentum

45 T. harzianum IMI 300082 Sri Lanka, soil

46 T. harzianum NBT 46 Brazil, soil

48 T. harzianum NBT 48 Brazil, soil

Trichoderma sect. Trichoderma

3 T. asperellum IMI 20179 France, soil

25 T. asperellum IMI 296237 Colombia, soil

51 T. asperellum NBT 51 Asia, unknown

53 T. asperellum IMI 20268 Spain, soil

11 T. atroviride IMI 352941 France, soil

33 T. atroviride IMI 352939 UK, soil

35 T. atroviride IMI 281112 Zimbabwe, soil

50 Trichoderma sp. NBT 50 Czech Republic, industrial oil

Trichoderma sect. Longibrachiatum

6 H. jecorina IMI 113135 Unknown, air

44 T. longibrachiatum IMI 304058 India, soil

52 T. longibrachiatum NBT 52 Slovakia, water treatment plant

a Trichoderma sp. codified as 36 and 37 are close to T. aureoviride, and Trichoderma sp. codified as 50 is close to T. viride.b IMI, CABI Bioscience, Egham (United Kingdom); and NBT, Newbiotechnic, Seville (Spain).

Juan A. Vizcaıno and others 1399

Kolgomorov-Smirnov test. The likeliness function tocompare joint differences between samples was calcu-lated with the likelihood ratio test and further studiedwith the unilateral Fisher test. The tests are includedin the SPSS 10.0 for Macintosh software (SPSS,Chicago, IL).

RESULTS

Evaluation of antimicrobial activity

The antimicrobial activities of single Trichodermaisolates against the entire spectrum of target microbesare shown in Tables 2A–C for the medium PDB, andin the Tables 3A–C for the medium CYS80. All 24representative isolates included in the antimicrobialtests showed at least one inhibitory activity against atleast one of the target microorganisms in at least one ofthe two culture media.

Tables 2A and 3A show the antibacterial activitiesdetected in the panel. In PDB (Table 2A), strains fromT. sect. Longibrachiatum were able to inhibit theenterobacteria E. coli MB4926 and P. vulgaris MB838but were not effective against P. aeruginosa MB979and M. smegmatis MB2233. Only T. sect. Pachybasiumstrains controlled this mycobacterium. In CYS80(Table 3A), strains from T. asperellum and T. longi-brachiatum had the best inhibition profiles withgood behaviour against Bacillus subtilis MB964,Enterococcus faecium MB5571 and Staphylococcusaureus MB5393 (Gram positive bacteria), and E. coliMB4926 and Proteus vulgaris MB838 (enterobacteria)tested in the panel. However, T. atroviride strains hadno consistent antibacterial inhibition profile. StrainsNBT 8, 18, 46 and 48, belonging to the genotype gt3 ofT. harzianum (Hermosa et al. 2004), had the highestinhibitory effect compared with the other members ofT. sect. Pachybasium.

Tables 2B and 3B show the anti-yeast profiles inPDB and CYS80, respectively. T. longibrachiatumstrains can inhibit the complete set of yeasts tested,except for NBT 44 against the basidiomycetousCryptococcus neoformans MY2062, in CYS80 medium.However, the inhibition rates for T. longibrachiatumNBT 44 against the four Candida spp. studied, con-firmed this strain to be a good candidate for furtheranti-yeast studies in clinical research. In a similar way,T. harzianum genotype gt3 members (NBT 8, 18, 46and 48) gave the best results against Candida in CYS80medium. By contrast, the two strains of Saccharomycescerevisiae were very resistant to most of the strainsassayed, and only T. longibrachiatum and T. atrovirideNBT 35 showed growth inhibition of this yeast.

