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TYLER AVIS
SPÉCIFICITE ET GÉNÉTIQUE DE P S E U D O Z W FLOCCULOSA, AGENT DE
LUTTE BIOLOGIQUE CONTRE LE BLANC
Thése
présentée
à la Faculté des études supérieures
de l'université Laval
pour l'obtention
du grade de Philosophiae Doctor (Ph. D.)
Département de phytologie
FACULTE DES SCIENCES DE L'AGRICULTURE ET DE L'ALIMENTATION
UNIVERSITE LAVAL
Q ~ B E C
AVRIL 2001
O Tyler Avis, 2001
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RÉsUMÉ COURT
La sélection d'une souche de Pseudozyrnaflocculoso la plus appropriée à introduire
dans le cadre d'un programme de lutte biologique repose sur une connaissance approfondie de
son mode d'action et de sa sp6cificité. Les acides gras inusités produits par P. flocculosa se
sont avérés être des principes actifs dans la répression des champignons de blanc par l'agent
antagoniste. Ces acides gras antifongiques s'insèrent dans les membranes cellulaires de
champignons et provoquent une desorganisation générale de ces dernières en haussant la
fluidité membranaire. La sensibilité des champignons de blanc est liée à un faible contenu en
stérols, facteur stabilisateur des membranes cellulaires. La production d'acides gras
antifongiques et la capacité de bio-contrôle du blanc sont des propriétés spécitiqws de l'espèce
P. jlocculosa, mais pas d'autres champignons du même genre. Chez les isolats connus de P.
flocculosa, trois souches sont identifiables sur la base d'études moléculaires de l'ADN
ribosomal et, en particulier, de microsatellites.
&SM LONG
L'objectif global de cette recherche etait d'etudier les propriétés et la spécificité des
mécanismes de l'activité antagoniste de l'agent Pseudozynrajlocculosa, afin de produire des
outils de sélectiondes isolats les plus appropriés à introduire dans le cadre &un programme de
lutte biologique contre le blanc des cultures semcoles.
La synthese chimique et la caractérisation biologique des acides gras antifongiques ont
montré que ces mttabolites étaient des principes actifs dans la répression des champignons de
blanc par P. flocculosri. Ce résultat pourrait permettre d'identifier des souches de P. jlocculosa
qui ont une production accrue en acides gras antifongiques et, de ce fait, cette caractéristique
pourrait servir à la sélection d'individus h haut potentiel antagoniste dans un programme de
lutte biologique.
Par ailleurs, le mode d'action spécifique de ces acides gras antifongiques repose sur
leur insertion dans les membranes cellulaires des champignons. Cette insertion cause une
désorganisation générale de la membrane fongique accompagnée d'une augmentation de la
fluidité membranaire. La sensibilité élevde de certains champignons face ii ces mttabolites est
liée à un faible taux de stérols, facteur stabilisateur des membranes biologiques. De plus, le
faible contenu en stdrols de S. fuliginea, agent causal du blanc du concombre, explique non
seulement sa sensibilitt aux acides gras antifongiques mais aussi sa grande susceptibilité face
B P. flocculosa. Ces rdsultats donnent un outil de prddiction de l'efficacitd de P. j?occulosa
basé sur son mode à'action et les propriétés intrinsèques des agents pathogénes ciblés.
L'étude physiologique et ghétique comparée des isolats connus de P. flocculosa et
d'autres champignons du genre Pseudozyma a montrC que différentes souches de P. jlocculosa
étaient identifiables sur la base d'analyses génetiques (rDNA et microsatellites), de
caractCristiques biochimiques (production d'acides gras antifongiques) et biologiques
(propriétés de bio-contrôle du blanc). Ces résultats donnent des outils précieux dans
l'identification et la sélection d'agents antagonistes appropries pour un programme de lutte
biologique.
iii
L'ensemble de ces resultats fait ressortir des découvertes importantes dans
Papprofondissement des connaissances fondamentales sur les propriétés de P. Jlocculosa. De
plus, cette recherche a produit des outils concrets qui aideront à prédire l'efficacité, la sélection
et le suivi des souches de P. flocculosa lors du processus de développement d'un bio-fongicide
pour le contrôle du blanc des cultures semcoles.
AVANT-PROPOS
Cette thèse comporte trois chapitres. Le premier porte sur la caractérisation biologique
d'acides gras antifongiques produits par l'agent de lutte biologique Pseudozymaj?occulosa. Le
deuxième chapitre se concentre sur les mécanismes sficifiques de l'activité biologique de ces
acides gras. Le troisjeme chapitre concerne la caractérisation génotypique et phhotypique des
différentes souches de P. flocnrlosa.
Tous ces chapitres ont étd rédigés en anglais sous forme de manuscrits afin de
permettre une diffision plus vaste des résultats. Cependant, un bref rdsume en français
précède chacun des chapitres. Les rdfdrences citées dans l'introduction et les conclusions
générales se retrouvent à la fin de la thèse, tandis que celles citées dans les sections rédigées
en anglais sont regroupées I la fui de ces dernières. En annexe, se trouve un manuscrit portant
sur les approches de caractérisation moléculaire d'agents de lutte biologique fongiques.
L'écriture de la présente thèse et la réalisation des travaux qui en sont à la base n'ont pu
être réalisées que par l'implication de certains individus.
Premièrement, je veux remercier mon directeur de thèse le Dr Richard Bélanger. II a
ma profonde reconnaissance pour m'avoir procuré toutes les facilités pour rendre terme cette
recherche, en plus de son soutien scientifique et fuiancier.
Je remercie également le Dr Richard Hamelin pour avoir mis a ma disposition le
materiel et l'expertise de son laboratoire.
Je suis également recomaissant au personnel du laboratoire de bio-contrôle, du Centre
de recherche en horticulture et du Centre de Foresterie des Laurentides. En particulier,
Caroline Labbe pour son soutien scientifique et le Dr Rénald Boulanger pour l'aide apportée
dans la partie chimique de la thtse. Mes remerciements s'aâressent aussi B Denise Auclair,
Hugo Germain et Nicole Lecours pour leur assistance technique.
Je tiens aussi à remercier le CRSNG, les Fonds FCAR et Plant Products Lt6e pour
avoir subventionné, en partie, mes Ctudes graduées.
Toute ma gratitude ii ma famille, Sophie Caron, Michel Maltais et la famille Gagnon
pour leur soutien moral tout au long de mes ttudes graduees.
RÉSUMÉ COURT ....... , ............................................................................................................................ i . .
&SU& LONG ... .... ... .. .. ....... ..... ... .. ... ...... . . . . . . . . ......... . . . . . . . . . . . . . ............. . . . . i i
AVANT-PROPOS .................................................................................................................................. iv
TABLE DES MATIERES. ................... ............................................. . ..................... ..... . . ................... v
. . . LISTE DES TABLEAUX ............................. . ..................... .... ..... .............. .. . ..... .............. ....~....~..~..~~...... ... . v111
LISTE DES FIGURES .......................................... . . - .............. . ............ ........................... ix
CHAPITRE 1 . P
iNTRODUCTION GENERALE .............. .... ... . . . ................ . . . . . . . . . . . . . . . . . . 1
1 .1. D~VELOPPEMENT D'UN BIOPESTICDE ..... ........ .......... ........,..... ... . . . .............. . . . . . . 2
1.2. P R O B L ~ M A ~ Q U E ........ ,, .........,........ . ........ . . . . . . . . . ........ . . . . . . . . . 3
1.3, MODE D'ACTION ............................................................................................................................... 4
1.3.1. Caructérisation biologique des acides gras antgongiques ..................................................... 4
1.3.2. Spécijicité des acides grac antifogique ....... . ... . . .. .. .. .. .. .. . .. .. . .. . .. .. . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . 6 1.4. CARACT~RISATION PHÉNOTYPIQUE ET G ~ O W P I Q U E DE P. FLOCCULOSA ............................. . . 7
Synthesis and biological characterization of (2)-9-hcptadecenoic and (2)-6-methyl-9-heptadecenoic acids, fatty acids with antibiotic activity prduced by Pseudozymafloccufoso ......................................... 10
ABSTRACT . . . ... .... ..... . .. . . . .... ... .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2
2.1 . INTRODUCTION ..................................................................................... ........................................... 1 3
2.2. METHODS AND MATERlALS ..... ........... ............. .... ............. . .......... ............... . . . .......... . 14
2.2.1. General chemical procedures and inimonents. .. .... ... .... .... ... ... .. . . . . . . .. ... .. ... .. . . . .. . . . ... . .. . .. ... . . . . . . .. 14
2.2.2. Synthesis of (2)-9-Heptadecenoic Acid.. ....... ... ... .. .. . ... .. ....... ... .. .. . .. . .. . .. .. . . . .. . .. . . ... ..... . .. ... . . ... .. . . . . 14
..................................................................... 2.2.3. Synthesis of (Z)-6-Methyl-9-Heptudecenoic Acid 16
2.2.4. Biological Activity of the S'thessUd Antibiotics on Fungal Material ................................... 20
............................................................................................................... 2.3. RESULTS AND DISCUSSION 2 1
REFERENCES ......................................................................................................................................... 2 5
CHAPITRE III
Specificity and mode of action of the antifungal fatty acid cis-9-heptadecenoic acid produced by Pseudozymajlocculosa: ............................................................................................................................. 31
ABSTRACT .............................................................................................................................................. 33
3.1 . WTRODUCTION ............................................................................................................................. 34
..................................................................................................... 3.2. MATEMALS AND METHODS 35
3.2.1. Fungal material ........................................................................................................................ 35
3.2.2. Synthesis of CHDA .................................................................................................................... 35
3.2.3. Effects of CHDA on /wgaf growth and spore germination ..................................................... 35
3.2.4. Exhoction of lipids .................................................................................................................... 36
3.2.5. Analysis of lipids ...................................................................................................................... 36
3.2.6. Evolution ufphospholipid unsaturotion ................................................................................... 38
3.2.7. Analysis of CHDA ...................................................................................................................... 38 . . 3.2.8. Stutzstical unulysis .................................................................................................................... 38
3.3. RESULTS .......................................................................................................................................... 39
3.3.1. General efects of CHDA on@@ ............................................................................................ 39
3.3.2. Total lipid composition ......................................................................................................... 39
3.3.3. Phospholipid fitty acid composition ......................................................................................... 40
3.3.4. Evohtion of phospholipid fa^ acid unsaturation .................................................................. 40
3.3.5. AnUlysis of CHDA ...................................................................................................................... 41
................................................................................................................................... 3.4. DISCUSSION 41
REFERENCES ......................................................................................................................................... 44
vii
CHAPITRE IV
Moleculai and physiological analysis of the powdery mildew antagonist Pseudozymajlocculosa and .................................................................................................................................................. related fiuigi 52
ABSTRACT .................................................................................................................................................. 53
............................................................................................................................. . 4.1 INTRODUCTION 55
..................................................................................................... 4.2. MATERIALS AND METHODS 56
................................................................................................................ 4.2.1. Biological Materiuls 56
.......................................................................................................................... 4.2.2. DIVA extraction 56
................................................................................................... 4.2.3. PCR ampl@catbn conditions 56
....................................................................................... 4.2.4. Nucleotide sequencing determination 57
.............................................................................................................. 4.2.5. Phylogenetic anolysis 58
.......................................................................................... 4.2.6. Production of antjfungal fatty acids 58
........................................................................................ 4.2.7. Biocontrol activiiy offugul isolates 59
4.3. RESaTS .......................................................................................................................................... 59
4.4. DISCUSSION ............................................................................................................................. 61
L I T E R A W CITED ............................................................................................................................ 63
CHAPITRE V . .