Antifungal profiles are shown in Tables 2C and 3C.Results demonstrate that T. harzianum NBT 37,T. atrovirideNBT 11 and T. virideNBT 50 have neitherantifungal nor anti-yeast activity. Fusarium oxysporumMF4014 was the most resistant filamentous fungus inthis test, since it was inhibited only by T. asperellum

strains NBT25, 51 and 53, and by T. harzianumNBT18, 40 and 41 in CYS80 medium. The inhibitionrates for other fungi were similar in both media used.The most efficient A. solani inhibitors were strains ofdifferent genotypes of T. harzianum. Strains of T.asperellum and T. atroviride showed good inhibitionof Aspergillus fumigatus and Botrytis cinerea ; T. longi-brachiatum strains inhibited A. niger, A. fumigatusand Colletotrichum acutatum in PDB but only of A.fumigatus and B. cinerea in CYS80.

The percentages of presence of total and partialantimicrobial activities against the target microorgan-isms were calculated for each Trichoderma strainin both media and are given in Table 4. The highestproportion of total antimicrobial activities occurredwith T. longibrachiatum NBT 52 and T. harzianumNBT 48 in PDB medium (75 and 70%, respectively),and with T. harzianum NBT 18 and 46, T. asperellumNBT 51, T. atroviride NBT 35 and T. longibrachiatumNBT 52 (75% in all cases) in CYS80 medium.The lowest proportion of these total activities wasfound in T. virideNBT 50 (no activity) and T. atrovirideNBT 11 (5.0%) in PDB; and in CYS80, the strainsT. harzianum NBT 37 (no activity), and NBT 39, 42and 45, T. viride NBT 50, and again T. atroviride NBT11 (5.0%). When we merged the data from the strainsthat belonged to the same taxonomic sections, thehighest percentage of total antimicrobial activity wasfound in the T. sect. Longibrachiatum with mean valuesfor aggregate antimicrobial activity of 65.0 and 71.7%,in PDB and CYS80 respectively.

Antimicrobial behaviour was separated intoantibacterial, anti-yeast and antifungal activities.The highest rate of antibacterial activities in PDBmedium was found in T. harzianum NBT 24, 39 and48, all of them belonging to T. sect. Pachybasium.In CYS80 medium, a 100% of antibacterial activitywas observed in T. harzianum NBT 46, showing thestrainsHypocrea jecorinaNBT 6,T. harzianumNBT 18,T. longibrachiatum NBT 44 and 52, and T. asperellumNBT 51 an 85.7%. Among the anti-yeast tests, thehighest activities were detected in strains of T. sect.Longibrachiatum : T. longibrachiatumNBT 52 (100% inboth media), H. jecorina NBT 6 (100% in CYS80)and T. longibrachiatum NBT 44 (85.7% in bothmedia). The highest antifungal activity was detected inT. harzianum NBT 18 and NBT40 in CYS80 medium(83.3%).

Statistical analysis

In PDB, there were no statistically significantdifferences (Kruskal-Wallis test) among the threeTrichoderma sections in the number of strains activeagainst bacteria (P=0.066) and yeasts (P=0.067).However, the P-values were very close to the 5% levelof significance and besides, there was a significantvariability between the three sections in the number ofstrains active against filamentous fungi (P=0.043).

Screening Trichoderma for antimicrobial activities 1400

Table 2. Antibiotic activities of the Trichoderma strains grown in PDB medium and sorted in taxonomic sections (Hermosa et al. 2004). Inside T. sect. Trichoderma the data are separated between