CONCLUSIONS GENERALES ............................................................................................................... 73
BIBLIOGRAPHIE ....................................................................................................................................... 7 9
ANNEXE A ........................ .. .................................................................................................................. 85
....................... Approaches to molecular characterization of fungal biocontrol agents: some case studies 85
Mii
LISTE DES TABLEAUX
Chapitre II
TABLE 1. Sphaerotheca jùligineognea conidial chain collapse following treatment with aqueous
.................................... solutions of antibiotic fatty acids fiom Pseudozyma flocculosa .30
Chapitre III
....................... TABLE 1. Inhibition and lipid analysis (percent dry weight) of selected h g i . 48
TABLE 2. Evolution of growth phase and phospholipid fatty acid unsaturation of selected b g i
.................... in absence (control) and presence (treated) of a sub-lethal dose of CHDA. 49
....................................................... TABLE 1. Fungal isolates and species used in this study. 66
..................................... TABLE 2. Polymerase chain reaction-arnplified regions and primers. 67
TABLE 3. Likelihood analysis of constrained and unconstrained trees fiom the combined
rDNA dataset. ............................................................................................................... 68
LISTE DES FIGURES
Figure 1. Étapes de développement d'un biopesticide ............................................................. 9
-- - - - - -
............................. Figure 1. Synthetic scheme for the synthesis of (2)-9-heptadecenoic acid 27
Figure 2. Synthetic scheme for the synthesis of (2)-6-methyl-9-heptadecenoic acid. ........... .28
Fipre 3. Growth of three-day liquid cultures of Cladosporium cucumerinum following
treatment with (2)-9-heptadecenoic and (2)-6-methyl-9-heptadecenoic acids at
concentrations ranging fiom O to 1 mg/rnl.. ................................................................... 29
CHAPITRE III
Figure 1. Proposed mode1 of activity of CHDA, an antifungai compound produced by P.
.............................................................................................................. occ cul osa. 50-5 1
. . . . . . . . . . . . . .. - - - .... - . .
Figure 1. Single most parsimonious tree infemd fiom partial sequences of the small nuclear
............................................................................................ subunit (nSSU) of rDNA.. -69
Fipre 2. Strict consensus of the five most parsimonious trees based on the combined dataset
............................................................................................. of three rDNA sequences. 7 0
Figure 3. Random amplified microsatellite fingerprints generated by GT primer. ................. 71
Figure 4. Random amplified microsatellite fingerprints generated by CCA primer. .............. 72
CHAPITRE 1
INTRODUCTION GENÉRALE
L'utilisation de microorganismes pour lutter biologiquement contre les maladies des
plantes est devenue une alternative B l'utilisation des pesticides chimiques en horticulture
(Baker et Cook, 1982; Whipps, 1997). Cependant, le processus du développement d'un agent
de lutte biologique est soumis B la rCglementation d'entités législatives telles le "Pest
Management Regulatory Agency" (PMRA) et le "United States Environmental Protection
Agency" (EPA), et aux contraintes commerciales inhérentes ih la mise en marche de produits
vivants (Cross et Polonenko, 1996; Fravel et al., 1999). C'est ainsi que l'emploi d'organismes
vivants dans un but de phytoprotection a gdnere de nouveaux ddfis pour les différents
intervenants du milieu horticole (compagnies, chercheurs, etc.) en ce qui concerne la
recherche, le d6veloppement et la commercialisation des produits phytosanitaires.
1.1. Développement d'un biopesticide
Le processus de ddveloppement d'un agent de lutte biologique, du laboratoire jusqu'à
son utilisation sur le marche, comporte plusieurs dtapes (Figure 1). En premier lieu vient la
découverte d'agents à potentiel antagoniste. La découverte se fait soit de façon empirique ou,
de façon plus courante, h la suite de recherches ciblées (Woodhead et al., 1990). Viennent
ensuite les tests d'efficacité, à petite et grande dchelles, visant à déterminer, in vitro et in vivo,
le potentiel biologique des agents antagonistes. Suite à ces dtudes, un certain nombre
d'organismes est retenu pour diriger le développement vers les candidats les plus prometteurs.
Les meilleurs candidats doivent alors être étudids quant h leur mode d'action. C'est également
à ce moment que les études toxicologiques sont mendes pour dvaluer les risques potentiels liés
à leur utilisation (Cross et Polonenko, 1996; Fravel et al., 1999). Ces deux dupes sont
déterminantes pour la continuation du processus de développement d'agents de lutte
biologique.
En cas de succts, on doit mettre sur pied un systhe de production du microorganisme
qui, de façon idéale, est efficace et peu coûteux (Amsellem et al., 1999; Fravel et al., 1998). Il
faut songer Cgalement au type de formulation qui permettra d'assurer la survie de l'organisme
et qui sera adequat pour l'utilisation dont on veut en faire. Ainsi, l'agent de lutte entre dans une
boucle de ddveloppement qui inclut la production massive de l'agent à i'echelle industrielle, la
formulation, qui assure la survie de l'agent, et les tests d'efficacité sur le terrain (Figure 1).
Cette boucle de développement vise B ddterminer (i) le meilleur système pour avoir un produit
B utilisation simple et compatible avec les pratiques culturales courantes et (ii) ks conditions
biologiques et environnementales requises qui permettront l'expression la plus complkte des
propriétés de l'agent de lutte. En parallèle A cette boucle, la stabilité en entreposage du produit
(Muller-Scharer et al., 2000; Rhodes, 1993) ainsi que sa stabilité ghdtique (Woodhead et al.,
1990) sont des paramétres qui doivent être ajustés afin d'assum l'eficacit& et la qualité du
produit à long terme.
Au-delà des aspects d'ordre technique ou technologique ci-haut mentionnés, le
développement complet d'un système de lutte biologique fait aussi appel i des considerations
légales et commerciales. La protection intellectuelle, par l'obtention de brevets couvrant les
technologies pertinentes, est une étape essentielle pour la continuation du développement
(Legget et Gleddie, 1995; Fravel et al., 1999). Ainsi, si une technologie ne peut être protégée,
il est probable que peu de compagnies soient interessees dans le développement d'un agent de
lutte.
Enfin, suite aux succès des diffdrentes dtapes énumérées, le produit pourra enfin être
homologué pour la mise en marché. Ces aspects ne seront réalises avec succès, que si les
expertises commerciale, légale et financiére existent, et que si les infrastructures pour la
production industrielle du produit sont disponibles ou peuvent être construites (Legget et
Gleddie, 1995).
Au laboratoire de Bio-contrôle du Centre de recherche en horticulture de l'université
Laval, un biofongicide, le SporodexTM, est en voie de ddveloppement et de commercialisation.
Ce biofongicide sera utilisé pour lutter contre le blanc (ou oïdium) d'espèces semcoles dont le
concombre et le rosier. Comme mentiorné prdcddemment, plusieurs Ctapes de recherche
fondamentale sous-tendent le succés d'une telle entreprise.
Des &des visant l'approfondissement des connaissances de base sur l'agent de lutte,
en l'occurrence, Pseudosymaflocculos<1, sont menées dans l'objectif d'appuyer l'efficacité et
l'utilisation sdcuritaire de cet organisme par des bases scientifiques solides. En effet, une
connaissance approfondie de l'efficacité d'un agent de lutte, de son mode d'action, de sa
promettew(s) pour une &entuelle utilisation dans le cadre d'un programme de lutte biologique
(Benyagoub, 1996). De plus, la production industrielle du microorganisme ainsi que son
relachement dans un systéme agricole, peuvent amener des changements inattendus, d'où
l'importance de développer des outils précis et sensibles pour faire le suivi de l'agent de lutte
dm d'assurer la qualit6 du produit et la sécuritt! d'utilisation (Markovic et Markovic, 1998).
L'objectif global de cette recherche &ait d'étudier les propriWs et la spécificitd des
mecanismes de l'activité antagoniste de ragent P. flocculosa, afin de produire des outils de
sélection des isolats les plus appropriés h introduire dans le cadre d'un programme de lutte
biologique contre le blanc des cultures ~emcoles
Les objectifs spécifiques de cette thèse de doctorat &aient donc (i) de cibler, à l'aide
d'études biochimiques precises, le mode d'action de P. jlocculosa et (ii) de différencier P.
/locculoso d'autres espèces voisines par des méthodes génotypiques et phdnotypiques.