T. asperellum (strains 3, 25, 51 and 53) and T. atroviride (strains 11, 33, 35 and 50).

(A) Antibacteriala (B) Anti-yeastb (C) Antifungalc

Strain

code BAC ENT ECO MYC PROT PSEU STAP CALB CGLA CRUG CTRO CRYP

SAC

2141

SAC

34 ALT ANIG AFUM BOT COL FUS

T. sect. Pachybasium

8 + x x x +++ x x x x x x x x x x x + x x x18 x x x x x x x ++ x +++ x x x x x x x x x x24 ++ + + + + + x x x x + + x x + x + + + x36 ++ x x x +++ x ++ +++ x +++ ++ x x x + x + + x x37 ++ x + + x x x ++ x x x x x x x x x x x x39 ++ + + + +++ x ++ ++ + + x x x x x x + + + x40 + + x x x x + x x x x x x x x x x x x x41 x + x x x x + x x x x + x x x x x + x x42 + x x x + x x x x x x x x x x x + x x x43 + x x + +++ x x ++ + +++ + x x x x x +++ + x x45 x + + x x x + x x x x x x x x x x x x x46 x x x x x x x x x + x x x x x x + x x x48 +++ + + + +++ x ++ ++ + + + + x x x x + + + x

T. sect. Trichoderma

3 +++ +++ + x x x ++ ++ + + ++ + x x x x + + x x25 x x x x x x x x + + x + x x x x + + x x51 x x x x ++ x x x x x x x x x x x + x x x53 + x x x x x x x x + x + x x x x x + x x11 x + x x x x x x x x x x x x x x x x x x33 +++ x + x x x ++ ++ + + ++ + x x x x + + x x35 + x x x x + x ++ x + ++ x + ++ x x ++ + x x50 x x x x x x x x x x x x x x x x x x x x

T. sect. Longibrachiatum

6 + x + x + x x x ++ + + x x x x x x x + x44 + + + x + x + +++ +++ +++ +++ x + + x + + x + x52 + x + x + x + + +++ + + + + + x + + + + x

Totald 16 9 10 5 11 2 10 11 9 14 10 8 3 3 2 2 15 12 6 0

Activities were classified according to the diameter of the inhibition zones around the point of application of the sample: +++, more or equal than 15 mm; ++, less than 15 mm; +, hazy/very hazy

inhibition zone; x, without activity.a BAC=Bacillus subtilis MB964, ENT=Enterococcus faecium MB5571, ECO=Escherichia coli MB4926, MYC=Mycobacterium smegmatis MB2233, PROT=Pseudomonas vulgaris MB838,

PSEU=Pseudomonas aeruginosa MB979, STAP=Staphylococcus aureus MB5393.b CALB=Candida albicans MY1055, CGLA=C. glabrata MY992, CRUG=C. rugosa MY1022, CTRO=C. tropicalis MY1012, CRYP=Cryptococcus neoformans MY2062, SAC 2141=Saccharomyces

cerevisiae MY2141, SAC 34=S. cerevisiae MY34.c ALT=Alternaria solani MF3550, ANIG=Aspergillus niger MF442, AFUM=A. fumigatus MF5668, BOT=Botrytis cinerea B05.10, COL=Colletotrichum acutatum IMI 364856, FUS=Fusarium oxy-

sporum MF4014.d Total number of active strains.

JuanA.Vizca

ınoandothers

1401

Table 3. Antibiotic activities of the Trichoderma strains grown in CYS80 medium and sorted in taxonomic sections (Hermosa et al. 2004). Inside the T. sect. Trichoderma the data are separated between

T. asperellum (strains 3, 25, 51 and 53) and T. atroviride (strains 11, 33, 35 and 50).

(A) Antibacterial (B) Anti-yeast (C) Antifungal

Strain

code BAC ENT ECO MYC PROT PSEU STAP CALB CGLA CRUG CTRO CRYP

SAC

2141

SAC

34 ALT ANIG AFUM BOT COL FUS

T. sect. Pachybasium

8 +++ + x + +++ x ++ ++ + + + x x x x x + + + x18 ++ + + + +++ x + + + + + x x x + x + + + +24 + x + x x x x x x x x x x x x x x x x x36 + + x x x x x + x + ++ x x x x x x + x x37 x x x x x x x x x x x x x x x x x x x x39 x + x x x x x x x x x x x x x x x x x x40 + x + x x + x ++ x + + + x x + x + + + +41 + + x x x x x x x + x + x x + x + x x +42 x + x x x x x x x x x x x x x x x x x x43 x + x x x x x x x x x x x x + x + x x x45 x x x x x x x x x + x x x x x x x x x x46 +++ + + + +++ + ++ +++ + + + + x x x x + + + x48 ++ x x + +++ x x + + + x x x x x x + x x x

T. sect. Trichoderma

3 ++ ++ x + + + x + + ++ x + x x x x + + x x25 +++ x + x + + + ++ + + ++ + x x x + + + x +51 +++ + + + +++ x ++ +++ + + + + x x x x + + + +53 ++ + + x x + + + + ++ x + x x x x + + x +11 x + x x x x x x x x x x x x x x x x x x33 ++ ++ x x + + x x + + x + x x x x + + x x35 + + + + + x x ++ x +++ ++ + x x + x + + + x50 x + x x x x x x x x x x x x x x x x x x

T. sect. Longibrachiatum

6 ++ + + x + + ++ ++ ++ +++ ++ ++ + ++ x x x + x x44 ++ + + + + x ++ ++ ++ + + x + ++ x x + + x x52 ++ + + + + x + + ++ + + + + ++ x x + + x x

Totala 17 18 11 9 12 7 9 14 12 17 11 11 3 3 5 1 15 14 6 6

See Table 2 for explanation of symbols and codes.a Total number of active strains.

Screen

ingTrich

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aforantim

icrobialactiv

ities1402

To the contrary, in CYS80, however statisticallysignificant differences were detected between the threesections in the number of strains active against bacteria(P=0.041) and yeasts (P=0.01), but they were notamong the strains active against filamentous fungi.

When the Kolgomorov-Smirnov test was applied, nostatistically significant differences were found in thenumber of active strains between the two culture mediafor the total and partial antimicrobial activities.

The likelihood ratio test compares jointprobabilities, to facilitate the detection of differencesbetween samples. When this statistical test was appliedto all the Trichoderma strains and both culture media,we found that the number of strains with antibacterialactivity was not comparable with the number of strainswith anti-yeast activity (P=0.812), but it was compar-able with the presence of active strains againstfilamentous fungi (P=0.029). In the same way, thepresence of active strains against yeasts and fila-mentous fungi was also comparable (P=0.000). Tocheck that this correlation was statistically significant,the unilateral Fisher test was applied, and this corre-lation was confirmed in both cases. When we separatedthe data corresponding to the two culture media, the

correlation was found only between the anti-yeast andantifungal activities (P=0.024 in PDB, P=0.000 inCYS80) and later confirmed using the unilateral Fishertest (P=0.038 in PDB, P=0.00 in CYS80).

When the data from T. sections Pachybasium andTrichoderma were separated, similar results wereobserved in CYS80 for joint antifungal and anti-yeastactivities : P=0.012 in T. sect. Pachybasium andP=0.023 in T. sect Trichoderma. However, the unilat-eral Fisher test (P=0.128) demonstrated that thecorrelation was not statistically significant in T. sect.Pachybasium. On the other hand, no correlation wasfound within these sections for the activities obtained inPDB medium.

Susceptibility of the target microorganisms

The susceptibility of the target microorganisms isalso shown in Tables 2A–2C and 3A–3C. The mostsusceptible bacteria were the Gram-positive Entero-coccus faeciumMB 5571 (inhibited by the extracts of 18strains of Trichoderma in CYS80) and Bacillus subtilisMB964 (inhibited by the extracts of 16 and 17 strains inPDB and CYS80, respectively). In contrast, only two

Table 4. Percentages of total and partial antimicrobial activities of 24 Trichoderma strains against the seven bacteria, seven yeasts and six

filamentous fungi included in the assays, in PDB and CYS80 media. Values in each column, corresponding to a partial antimicrobial activity

on a given medium, that are followed by the same letter do not differ significantly (Kruskal-Wallis test, 95%).