1.3. Mode d'action
1.3.1. Caractdrisation biologique des acides gras antifongiques
Pseudozyn>ajiocculosa (Traquair, L. A. Shaw Br Jarvis) Boekhout & Traquair
(Boekhout, 1995) est un champignon de type levuroide qui posshde une forte activité
antagoniste contre les agents pathoghnes responsables du blanc (Hajlaoui et Bélanger, 1991,
1993; Jarvis et al., 1989). Des expériences en serres commerciales ont montré qu'une
formulation à base de P. flocculosa procurait un haut taux de répression du blanc du rosier
(Sphaerotheca pannosa (Waller.:Fr.) Lev. var. rosae Woronich.) (Bureau, 1999) et du
concombre (Sphaerothecafulginea (Schlecht.) Pollaci) @ik et al., 1998).
Des btudes concomitantes en laboratoire ont r&eie que P. flocculosa &ait efficace
contre le blanc en causant la fuite de protdines et d'Clectrolytes des cellules fongiques
(Hajlaoui et al., 1994), une désorganisation du cytoplasme et la desintegration des membranes
cellulaires de mycdlium et des spores fongiques (Hajlaoui et al., 1992). Il a CtC Cgalement
montrd que cette activite antifongique se produisait sans activitd chitinolytique ni pénCtration
des structures fongiques par P. jZocculosa (Hajlaoui et al., 1992). Ces résultats suggéraient que
l'antagoniste agit par antibiose i s . par le relâchement de mbtabolites toxiques (Fravel, 1988).
Rdcemment, trois molécules & activité antifongique ont ttd isolées de filtrats de culture de P.
flocculosa. Ces molécules sont des acides gras insaturés inusités avec une chaîne principale de
dix-sept atomes de carbone dont certains comportent un groupement méthyle sur la chaîe
principale. Les molécules antifongiques identifiées sont les suivantes: l'acide 4-méthyle-7,110
heptadécadienoique (Choudhury et al., 1994)' l'acide 6-mdthyle-9-heptaâécenoïque et l'acide
9-heptadécenoïque (Benyagoub et al., l996a).
Dans le cas des champignons de blanc, agents pathogénes ciblés pour la lutte
biologique par P. flocculosa, il est trés dif'fïcile de cibler les rndcanismes par lesquels
P. flocculosa les répriment. En effet, &nt des parasites obligatoires, donc dépendant du
métabolisme de la plante hôte pour leur croissance, ils ne peuvent être cultivds sur des milieux
artificiels (Agrios, 1988). Pour cette raison, la plupart des études conduites sur les propriétés
antifongiques de P. flocculosa contre l'oïdium, jusqu'ii ce jour, ont été réalisees de façon
indirecte à l'aide de champignons modeles. Les résultats obtenus ont ensuite dté inférés aux
champignons de blanc. Par exemple, l'identification des acides gras toxiques a dte conduite
avec le champignon Cladosporium cucumerinum Ellis & Arh. (Benyagoub et al., 1996a).
Cependant, l'identification de tels composds ne signifie pas nécessairement que ces derniers
soient responsables de l'eflicacité de P. j7occuloso contre les oïdiums. En effet, des études ont
déjà montré que les fonctions ou propriétés identifiées d'un agent antagoniste n'étaient pas
necessairement les caractéristiques principales permettant d'expliquer leur potentiel de lutte
biologique. Par exemple, la production de chitinase chez Trichoderma sp., un autre agent de
lutte biologique, n'est aucunement impliquée dans son activité antagoniste chez les agents
pathogènes qui ne contiennent pas de chitine tels les Oomycétes (Howell et al., 2000; Thrane
et al., 2000; Deshpande, 1999). Il a aussi 6tt montré que ni la production de sidérophores, ni la
production d'antibiotiques par l'espèce Pseudomonas fluorescens n'&aient responsables de
l'activité biologique de ces bacteries contre Pythium aphanidermatum chez le concombre
(Ongena et al., 1999). 11 reste alors ii savoir si ces acides gras sont toxiques chez les
champignons de blanc et donc si les acides gras sont réellement les p ~ c i p e s actifs dans la
répression du blanc par P. flocculosa.
La premiere partie de cette thèse s'intdresse donc & la caractdrisation biologique des
acides gras antifongiques potentiellement impliques dans l'activite antagoniste de P.
flocculosa. La difficuht! il isoler quantitativement et h purifier les acides gras des filtrats de
culture est un des facteurs lirnitants dans l'étude de leun propriétds et la compr6hension de
leur mode d'action contre les champignons de blanc. Pour faciliter l'étude de l'implication
biologique de ces m&abolites, nous avons réalisé la synthèse de deux des acides gras actifs par
des mCthodes standards de chimie organique. Ces synthèses ont donné des quantités
suffisantes et la puretd requise pour la caractérisation biologique. Les acides gras
nouvellement synthCtisés ont CtC dtudies pour quantifier leurs activitds antifongiques
respectives en particulier en ce qui concerne filiginea, agent causal du blanc du concombre.
Cette étude a aussi étd mende pour Clucider la relation entre les différences structurales des
acides gras et leurs activites biologiques propres.
1.3.2. SpécificitC des acides gras antifongiques
La co~aissance du mode d'action spécifique de P. flocculosa demeure un atout
important dans la prédiction de l'efficacité de l'antagoniste A court et à long terme (Bélanger et
Deacon, 1996). La sensibilité aux acides gras fongiques dicterait ainsi la gamme d'hôtes de
l'agent antagoniste et devrait permettre de conclure sur la spécificitd de l'antagoniste quant aux
champignons ciblds pour la lutte biologique. Des Ctudes ont déjà montré que l'issue de la
relation agent pathog&ne/agent antagoniste dépend des propriétés intrinsèques de l'agent
pathogéne (Benyagoub et al., 1996b; Jones et Hancock, 1988) et des attributs physiologiques
de l'agent antagoniste (Bdlanger et Deacon, 1996). La sensibilitd relative d'un nombre restreint
de champignons aux acides gras produits par P. flocculosa a récemment Ct6 corrélée avec la
composition lipidique de leurs membranes cellulaires (Benyagoub et al., 1999). 11 a été
observé qu'un faible contenu en stérols ainsi qu'une insahiration ClevCe des chaînes acyles des
phospholipides membranaires semblaient responsables d'une sensibilité accrue des différents
organismes ib l'étude face aux acides gras toxiques. Dans le cadre de ces mêmes travaux, la
sensibilité des champignons semblait lite A une ddsorganisation plus importante de leurs
membranes cellulaires. Cette dernikre hypoth&se a dtt confhde subséquemment en mesurant
les changements de la fluiditd membranaire (degr6 de désorganisation) dans des membranes
artificielles (liposomes) (Benyagoub et al., 1996b). Cette perte d'intégritd membranaire serait
donc responsable de la fuite d'électrolytes, de la desorganisation du cytoplasme et, halement,
de la desintégration complète des cellules fongiques.
La deuxitme partie de cette recherche s'intdressait donc aux mécanismes moleculaires
de l'activitt! antifongique des acides gras toxiques, au site prkcis d'activité et A l'implication des
composantes lipidiques des champignons dans l'issue de la relation de sensibilitd/résistance.
Ainsi, des études ont dtd entreprises sur les membranes cellulaires d'une gamme plus
importante de champignons avec des caractCristiques connues en ce qui a trait h leurs
compositions lipidiques. Un champignon de blanc (S. fuliginea) a 6té inclus dans ces
expdriences afin d'établir la relation entre sa composition lipidique, sa sensibilité aux acides
gras toxiques et sa susceptibilité à P. flocculoso. De plus, une Localisation des acides gras
toxiques dans les fractions lipidiques de champignons traites a Ct6 conduite pour déceler leur
mode et site d'action dans les membranes fongiques.
1.4. Caract6risation phdnotypique et gkaotypique de P.jlocculloso
Plusieurs études ont montré que la sdlection de l'agent antagoniste le plus approprie
dépendait de l'&olution du contexte agronomique d'utilisation (Kiss et Nakasone, 1998;
Grondona et al., 1997). Par exemple, un agent antagoniste efficace dans des conditions
agronomiques précises peut être moins efficace lorsque certains facteurs changent tels l'agent
pathogène ciblé, les conditions culturales et les régions géographiques d'utilisation. Ceci est
d'autant plus vrai en ce qui concerne les propribtt!~ et fonctions spécifiques que l'agent
antagoniste doit posséder dans la manifestation de son pouvoir antagoniste. En somme, l'agent
de lutte le plus approprie doit être évaluC sur une base de cas par cas (Burns et Benson, 2000).
Il faut donc avoir des outils de sélection précis pour identifier et sélectionner le(s) individu(s)
le(s) plus efficace(s) et approprid(s) dans le cadre du ddveloppement d'un produit de lutte
biologique (Avis et al., 2001).
Dans ce contexte, la dernih partie de cette these s'intdressait ih l'&ude de la variabilitd
des isolats connus de P. flocculosa en ce qui a trait B leur potentiel antagoniste ainsi qu'à leur
pertinence pour leur d6veloppement dans le cadre d'un programme de lutte biologique contre
le blanc. Un maange de caractdristiques phhotypiques et génotypiques a CtC &tudie pour
évaluer la diversW des isolats connus de P. flocculosa et pour discriminer P. jlocculoso
d'autres champignons semblables. Au niveau gtnotypique, les etudes ont reposé sur
i'dvaluation de l'ADN ribosomique et les microsatellites. Au niveau phdnotypique, la
production des acides gras antifongiques et les propnetCs antagonistes des organismes contre
l'oïdium du concombre ont été testées.
Les recherches qui suivent tentent d'éclaircir un certain nombre de concepts
fondamentaux en ce qui a trait à l'agent de lutte biologique Pseudozyma f7occuloso.
L'exploitation pratique de cet agent antagoniste repose sur une connaissance approfondie de
son mode à'action et sur la production d'outils nCcessaires pour la sélection des isolats les plus
prolifiques face aux conditions changeantes d'utilisation de l'agent antagoniste. Seulement a
travers ces connaissances sera-t-il possible d'avoir une efficacité constante et une sélection des
candidats les plus approprids pour un programme de lutte biologique contre le blanc.
OK Wcobgie Mode d'actiofi
h* Stabi lit&* Essaidexpériences génétique
@s - Production de masse
Formulation
Analyse de. marché Brevet
Homologation 4 Commercialisation
Figure 1 . Étapes de développement d'un biopesticide. (a ) Points à l'étude lors de ces
travaux.
CHAPITRE II
SYNTHESIS AND BIOLOGICAL CHARACTENZATION OF
(Z)3-HEPTADECENOIC AND (+6-METHYL-9-HEPTADECENOIC ACIDS, FATTY
ACIDS WITH ANTIBIOTIC ACTIVITY PRODUCED BY PSEUDOZYUA FLOCCULOSA
T. J. AVIS, R. R. BOULANGER, and R. R. BÉLANGER
Departement de phytologie, Centre de recherche en horticulture, Université Laval
Ce chapitre a &te publié dans la revue Journal of Chernical Ecology (2000) 26:987-1000
Dans le cas des champignons de blanc, agents pathogènes ciblés pour la lutte
biologique par Pseudozyma flocculosa, il est trés difficile de cibler les mécanismes par
lesquels l'agent antagoniste les répriment. En effet, étant des parasites obligatoires, donc
dépendants du métabolisme de la plante hôte pour leur croissance, ils ne peuvent être cultivés
sur des milieux artificiels (Agios, 1988). Pour cette raison, la plupart des &tudes conduites sur
les propridtés antifongiques de P. Jlocculosa contre le blanc telle l'identification des acides
gras toxiques (Benyagoub et al., 1996a), ont CtC réalisées de façon indirecte h l'aide de
champignons modeles. Cependant, l'identification de tels composés ne signifie pas
ndcessairement que ces derniers soient responsables de l'efficacité de P. j7occulosa contre les
champignons de blanc. En effet, des études ont déjii montré que les fonctions ou propriétés
identifiées d'un agent antagoniste n'&aient pas nécessairement les cmctéristiques principales
permettant d'expliquer leur potentiel de lutte biologique (Howell et al., 2000; Deshpande,
1999; Ongena et al., 1999).
Cette partie du projet de recherche visait l'ktude de la toxicité des acides gras
antifongiques chez les champignons de blanc et donc de vérifier si ces acides gras etaient
rdellement des principes actifs dans la répression du blanc par P. flocculoso. Pour ce faire, la
difficulté 21 isoler et h purifier les acides gras antibiotiques de l'agent de lutte biologique P.
jlocculosa ii partir de milieux de culture était l'un des facteurs limitants. Dans le cadre de ce
travail, un protocole de synthèse chimique pour deux molécules A activité antibiotique
préalablement identifides chez P. jlocculosa, soit l'acide (2)-9-heptadécenoïque et l'acide (2)-
6-méthyle-9-heptadécenoïque, a CtC mis au point et ddcrit. Ainsi, la synth&se chimique de ces
acides gras a permis de quantifier l'activité biologique de ces moldcules et ce, de façon
reproductible. Lors de bio-essais, les deux molCcdes de synthèse ont montd une activité
antifongique correspondant B leur potentiel appr6hendd. Contre Sphaerotheca f i i e a , agent
causal du blanc du concombre, les acides gras avaient une forte activitd inhibitrice. En fait, ces
acides gras induisaient le même effet inhibiteur (l'effondrement des chaînes de conidies du
blanc) que P. jloccirlosa lui-même (Hajlaoui et Bdlanger, 1991) ce qui a permis de conclure
que les acides gras &aient des principes actifs dans la dpression du blanc par P. flocculosa-
Enfn, grâce h ces travaux, il etait dés lors plus facile d'dtudier les propriétés
écologiques de P. flocculosa en relation avec d'autres organismes.
Difficulties in isolating and purifying antibiotic fatty acids fiom culture filtrates of
Pseudozyma flocculosa, a biocontrol agent against powdery mildewq have been limiting
factors in studying the properties and understanding the mode of action of the biocontrol
agent. We report a new protocol for synthesizing (2)-9-heptadecenoic and for the fvst time
synthcsis of (2)-6-methyl-9-heptadecenoic acids, two antibiotic fatty acids produced by P.
flocculosa. This allowed reproducible and quantifiable means of assaying biological activity of
the molecules. In these bioassays, both molecules exhibited antifiingal activity corresponding
to their expected potency. These new developments should facilitate M e r studies aimed at
deciphering the ecological properties of P. flocculosa.
2.1. INTRODUCTION
Pseudorymoj?occuloso (Traquair, L. A. Shaw & Jarvis) Boekhout & Traquair (syn.:
Sporothrixflocculosa Traquair, Shaw & Jarvis) (Boekhout, 1995) is a yeast-like fungus with
antagonistic activity against powdery mildew h g i (Jarvis et al., 1989: Hajlaoui and Bélanger,
1 99 1, 1993; Bélanger et al., 1994). Microscopie and cytochemical observations revealed that
the antagonist induces rapid collapse of powdery mildew conidial chahs and cytoplasmic
disintegration of fimgal cells without hyphal pemtration (Hajlaoui et al., 1992). These fndings
suggested that antibiosis was the main mode of action by which P. jZocculosu exerted its
biocontrol activity.
Recently, our laboratory has isolated extracellular fatty acids with antimicrobial
properties produced by P. jloccufosa, two of which were reported for the first tirne
(Benyagoub et al, 1996a). These molecules, identified as 9-heptadecenoic acid and 6-methyl-
9-heptadecemic acid, exhibited a toxic activity against several fungi, but had little eEect on P.
jlocculosa itself (Benyagoub et al., 1996b). Bélanger and Deacon (1996) found a correlation
between fimgal sensitivity to the fatty acids and fimgal susceptibility to P. jlocculosa,
suggesting that these fatty acids are the active principles behind the biocontrol potential of the
antagonist .
Isolation and manipulation of these fatty acids fiom culture filtrates has proven
dificult as extraction and purification procedures resulted in low and unreiiable yields. This
has hampered o u efforts to learn more about the ecological and molecular pmperties of these
compounds. As a result, we sought a way to obtain large and stable amounts of the antibiotics.
Therefore the objective of this work was to develop a synthesis method for both (2)-9-
heptadecenoic and (Z)a-methyl-9-heptadecenoic acids in order to facilitate biological studies
of the antibiotics.
2.2. METHODS AND MATERIALS
2.2. l . General chernical procedures and instruments.
Al1 oxygen or moisture-sensitive reactions were perfonned in flame or oven-dried
glassware under pressure of nitrogen or argon. Au-sensitive liquids and solutions were
transferred by syringe or cannula. Ether, tetrahydrofûran and 1,4-dioxane were dried before
use. 1,7-dibromoheptane, 3-methylglutaric anhyàride, and 1 -nonyne were obtained fiom
commercial sources (Sigma-Aldrich, Mississauga, Ontario, Canada). Flash column
chromatography was performed with silica gel EM science 200-400 mesh (40-63 pm). Al1
nuclear magnetic resonance spectroscopy ('H, ')c) was recorded in deuterium chloroform
using a Bniker 300 MHz. Peaks positions are expressed as domifield shifts in parts per million
(6) fiom tetramethylsilane interna1 standards. Split patterns are designed as singlet (s); doublet
(d); doublet-triplet (dt); triplet (t); quartet (q); multiplet (m); broad signal (br); J is coupling in
Hertz. GC-MS data were recorded with a Hewlett Packard 5890 series II gas chromatography
coupled to a Hewlett Packard 5972 mass selective detector. Mass spectra were obtained with
electronic impact (EI) (70 eV, energy bearn).
2.2.2. Synthesis of (2)-9-Heptadecenoic Acid.
Preparation of various homologs and positional isomers of the C- 1 3-C- 19 odd-carbon
ethenoic acids were synthesized according to Gr imer and Kracht (1963) and Jacob and
Grimmer (1966) (Figure 1). These acids were prepared by reaction of the appropriate lithium
acetylide and dihaloalkane in 1,4-dioxane and followed by reaction of the tesulting acetylenic
halide with sodium cyanide to obtain the conespondhg acid 7, which was half-hydrogenated
to the ethenoic according to Jacob (1966).
I,7-Diiodoheptcme (3). A solution of 1.7-dibromoheptane 1 (5.17 g, 0.022 mol) in 20
ml of dry acetone was added dropwise to a 250-ml flask equipped with a condenser,
containing a suspension of sodium iodide (13.3 g, 0.09 mol) with 100 ml of dry acetone at
room temperature. The mixture was heated to reflux for 3 hr. After cooling, the reaction
mixture was filtered and the solvent was evaporated in vacuo. The crude cesidue was purified
by flash chomatography with hexane as eluent. The yield was 96%, 7.6 g. C7Hi412: 'H NMR
(CDC13): S 1.30 (m, 6H), 1.39 (m, 4H), 3.17 (t, 4H) ppm; 13c NMR (CDC13): 6 6.91 (C-4).
27.32 (C-3), 27.81 (C-3'),30.14 (two carbons) (C-2), 33.21 (two carbons) (C-1); EI-MS (dz,
loss fiagment): 352 (m, 225 (M'+-HI), 183 (M+-HI-c&), 155 (MIc~HI~I), 97 (C,H13).