Strain code Antibacterial activities Anti-yeast activities Antifungal activities Total activities

T. sect.

Pachybasium

PDBA CYS80A PDBA CYS80A PDBA CYS80A PDB CYS80

8 28.6 71.4 0 57.1 16.7 50.0 15.0 60.0

18 0 85.7 28.6 57.1 0 83.3 10.0 75.0

24 85.7 28.6 28.6 0 66.7 0 60.0 10.0

36 42.9 28.6 42.9 42.9 50.0 16.7 45.0 30.0

37 42.9 0 14.3 0 0 0 20.0 0

39 85.7 14.3 42.9 0 50.0 0 60.0 5.0

40 42.9 42.9 0 57.1 0 83.3 15.0 60.0

41 28.6 28.6 14.3 28.6 16.7 50.0 20.0 35.0

42 28.6 14.3 0 0 16.7 0 15.0 5.0

43 42.9 14.3 57.1 0 33.3 33.3 45.0 15.0

45 42.9 0 0 14.3 0 0 15.0 5.0

46 0 100 14.3 71.4 16.7 50.0 10.0 75.0

48 85.7 42.9 71.4 42.9 50.0 66.7 70.0 50.0

Total 42,9 36,3 24,2 28,6 24,4 29,5 30,8 31,5

T. sect.

Trichoderma

PDBA CYS80B PDBA CYS80B PDBB CYS80A PDB CYS80

3 57.1 71.4 71.4 57.1 33.3 33.3 55.0 55.0

25 0 71.4 42.9 71.4 33.3 66.7 25.0 70.0

51 14.3 85.7 0 71.4 16.7 66.7 10.0 75.0

53 14.3 71.4 28.6 57.1 16.7 50.0 20.0 60.0

11 14.3 14.3 0 0 0 0 5.0 5.0

33 42.9 57.1 71.4 42.9 33.3 33.3 50.0 45.0

35 28.6 71.4 71.4 57.1 33.3 66.7 45.0 75.0

50 0 14.3 0 0 0 0 0 5.0

Total 21,4 57,1 35,7 44,6 16,7 35,1 25,6 47,5

T. sect.

Longibrachiatum

PDBA CYS80C PDBA CYS80C PDBC CYS80A PDB CYS80

6 42.9 85.7 42.9 100 66.7 16.7 50.0 70.0

44 71.4 85.7 85.7 85.7 50.0 33.3 70.0 70.0

52 57.1 85.7 100 100 66.7 33.3 75.0 75.0

Total 57,1 85,7 76,2 95,2 44,4 27,8 60,0 71,7

Juan A. Vizcaıno and others 1403

strains, T. harzianum NBT 24 and T. atroviride 35,showed activity against Pseudomonas aeruginosaMB979 in PDB (Table 2A).

The most susceptible yeast was Candida rugosaMY992 (inhibited by the extracts of 14 and 17 strains,in PDB and CYS80 media respectively). The most sus-ceptible filamentous fungi were Aspergillus fumigatusMF5668 (inhibited by the extracts of 15 strains in bothmedia) and B. cinerea (inhibited by the extracts of12 and 14 strains in PDB and CYS80, respectively).In contrast, no activity was found against Fusariumoxysporum MF4014 in PDB (Table 2C), and onlyT. asperellum NBT 25 was active against A. nigerMF442 in CYS80 (Table 3C).

DISCUSSION

In previous studies, we have explored antibioticproduction by strains of T. harzianum and T. atroviride(Keszler et al. 2000, Kiss et al. 2000) and identifiedputative peptide synthetase genes in T. harzianum,T. asperellum, T. atroviride and T. longibrachiatum(Vizcaıno et al. 2005). However, the characterizationof metabolites involved in antimicrobial activities ofTrichoderma is not an easy task, and there is a need tohave a better knowledge of antimicrobial activities,corresponding to a species or genotype before under-taking the chemical identification of compoundsresponsible of such activities. Besides, the cloning ofthe gene or genes coding for a given metabolite can bevery hard due to several factors like the large size ofthese genes, their modular and repetitive structure andsometimes their low level of expression. The objectiveof this study was to evaluate and to compare the abilityto produce bioactive secondary metabolites of a selec-ted set of 24 strains, representing Trichoderma speciesand genotypes previously characterized in our labora-tory (Hermosa et al. 2004, Sanz et al. 2004). Thefrequency of the resultant antimicrobial activities wasconsidered an indicator of the capability of the differenttaxa to produce bioactive secondary metabolites ofpotential therapeutic interest.