9-Hexadecyne Iodide (4). Under an atmosphere of argon, 1-nonyne 2 (2.5 g, 0.02 mol)
in 500 ml of dry 1,Cdioxane was reflwed with lithium amide (0.414 g, 0.01 8 mol) for 3.5 hr
in a tricol equipped with a condenser and a dropping fùnnel with pressure equalization am. To
the organolithium compound thus formed, l,7-diiodoheptane 3 (8 g, 0.022 mol) in 50 ml of
dry 1,4-dioxane was added dropwise. The mixture was refluxed an additional 2 hr, allowed to
cool to room temperature, and then poured into a mixture of ice-water and stirred 15 min. The
mixture was extracted with diethylether (3 X 50 ml). The combined organic fractions w e n
dried over magnesium sulfate, filtered, and the solvent was evaporated in vacuo. The residue
was purified by flash chromatography with hexane as eluent to give the corresponding
iodoalkyne 4 with a 95% yield (6.6 g). C I ~ H ~ ~ I : 'H NhlR (CDCli): 6 0.87 (t, 3H), 1.27-1.54
(m, 18H), 1.79- 1-92 (m, 2H), 2.1 3 (t, 4H), 3.18 (t, 2H) ppm; "C NMR (CDC13): 6 7.1 1
(CHjCHz), 14.1 1, 18-72, l8.78,22.65, 28.07, 28.58, 28.85 (2 carbons), 29.00, 29.19, 30.42,
3 1.79,33 S2, 80.00 (C, alkyne), 80.42 (C, alkyne) ppm; EI-MS (nJr, loss fragment): 348 (m, 305 (M+-c,H~), 291 (W-GHg), 263 ( W - C ~ H ~ ~ ) , 249 (W-c.1~15).
9-Heptadecyne Cyanide (5). A solution of iodide compound 4 (4 g, 0 .O 1 1 5 mol) with
200 ml of 92% ethanol was added in a 250-ml flask equipped with a condenser and was
refluxed 5 hr with sodium cyanide (2.8 g, 0.06 mol). After cooling to room temperature the
mixture was poured into 50 ml water and extracted with hexane (3 X 50 ml). The combined
organic fraction was dried over magnesium sulfate, filtered, and the solvent was evaporated in
vacuo. The residue was purified by flash chromatography with hexane as eluent. The yield
was 2.5 g (88%). Cl7HZ9N: 'H NMR (CDC13): 6 0.86 (t, 3H), 1.26-1.49 (m, 18H). 1.59-1.69
(m. 2H), 2.1 1 (t,4H), 2.31 (t, 2H) ppm; 13c NMR (CDC13): S 13.92 (çH3CH2), 16.95, 18.52,
18.59, 22.97, 25.19,28.14,28.27 (two carbons), 28.42, 28.68,28.77,29.01,3 1.63, 79.71 (C,
alkyne), 80.31 (ç, aikyne) 119.59 (ç, nitrile) ppm; EI-MS (mk, loss fragment): 247 (m, 176
@f%H~i), 162 (W-Cdh), 148 ( M * - ~ i h ) , 134 (M+-GHi7), 8 1 (CSH~W, 67 (CSIN.
9-Hepiadecynoic Acid (6). In a 250-ml flask equipped with a condenser, the cyanide 5
(2.5 g, 0.0094 mol) was diluted with 50 ml of 92% ethanol. Sodium hydroxide (2.28 g, 0.06
mol) in 25 mi of water was added in one portion, and the mixture was heated to reflux 10 hr.
The reaction was cooled to room temperature, diluted with 200 ml of water, acidified with
hydrochloric acid 2N, and extracted with diethylether (3 X 50 ml). n i e cmde residue 3.8 g
was purified by flash chromatography using a gradient of mixture of 10-30% ethylacetate-
hexane as eluent to yield 2 g (66%) of 9-heptadecynoic acid 6. CL7HI02: 'H NMR (CDC13): 6
0.81 (t, 3H), 1.21-1.40 (m, 18H). 1.52-1.60 (m, 2H), 1.98-2.09 (t, 4H), 2.26-2.31 (t, 2H) 8.75
(br, COOH, 1H) ppm; I3c NMR (CDCli): 6 13.93 (CH3CHI), 18.58, 22.48, 24.48, 28.46,
28.62 (two carbons), 28.68 (two carbons), 28.81, 28.92, 29.03, 31.63, 33.81, 79.92 (ç,
alkyne), 80.22 (C, alkyne) 179.46 (çOOH) pprn; EI-MS (A, loss fragment): 248 (M"), 195
(*-C5~i 11, 1 78 (M+-cIH~Q), 164 (M+GHio9). 128 (M%~i202) , 95 (GHi i), 8 1 (Cs&),
67 (C5H7).
(2)-9-Heptadecenoic Acid (7). 9-Heptadecynoic acid 6 (1.35 g, 0.005 1 mol) was
dissolved with 50 ml of methanoVpyridine (5: l), poured into a hydrogenation tube, and 0.03 g
of Raney nickel powder was added to the mixture. The tube was closed and shaken under
hydrogen atmosphere until adsorption ceased (30 min). The naction mixture was filtered on
celite, the solvent was evaporated in vacuo and the residue was purified by flash
chromatography using a gradient of 10-30% ethylacetate-hexane as eluent. Pure (2)-9-
heptadecenoic acid 7 was obtained. The yield was 1.1 g (81%). C17H3202: 'H NMR (CDCl,): 6
0.81 (t, 3H), 1.18-1.24 (m, 19H), 1.51-1.59 (m, 2H), 1.91-2.07 (t, 4H), 2.25-2.30 (t, 2H) 5.22-
5.33 (m, 2H, J= 5.80 Hz) ppm; "C NMR (CDC13): 6 13.99 (ÇH3CH2), 22.56, 24.54, 27.09,
28.94 (two carbons), 29.02, 29.15 (two carbons), 29.56 (two carbons), 29.65, 3 1.76, 33.96,
129.61 (C, alkene), 129.90 (ç. alkene) 180.20 (COOH) ppm; EI-MS (&, loss kgment): 268
(m, 250 (W-H~o), 196 (M+-c3)402), 182 (W-C&02), 138 (C7HMO2), 95 (CiH, I), 81
(C6Hd, 67 (C5H7)-
2.2.3. Synthesis of (Z)aMethyl-9-Heptadecenoic Acid.
Heptadecenoic acid methyl substituted at the 6 position was synthesized by a
modification of Figure 1. The comsponding a,ci>-dibromopentane was prepared by reduction
of methyl-3-glutaric anhydride followed by a bromation with phosphorus tribromide or a
Vonbraun degradation of substituted piperidi (Nguyen and Cartledge, 1986) (Figure 2).
3-Methyl-1.5-Pentanediol(10). A solution of 3-methylglutaric anhydride 9 (1.48 g,
0.008 mol) in 50 ml of anhyârous tetrahyârofursn was added dropwise to a suspension of
lithium aluminum hydride (0.59 g, 0.96 mol) in 200 ml of anhydrous tetrahydrofiiran. After 2
hr of stimng at room temperature, hydrolysis was accomplished by pouring the reaction
mixture in an ice-cold saturated ammonium chioride solution. The mixture was extractcd with
diethylether (3 X 50 ml). Drying of the combined organic phase was performed over
magnesium sulfate, and evaporation of solvent gave pure 3-methyl-1,s-pentanediol 10,0.602
g (44% yield). C6H14O2: 'H NMR (CDC13): 6 0.92 (d, 3H, J= 6.63 Hz), 1.36- 1.45 (dt, 2H),
1.53-1.64 (dt, 2H), 1.72-1.83 (m, lH), 3.39 (br, ZH), 3.59-3.74 (m, 4H) ppm; "C NMR
(CDCli): 6 19.71, 25.69, 39.24 (two carbons), 60.18 (two carbons) ppm; EI-MS (dz, loss
fragment): 119 (M?+l), 99 (M'-H~o), 82 (Cdlo), 67 (C5Hi).
3-Methyl-1.5-dibromopentane (1 1). Phosphorus ûibromide, 1.4 1 g (0.0052 mol) was
slowly added to a 250-ml flask equipped with a condenser, containing a 0.6 g (0.0051 mol) of
3-methyl-1,s-pentanediol 9, at -lO°C. The mixture was brought to room temperature and
stirred 20 hr. Ethylacetate (20 ml) was added to precipitate the oxides, followed by filtration
and evaporation of solvents in vacuo. The crude residue was purified by flash chromatography
with hexane as eluent or by distillation (bp 8OC6 mm Hg), to give pure dibromide 11. The
yield was 0.9 g (73%). C6Hl2Br2: I H NMR (CDC13): 6 0.93 (d, 3H, & 6.26 Hz), 1.65-1.77 (m,
2H), 1.82-1.97 (m, 3H), 3.36-3.49 (m, 4H) ppm; I3c NMR (CDC13): 6 18.05, 30.47, 31.12
(two carbons), 39.28 (two carbons) ppm; EI-MS (m/z, loss fragment): 244 (M+, 79~r ) , 246
(M', " ~ r ) , 162 (Mt-%), 164 (bf-'%r), 135 (M+-c~&"B~), 137 (M'-c2b7%r), 83 (C6Hi ,).
3-Methyl-1,s-diiodopentune (12). A solution of 3-methy l- 1,s-dibromopentane 11
(1.15 g, 0.045 mol) in 20 ml of dry acetone was added dropwise to a 250-ml flask equipped
with a condenser and containhg a suspension of sodium iodide (1.8 g, 0.012 mol) with 25 ml
of dry acetone at m m temperature. The mixture was heated to reflux for 3 hr. mer cooling,
the reaction mixture was filtered and the solvcnt was evaporated in vacuo. The crude residue
was purified by flash chromatography with hexaae to give pure diiodide 12. The yield was
1.65 g (89%). &H1212: 'HNMR (CDC13): 6 0.90 (d, 3H, & 5.32 Hz), 1.71 (m, 3H), 1.90 (m,
2H), 3.30 (m, 4H) ppm; "C NMR (CDCI,): S 3.87, 17.69, 34.91,4O.OO (two carbons) ppm;
EI-MS ( d z , loss fragment): 338 (m, 21 1 (&- 1). 169 (M*-c~&,I), 155 (MI-CIH~), 127
(M*-C6Hl21), 83 (C6Hll), 55 (C4H7).
3- Methyl-6-tetrodecyne Brornide (1 3) or 3-Methyl-6-tetradecyne lodide (II). Under an
atmosphere of argon, 1-nonyne 2 (3.72 g, 0.03 mol) in 100 ml of dry 1,4-dioxane was reflwed
with lithium amide (0.762 g, 0.033 mol) for 3.5 hr in a tricol equipped with a condenser and a
dropping h e l with pressure equalization am. To the organolithium compound thus formed,
0.0047 mol of the 3-methyl- 1,s-halopentane 11 (1.14 g) or 12 (1.59 g) in 50 ml of dry 1,4-
dioxane was added dropwise, and the mixture was heated to reflux another 2 hr. The mixture
was allowed to cool to room temperature and poured into a mixture of ice-water and stined 15
min. The mixture was extracted with diethylether (3 X 50 ml). The combined organic fractions
were dried over magnesium sulfate, filtered, and the solvent was evaporated in vacuo. The
residue was purified by flash chromatography with light petroleum ether as eluent to give the
corresponding haloalkyne 13 (0.897 g, 76% yield), or 14 (1 .O0 g, 64% yield). CISH2,Br: IH
NMR (CDCI,): 6 0.89 (d, J = 6.47 Hz, 3H), 0.92 (t, 3H), 1.10- 1.49 (m, 1 1 H), 1 S2- 1.94 (m,
4H), 2.10-2.27 (m, 4H), 3.47 (m, 2H, CH2-Br) ppm; "C NMR (CDC13): 6 13.94 (CH3CH2),
16,22, 1 8 3 , 18,61, 22,49, 28,70, 2899 (two carbons), 30,49, 3 1,56, 31,64, 3537, 3937,
79.46 alkyne), 80.45 (C, alkyne) ppm; EI-MS (dz, loss fragment): 288 (M'), 207 (M'- 8 1 Br), 1 89 ( C S H I ~ B ~ ) , 187 ( c I I H I ~ ~ ~ B ~ ) , 125 (C9H17). 95 (C7H1 I), 8 1 (C6H9), 67 (C5Hï).
C I ~ H ~ ~ I : IH NMR (CDCI3): 6 0.89 (d, J = 6.10 Hz, 3H), 0.92 (t, 3H), 1.25 (m, 14H), 1.55 (m,
2H), 1.63-1.80 (m, lH), 1.83-2.20 (m, ZH), 3.20 (t, 2H, CH2-1) ppm; 13c NMR (CDC13): 6
4.53 (ÇH~CHI), 13.97, 16.22, 18.18, 18.63, 22.51, 28.71 (two carbons), 29.00, 31.65,32.98,
35.32,40.50,79.48 Cç, alkyne), 80.58 (ç, alkyne) ppm; El-MS ( d z , loss hgment): 334 (m, 207 (W-I), 155 (M+-c~H~), 123 (C6H&, 55 (C4H7), 41 (CIH 5).
5-Merhyl-8-hexudecyn-lel(I5): Under an atmosphere of argon, 0.087g (3.6 rnmol) of
magnesium W n g s were introduced into a three-necked 100-ml round-bottomed flask,
equipped with a pressure-equalizing addition h e l , a condenser, and a magnetic stir bar. The
magnesium was covered by 10 ml of anhydrous diethylether (fkshly distilled on sodium). A
solution of 3-methyl-6-tet~adecyn iodide 14 (0.800 g, 0.0024 mol) in 20 ml of anhydrous
tetrahydrofiuan was added dropwise at 20'C. The formation of the Grignard reagent was
accomplished afler 2 hr of stirring. The solution was then cooied to O'C in an ice water bath
and a flow of ethylene oxide was bubbled into the Grignard solution for a period of 45 min.
The resulting suspension was stirred for an additional 45 min at O'C. The reaction was allowed
to warm at room temperature and treated with cold saturated ammonium chloride. The
resulting mixture was extracted with diethylether, and the combined ether extract was washed
with b ~ e and dried over anhyàrous magnesium sulfate. Removal of the solvent by distillation
under pressure afforded of the desired product. The alcohol was purified by flash
chromatography and eluted with light petroleum ether-ethylacetate (70:30). Pure
hydroxyalcyne 14 was obtained (0.100 g, 13%). C i7H120: 'H NMR (CDC13): 6 0.89 (t, 3H),
0.92 (d, J = 6.57 Hz, 3H), 1.1 1 - 1.75 (m, 20H), 2.09-2.27 (m, 4H), 3.62-3.76 (m, 2H, CkOH)
ppm; "C NMR (CDC13): S 13.93, 16.32, 18.60, 19.00, 19.21, 22.95, 28.62 (two carbons),
28.69,29.01,29.55, 31.63, 36.26,39.39,60.91,79.81 (C, alkyne), 80.26 (C, alkyne) ppm; EI-
MS ( d z , loss fiament): 223 (@- C2H5), 209 (M'- C3%), 193 (w-C~H~O), 179 (C4H90), 95
(C~HI l), 80 (C~HS), 67 (CSH7).
5-Methyl-8-hexudecyne Bromide (16). Freshly recrystallized from hexane,
tnphenylphosphine (0.1 55 mg, 0.00059 mol) was added in one portion to a cold solution of
hydroxyalcyne 15 (0.135 g, 0.00053 mol) with 5 ml of dry dichloromethane. AAer a 1-hr
reaction period, carbon tetrabromide, 0.197 mg (0.00059 mol) was added in one portion and
the mixture was stirred at O'C for 5 hr. A mixture of ethylacetate and petroleum ether (20:80)
was added, and the mixture was filtered on a bed of silica gel and washed with the sarne
solvent. After evaporation of the solvent in vacuo, the bromoalkyne 16 was purified by flash
chromatography with light petroleum ether as eluent to give 0.138 mg, 82% yield. CIW31Br: t H NMR (CDCl,): 6 0.87 (t, 3H), 0.98 (d, J = 6.40 Hz, 3H), 1.10-1.96 (m, 19H), 2.1 1-2.25 (m,
4H), 3.36-3.5 1 (m,2H, CbBr) ppm; "C NMR (CDCh): 6 13.94, 16.22, 18.38 (two carbons),
18.61, 22.49, 28.71, 28.99 (three carbons), 30.79, 31.60, 31.64, 35.57, 39.57, 79.48
alkyne), 80.48 (ç, alicyne) ppm; EI-MS ( d z , loss hgment): 273 (M'- CI&), 235 (M'- "~r ) ,
1 79 (M'-GH,B~), 95 (C7H 1 i),8 1 (C6H9).
6-Methyl-9-heptadecynoic Acid (1 7). A solution of bromide compound 16 (0.120 g,
0.00038 mol) in 20 ml of 92% ethanol was added to a 50-ml Bask equipped with a condenser
and was heated to reflux 5 hr with sodium cyanide (0.093 g, 0.00191 mol). The cyanide
formed was not isolated and was immediately treated with sodium hydroxide (0.0076 g,
0.00199 mol) in 25 ml of water. The mixture was heated to reflux for 14 hr. The naction was
cooled to room temperature, diluted with 200 ml of water, acidified with hydrochloric acid
2N, and extracted with diethylether (3 X 50 ml). The cmde residue was purified by flash
chrornatography with a gradient of mixture of 10-30% ethylacetate-light petroleum ether as
eluent to give 0.060 g, 57% yield of 6-methyl-9-heptadecynoic acid 17. C18H3202: 'H NMR
(CDCI,): S 0.79 (t, 3H), 0.82 (d, J = 6.47 Hz), 1.19-1.68 (m, 19H). 2.03-2.17 (t, 4H), 2.26-2.39
(t, 2H) 9.75 (br, COOH, 1H) ppm; 13c NMR (CDC13): 6 13.95 (çH3CH2), 18.59, 18.59, 18.63,
22.49,28.69 (three carbons), 28.99,29.57,31.11 (two carbons), 3 1.37.3 1.64,35.70,79.62 (C,
alkyne), 80.31 (C, alkyne) 180.25 COOH) ppm; EI-MS ( d z , loss fragment): 281 (Mf+ l), 236 (M'-COI), 168 (W-C~HIO~), 108 (M'-C~HI~), 95 (C7Hii), 81 (C6H9), 67 (CsH7).
(2) -6-Methyf-9-heptadecenoic Acid (1 8). 6-Methy l-9-heptadecynoic acid 17 (0.1 27 g,
0.00045 mol) was dissolved in 50 ml of methanol-pyridine (5:l) and poured into a
hydrogenation tube, and 0.03 g of Raney nickel powder was added to the mixture. The tube
was closed and shaken under hydrogen atmosphere until adsorption ceased (15 min). The
reaction mixture was filtered on celite, the solvent was evaporated in vacuo, and the residue
was purified by flash chromatography with a gradient of 10030% ethylacetate-light petroleum
ether as eluent. Pure (2)-6-methyl-9-heptadecenoic acid 18 was obtained (0.1 17 g, 9 1% yield).
CI8HJ4O2: 'H NMR (CDCli): 6 0.86 (t, 3H), 0.89 (d, J = 6.19 Hz, 3H), 1.12-1.76 (m, 17H),
1.99-2.1 8 (t, 4H), 2.27-2.45 (m, 2H) 5.28-5.40 (m, 2H, J = 5.80 Hz) 9.81 (CHgOOH) ppm;
"C NMR (CDC13): S 13.94 (CH3CH2), 18.96, 19.1 1,22.53,24.48,27.06,29.08,29.13,29.22,
29.56, 29.61, 31.45, 31.73, 31.87, 36.51, 129.39 (C, alkene), 129.97 (ç, alkene), 180.08
(COOH) ppm; EI-MS ( d z , loss hgment): 281 (M+-1). 267 (MICB), 254 (Mç- C2&), 238
(M+-C@), 236 ( w - ~ 2 0 , -C2&), 221 (M+-C~HS~) , 114 (CsHio02), 97 (CiHi,), 83 (CsHii).
2.2.4. Biological Activity of the Synthesized Antibiotics on Fungal Material.
Cladosporium cucumerinum EUis and Arth. was obtained from Biosystemics Research
Centre (Ottawa, Ontario, Canada) and maintained on potato dextrose agar (PDA).
Sphaerotheca fuliginea (Schlechtend.:Fr.) Pollacci was maintained in axenic conditions on
long English cucumber (Cucumis sativus L. cv. Corona).