Samples with bioactive metabolites were extractedand concentrated using 100% methanol in order to getlow molecular weight metabolites, reducing to a mini-mum the probability of having large molecules such asenzymatic proteins with cell-wall-degrading activities inthe samples to be tested. As for the effectiveness of theextractionmethods, some studies showed that methanolextraction yielded higher antimicrobial activity thann-hexane and ethyl acetate (Febles et al. 1995) whereasin others chloroform was better than methanol andbenzene (Sastry & Rao 1994). It is clear that the use oforganic solvents always provides a higher efficiency inextracting antimicrobial activities compared with waterextraction (Rossel & Srivastava 1987). We selectedmethanol extraction for this study based on ourprevious experience (Suay et al. 2000, Gonzalez del Valet al. 2001).

In order to study the influence of the culture mediumin antimicrobial activities, two different media, PDBand CYS80, were used. However, no statistically sig-nificant differences were found in the number of activestrains when the total and three partial antimicrobialactivities were compared in the two culture media(Kolgomorov-Smirnov test). The three Trichodermasections tested, contained isolates with antimicrobialactivities against bacteria, yeasts or filamentous fungi.T. sect. Longibrachiatum includes excellent cellulaseproducing strains more than biocontrol agents.The isolates selected for the present study are rep-resentative of the two main species, T. longibrachiatumand H. jecorina (anamorph: T. reesei), of this sectionthat has eleven more, but less common, species (Kuhlset al. 1997).

All of the highest partial activities in both media,excepting the antifungal activity in PDB (higher inT. sect. Trichoderma), were found in the sect.Longibrachiatum, although it must have taken intoaccount the low number of isolates from this sectionincluded in the study since, compared with the othertwo sections, the number of genotypes referable toT. longibrachiatum found in nature is limited.

The isolates included in the T. sect Pachybasiumshowed a wide distribution of antimicrobial activities.This is a confirmation of the difficulty of clearly defin-ing species in this section, using biological activities.In contrast, a study based on biochemical attributesrevealed that someT. sect Pachybasium species exhibit ahigh stability of physiological characters (i.e., T. virensor T. spirale), whereas others such as T. harzianumexhibit a considerable diversity (Kubicek et al. 2003).In the Trichoderma sect Trichoderma, strains ofT. asperellum showed higher antimicrobial activitiesgenerally than those of T. atroviride. However, theopposite was demonstrated when other biocontrolactivities, including cell-wall-degrading enzymes (Sanzet al. 2002) and sporulation on the pathogen (Hermosaet al. 2000), were also involved.

In a previous study (Suay et al. 2000), the anti-microbial activity of 317 isolates of basidiomycetes wasevaluated against nine target microorganisms (eight ofthem are included in the present study). The culturemedium used was CYS80. Although the number of theTrichoderma strains in this study is much lower, wecompared the results from both studies, and in all casesthe percentages of activity were considerably higher forthe Trichoderma extracts in CYS80. The most suscep-tible strain in both studies was Bacillus subtilis MB964(70.8% of the extracts from Trichoderma, Table 4, and26.5% of the extracts from basidiomycetes). This strainwas also the most susceptible in a study with extractsfrom 44 species of seaweed (Gonzalez del Val et al.2001). On the other hand, the least susceptible strainin the assay with basidiomycetes and in the seaweedstudy, Mycobacterium smegmatis MB2233, did notbehave the same way with the Trichoderma extracts(37.5%). The percentages of activity of the extracts