Two agar disks (1 5 mm) of C. cucumerinum were suspended in 100 ml of potato
dextrose broth (PDB). The antibiotics were tested at concentrations of 0.1, 0.2, 0.4, 0.6, 0.8,
and 1 mg/ml. The fatty acids were solubilized in Na-dimethylfomarnide @MF) prior to
king added to the medium. DMF alone (0.5%) was added to the culture media to serve as
control. Growth was quantified d e r a three-&y culture at 2S°C on a rotary shaker (150 rpm)
by dry weight measurement following lyophilization. Further concentrations of each antibiotic
were tested to detennine minimum inhibitory concentration (MIC) of C. cucumerinum. MIC
was defmed as the lowest concentration of each antibiotic at which no macroscopic evidence
of growth was obsmed. For each concentration, the expriment was repeated three times. In
order to determine the dose of each antibiotic which reduced growth of C. cucumerinum by
50%, probit analysis was performed with the PROC Probit procedure in SAS System (SAS
Institute Inc., Cary, North Carolina).
Foliar disks (5 cm) were cut from S. fulginea-infected cucumber leaves and placed on
20-20-20 agar containhg 2 d i te r 20-20-20 and 8 glliter bacto-agar. Disks were cut fiom the
leaf portion covered by at least 95% with colonies of S. fiiiginea. The antibiotics were sprayed
on leaf disks as aqueous solutions of 0.1 and 0.2 mghl. The antibiotics were solubilized in
N , N - d i m e y l f o d e (DMF) prior to adding to sterile water. DMF alone (0.5%) was added
to the sterile water to serve as control. Evaluation of antibiotic effects on S. firliginea was
detemined following incubation periods of 0, 12, and 24 hr at 2S°C. Further evaluation of the
antibiotics was not possible because of their rapid degradation under these conditions.
Antifùngal activity was detemined using an arbitrary scaie of O to 4, where O = no effect on S.
fuiiginea conidial c h a h , 1 = 1 to 25% collapse of conidial chahs, 2 = 26 to 50%, 3 = 5 1 to
75%, and 4 = 76 to 100% collapse. For each treatment, the experiment was repeated three
times with two replicates per expriment.
23. RESULTS AND DISCUSSION
By following the synthetic route of Figure 1, it was possible to obtain pure (2)-9-
heptadecenoic acid. Ames and Bowman (1951) and Broughton et al. (1952) described the
synthesis of ethylenic acids starting with an acid chloride and tribenzyltricarboxylate, which
can also lead to synthesis of this fatty acid. However, that synthesis was not explored because
the protocol in this study was more efficient as it reduced the nurnber of steps. Figure 2 gave
pure (2)-6-methyl-9-heptadecenoic acid. Global yield of a,o-dibromopentane was low when
prepared by reduction of 3-methylglutaric anhydride followed by a bromation with
phosphorus tribromide. The Vonbraun degradation of substituted piperidine gave better
results. In order to obtain the organolithium reagent, Branhge et al. (1984) proposed a new
technique by using lithium 4,4'-di-tert-butylbiphenylide (LDBB) as an intermediate reagent at
-60°C in tetrahydrofuran. However, Our attempts to use this synthetic route were not
conclusive (Figure 2). On the other hand, the y ield of 5-methyl-8-hexadecyn- 1-01 (1 S),
although not optimal, was obtained reproducibly through Figure 2. For large-scale endeavors,
this step could possibly be optirnized with a modification of the halogenue structure such as
hydrogenation of the haloalcyne to haloalcene prior to the Grignard reaction.
Biological testing of the synthesized fatty acids was perfonned to verify theu toxic
activity on hngi and to determine the specific characteristics of each molecule. Both
compounds were assayed against C. cucumerinum, a cucumber pathogen that inhabits the
sarne ecological niche as P. flocculosa and is sensitive to the antifùngal fatty acids produced
by the antagonist. Growth of C. cucumerinum was inhibited by both fatty acids, with 6-
methyl-9-heptadecenoic acid demonstrating greater biological activity (Figure 3).
Determination of MICs resulted in no growth of C. cucumerinum at 0.6 and 0.35 mgml for 9-
heptadecenoic acid and 6-methyl-9-heptadecenoic acid, respectively. Probit analysis revealed
that the dose of each antibiotic expected to reduce growth of C. cucumerinum by 50% is 0.16
and 0.10 mg/ml for Pheptadecenoic acid and 6-methyl-9-heptadecenoic acid, respectively.
These results support previous qualitative reports that 6-methyl-9-heptadecenoic acid has the
higher biological activity of the two when assayed against C. cucumerinum (Benyagoub et al.,
1996a).
The antifungal fatty acids were also tested against S. filiginea, the causal agent of
cucumber powdery mildew for which P. jlocculosa is under study as a biocontrol agent. When
assayed against S. fuliginea, both antibiotics induced a rapid collapse of the conidial chahs of
the pathogen, with once again a xemingly greater effect of 6-methyl-9-heptadecenoic acid
(Table 1). This collapse of conidial chains was similar to the one induced by P. flocculosa
itself. This suggests that the two fat@ acids are, at least in part, responsible for the biocontrol
activity. When assayed at 0.1 mgM, 6-methyl-9-heptadecenoic acid caused a signEcantly
higher degm of collapse compared to Bheptadecenoic acid 12 hr following the treatrnent. At
24 hr, these same treatments no longer showed differences in biological activity. This
indicates that 6-methyl-9-heptadecenoic acid initially acts more rapidly on S. fuliginea than its
non methylated counterpart. The fatty acids used under these conditions lost their activity
when assayed at 36 hr (data not shown). This is a result of their degradation, as previously
demonstrated, when they are not stored under suitable conditions (appropriate temperature and
protection h m oxidation) (Kates, 1986). This rapid degradation indicates that P. flocculosa
itself must be applied as the control measure of S. fuliginea because the fatty acids alone could
not provide continuous control. Because the fatty acids degrade rapidly, there is less seiection
pressure on powdery mildew populations as a result of limited exposure to the compounds.
This implies that the development of resistant strains of powdery mildew fungi to the
antibiotics may be reduced or retarded on the leaf sunace. Along these lines, it is apparent that
residual activity would not be an issue, which makes P. flocculosa an environrnentally fnendly
agent of powdery mildew biocontrol.
Frorn an ecological perspective, P. jlocculosa is considered a weak cornpetitor for
nutrients (Hajlaoui et al., 1992). Therefore, these antibiotics would provide this antagonist
with a means to protect its ecological niche against powdery mildew fungi. This in turn
explains its rather selective biological control efficiency against powdery mildew fungi, which
compete for the same ecological niche on the leaf surface. The cornpetition is not based on
nutrients because they are biotrophs, i.e., organisms that develop only on another living
organism.
We consider the synthesis of these two fatty acids produced by P. flocculosa as a major
breakthrough in our system because it allows further experimentation of their antibiotic
activity without the underlying difficulties associated witb extraction and purification
procedures fiom culture filtrates. The newly synthesized molecules may be used as standards
to follow kinetics and quanti@ biosynthetic productions by P. flocculosa. This should give a
better understanding of the agent's antagonistic activity on leaf d a c e s and provide a tool for
screening and selecting P. flocculosu strains with superior biocontrol potential.
It has k e n s h o w that the antibiotic fatty acids induce leakage of electrolytes and
proteins (Hajlaoui et ai., 1992). These and other results have led to the hypothesis of a
structural mode of action of P. jloccuiosa antibiotics, w h e ~ fungai membranes are the primary
target of action. Briefly, the antibiotics would partition into fungal membranes and induce
disorder due to their bulkiness caused by the cis double bond located near the center of these
fatty acids. This in tum would cause changes in the physical properties and function of fimgal
membranes which would induce electrolyte leakage and increased fluidity as show by
Benyagoub et al. (1996b). It is now apparent that the antifungal fatty acids do not directly or
indirectly act upon membrane sterols, factors that influence membrane fiuidity, because they
are toxic to pythiaceous fungi that do not contain membrane sterols (unpublished results).
Furthemore, the dose-dependent increase in both toxicity (this study) and fluidity (Benyagoub
et a!., 1996b) by the fatty acids would suggest that they interfere with the general membrane
lipid domain rather than a localized effect on one of its components. This model is also
consistent witb results in this study, in which the two antibiotics show differential activity in
exposed b g i . An additional methyl branch would occupy a larger cross-sectional area in
fungal membranes, thereby inducing greater disorder and increased fluidity. This would
support the higher biological activity of 6-methyl-9-heptadecenoic acid.
The newly synthesized molecules should facilitate elucidation of the specific mode of
action of this biocontrol agent. For example, immunolocalization and cytochemical
observations could pinpoint sites of action of the fatty acids within fùngal cells, thus providing
a valuable tool to validate this model and eventually help in predicting the e@cacy of the
antagonist by analysis of cellular membranes of powdery mildew fungi.
Overall, the synthesis of ( 2)-Pheptadecenoic and (a-6-methyl-9-heptadecenoic acids
fiom P. flocculosa will give stable and suffifient quantities of the products for M e r testing
with regards to specific mode of action of the antibiotics. This will be helpful in elucidating
the molecular and ecological phenornena behind the antagonism by P. floccufosu and in
helping the prediction of its biocontrol eficiency. Finally, the antibiotics c m be used in
repeated selection pressure experiments to determine whether target fungi can eventually
develop resistmce.
ACKNOWLEDGMENTS
Financial assistance fiom NSERC and Piant Products Co. Ltd.
REFERENCES
AMES, D. E. and BOWMAN, R. E. 195 1. Synthetic long-chah aliphatic compound. Part III.
A critical examination of two methods of synthesis of olefinic acids. J. Chem. Soc.1079-1086.
BÉLANGER, R. R. and DEACON, J. W. 1996. Interaction specificity of the biocontrol agent
Sporothrir flocculosa : A video microscopy study . Phytopathology 86: 1 3 1 7- 1 3 23.
BÉLANGER, R. R., L A B B ~ , C., and JARVIS, W. R. 1994. Commercial-scale control of rose
powdery mildew with a fimgal antagonist. Plant Dis. 78:420-424.