Screening Trichoderma for antimicrobial activities 1404

from Trichoderma were always higher : Enterococcusfaecium MB5571 (75% from Trichoderma extracts,5.05% from basidiomycetes extracts), PseudomonasaeruginosaMB979 (29.2% and 4.73%), Staphylococcusaureus MB5393 (37.7% and 20.8%), C. albicansMY1055 (58.3% and 8.8%), and A. fumigatusMF5668 (62.5% and 12.9%, respectively). Althoughthe number of the Trichoderma strains in our study ismuch lower, these results are indicative of the abilityof these strains to produce bioactive metabolites. Forinstance, three T. harzianum, three T. asperellum, threeT. atroviride and one Hypocrea jecorina strains wereable to inhibit the growth of P. aeruginosa MB979, themost resistant bacterium of the MSD panel.

A low antimicrobial activity does not mean a badbiocontrol effect since a given strain can be moremycoparasitic than antibiotic, and this aspect canfacilitate its registration for agronomical use. The lowantimicrobial activity detected in T. atroviride NBT 11is remarkable, since it inhibits only E. faeciumMB 5571but it does have an excellent cell-wall-degradingenzyme production (Castillejo et al. 2001, Santorumet al. 2001), a good variety of protease, chitinase andb-1,3-glucanase isoenzymes (Sanz et al. 2004), and anaggressive mycoparasitic behaviour as biocontrol agentof phytopathogenic fungi at laboratory (Hermosa et al.2000) and field level (Perez de Algaba et al. 1992). Theidentification of volatile components in the fermen-tation broth of T. atrovirideNBT 11 was carried out byour group (Keszler et al. 2000) : nineteen pyrone anddioxolane derivatives and two aliphatic esters wereidentified. Although the culture broth was different, agood agreement is observed between the low anti-biotical activity and the volatile pattern of this strain.

Using the likelihood ratio tests, a correlation wasfound only between the strains that were active againstyeasts and filamentous fungi in the two culture media.The fungal wall is a complex structure composedtypically of chitin, b-1,3- and b-1,6-glucan, mannanand proteins, although wall composition frequentlyvaries markedly between species of fungi and yeasts(Adams 2004). The differences in the structuresbetween the bacterial and fungal walls make us tothink that the bioactive compounds produced by theseTrichoderma strains seem to affect mainly somecomponents of the fungal walls.

Some attempts have been done in order to find acorrelation between the structures of the Trichodermasecondary metabolites and the species that producethem (Ghisalberti & Sivasithamparam 1991, Siva-sithamparam & Ghisalberti 1998). In the present study,this correlation has not been found at the level of theantimicrobial activities, so we could imagine that it doesnot exist either at the level of the chemical structures.

In summary, our results indicate that the species ofTrichoderma considered in the present study have asignificant capacity to show a variety of antimicrobialactivities, which makes them interesting on naturalproduct screening programs mainly because an

important number of the isolates screened could inhibitthe most resistant microorganisms of our panel.The maximum number of active strains against bacteriaand filamentous fungi were detected in strains fromT. sect. Pachybasium. Those included in T. sect.Longibrachiatum showed the highest number of activestrains against yeasts. Taking into account the vari-ation detected for the antimicrobial activities at strainlevel within the different sections and species studied,the antimicrobial spectrum of a given strain mustbe evaluated individually. However, strains fromT. asperellum, mainly in CYS80, and T. longi-brachiatum showed the best non-enzymatic anti-microbial profiles against the MSD panel of targetmicroorganisms.

ACKNOWLEDGEMENTS

We would like to thank Antonio Llobell for his support in setting up

the NBT Trichoderma culture collection, to the CIBE-MSD tech-

nicians involved in this study, Fernando Pelaez for the facilities to

carry out the antimicrobial tests ; and Alicia Enguix for her support in

statistical studies. This work has been funded in part by the EU

project FAIRCT98-4140, the Spanish project INIA-RM03-008-C3-2

and the ‘Fundacion Andaluza de I+D’.

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