BENYAGOUB, M., BEL RHLID, R., and BÉLANGER, R. R. 1996a. Purification and
characterization of new fatty acids with antibiotic activity produced by Sporothrixflocculosa.
J . Chem. Ecol. 22:405-413.
BENYAGOUB, M., WILLEMOT, C., and &LANGER, R. R. 1996b. Influence of a
subinhibitory dose of antifungal fatty acids from Sporothrix flocculosa on cellular lipid
composition in fimgi. Lipidr 3 1 : 1077-1 082.
BOEKHOUT, T. 1995. Pseudozyma Bandoni emend. Boekhout, a genus for yeast-like
anamorphs of Ustilaginales. J . Gen. Appl. Microbiol. 4 1 :359-366.
BRANDANGE, S., DAHLMAN, O., LINDQUIST, B., MAHLEN, A., and MORCH, L. 1984.
Absolute configuration and enantioselective synthesis of spiculisporic acid. Acta chemica
scand B 38:837-844.
BROUGHTON, B. W., BOWMAN, R.E., and AMES, D. E. 1952. Synthetic long-chain
diphatic compound. Part VIL Some Mono-olefuiic acids. J Chem. Soc. 671-677.
GRIMMER, G. and KRACH?, J. 1963. The synthesis of odd-numbered unsaturated fatty
acids. Berichte 96:3370-3373.
HAKAOUI, M. R., AND BÉLANGER, R. R 1991. Comparative effects of temperature and
humidity on the activity of three potential antagonists of rose powdery mildew. Neth. J Plant
Pafhol. 97:203-208.
HAJLAOUI, M. R., AND BELANGER, R. R. 1993. Antagonism of the yeast-like phylloplane
fimgus Sporothrix flocculoso against Erysiphe grominis var. tritici. Biocont. Sci. Technol.
3 :427434.
HAJLAOUI, M. R., BENHAMOU, N., and &LANGER, R. R. 1992. Cytochemical study of
the antagonistic activity of Sporothrix jlocculoso on rose powdery mildew, Sphaerotheca
pannosa var. rosae. Phytoputhology 82: 5 83 -589.
JACOB, V. J. and GRIMMER, G. 1966. Bildung doppelbindungsisomenr alkencarboasiiuren
bei der hydrierung von 9-alkincarbo~wen. Te~ahedron Lett. 24:2687-2688.
JARVIS, W. R., SHAW, L. A., and TRAQUAIR, J. A. 1989. Factors af5ecting antagonism of
cucumber powdery mildew by Stephanoascus jlocculosus and S. rugulosus. Mycol. Res.
92: 162-1 65.
KATES, M. 1986. Techniques of lipodology: isolation, analysis and identification of lipids,
pp. 100-1 11, in T. S. Burton and P. H. van Knippenberg (eds.). Laboratory Techniques in
Biochemistry and Molecular Biology. Elsevier, Amsterdam.
NGUYEN, B. T. and CARTLEDGE, F. K. 1986. A convenient synthetic route to methylated
silacyclohexanes. J. Org. Chem. 5 1 :22O6-22 10.
Figure 1 . S ynthetic scheme for the synthesis of (2)-9-heptadecenoic acid.
4, UAIH, - THF anhydm
O
1- M C N / EtOH 2- M O H - HXi
Figure 2. Synthetic scheme for the synthesis of @)a-methyl-9-heptadecenoic acid.
E 8
--C- 9-heptadecenoic acid
O 0 2 0,4 0,6 03
Antibiotic concentration (mglml)
Figure 3. Growth of threeday liquid cultures of CZadosporium cucumerinum following
treatment with (2)-9-heptadecenoic and (2)-6-methyl-9-heptadecenoic acids at concentrations
ranging from O to 1 mglml. Values are means SD.
Table 1 . Sphaerotheca fuliginea conidial chah collapse following treatment with aqueous solutions of antibiotic fatty aciàs from
Pseudozyma flocculosa '
Control (2)-9- heptadecenoic acid (2)-6-methyl-9-heptadecenoic acid
Time (hr) 0.0 mglml 0.1 mglml 0.2 mg/ml 0.1 mg/ml 0.2 mglml
' Values are means of conidial collapse where O = no effect on S. fuliginea conidial chains, I = 1-25% collapse of conidial chains, 2 =
26-50%. 3 = 51-75%. and 4 = 76-100% collapse. Within a line, means followed by same letters are not significantly different by
Fisher's protected LSD (P = 0.01).
CHAPITRE III
SPECIFICITY AND MODE OF ACTION OF THE ANTIFUNGAL FATTY ACID CI&
9-HEPTADECENOIC ACID PRODUCED BY P S E U D O Z M FLOCCULOSA
T. L AVIS AND R. R. BÉLANGER
Departement de phytologie, Centre de recherche en horticulture, Université Laval.
Ce chapitre a été publié dans la revue Applied and Environmental Microbiology (2001)
67:956-960
Connaître le mode d'action spécifique de Pseudozymo flocculosa est un atout
important dans la prédiction de son efficacité à court et & long terme dans un contexte de lutte
biologique. De même, la co~aissance de la nature exacte de son mode d'action peut aussi
permettre de determiner l'impact écologique que son application massive comme biofongicide
pourrait avoir en milieu horticole. Sachant que le mode d'action de P. flocculosa est
l'antibiose (voir chapitre prkcédent), il serait important de savoir par quel rnbcanisme les
acides gras ont une activité antifongique sur les organismes cibles. Suite à des travaux
précédents, l'hypothèse que la sensibilité de divers organismes aux acides gras antifongiques
de P. jlocculosa dicterait la gamme d'hôtes de cet agent antagoniste a Ct6 émise. Cependant,
l'issue de la relation agent pathogènefagent antagoniste dépend également des facteurs
environnementaux requis pour leur croissance respective.
Cette recherche s'intéressait donc aux mécanismes mol6culaires de l'activité d'un des
acides gras toxiques, l'acide cis-9-heptadécenoïque (ACHD), contre divers organismes
possédant différentes compositions lipidiques membranaires. En effet, suite aux travaux de
Benyagoub et al. (1996b)' il avait et6 montré que la sensibilité de certains champignons aux
acides gras toxiques semblait liée à la composition lipidique de leur membrane cellulaire. De
ces mêmes travaux, I'hypoth&se retenue etait qu'un contenu faible en sterols membranaires
ainsi qu'un haut taux d'insaturation des chaînes acyles des phospholipides conféreraient une
sensibilité accrue des champignons aux acides gras toxiques.
Ainsi, des études ont Cté entreprises sur une gamme plus importante de champignons
ayant des compositions lipidiques membranaires différentes. Un champignon de blanc
(Sphaerotheca filiginea) a dte inclus dans ces expériences afîn d'dtablir la relation entre sa
composition lipidique, sa sensibilitt aux acides gras toxiques et sa susceptibilitd à P.
jlocculosa. De plus, une localisation de I'ACHD dans les fractions lipidiques de champignons
traités a étt conduite pour déceler son site d'action dans les membranes fongiques.
L'inhibition de la croissance et/ou de la germination a varit considdrablement selon les
organismes à l'étude. Ce rCsultat a permis de révéler des groupes de diffërentes sensibilitds
face à I'ACHD. L'analyse de la composition lipidique membranaire des champignons cibles a
montré que la sensibilité de certains &ait reliée B un taux faible de sterols membranaks.
Contrairement B ce qui avait et6 supposC lors de travaux pdcedents, le taux d'insaturation des
chaines acyles des phospholipides membranaires n'a pas semble être reliC la sensibilité. Nos
rdsultats indiquent que l'ACHD n'agit pas directement sur les stérols membranaires, n'est pas
métabolisé ou encore modifie par les champignons soumis h son contact. Un mécanisme
structural du mode d'action de I'ACHD, tenant également compte des autres acides gras
antifongiques produits par P. jlocculoso, est propose afin d'expliquer l'activité de ces acides
gras et leur spkificité. La séquence probable des evhements moléculaires impliquds serait (i)
l'incorporation de I'ACHD dans les membranes fongiques, (ii) une élévation de la fluidité
membranaire plus ou moins variable en regard avec la capacitti de la membrane B tamponner
(contenu en stérols) l'arrivée des acides gras toxiques dans les champignons, (iii) une
désorganisation de la membranes causant des désordres de conformation des protéines, une
augmentation de la perméabilité membranaire et, ultimement, la désintdgration du cytoplasme.
ABSTRACT
cis-9-heptadecenoic acid (CHDA), an antifungal fatty acid produced by the biocontrol
agent PseudozymajloccuIosa, was studied for its effects on growth andor spore germination
in h g i . Inhibition of growth and/or germination varied considerably and revealed CHDA
sensitivity groups within tested fungi. Analysis of lipid composition in these fungi
demonstrated that sensitivity was related primarily to a low intrinsic sterol content and chat a
high level of unsahiration of phospholipid fatty acids was not as involved as hypothesized
previously. Our data indicate that CHDA does not act directly with membrane sterols, nor is it
utilized or otherwise modified in fbngi. A structural mechanism of CHDA, consistent with the
other related antifungal fatty acids produced by P. jlocculosa, is proposed in light of its
activity and specificity. The probable molecular events hplicated in the snisitivity of fhgi to
CHDA are (i) partitionhg of CHDA into fûngal membranes; (ii) a variable elevation in
fluidity dependent on the buffering capability (sterol content) in fungi; and (iii) higher
membrane disorder causing conformational changes in membrane proteins, increased
membrane permeability and, eventually, cytoplasmic disintegration.
3.1. INTRODUCTION
Pseudozyma jlocculosa (Traquair, Shaw et Jarvis) Boekhout et Traquair (=Sporothrk
flocculosa Traquair, Shaw et Jarvis) (8) is a yeast-like fùngus with biocontrol properties
against powdery mildew fhgi (3, 14, 15, 20). Cytochemical observations revealed that it
induces a rapid collapse of powdery mildew conidial chains and a cytoplasmic disintegration
of the cells (16) through the production of unusual extracellular fatty acids with antifungal
properties (1, 4, 11). These antifùngal fatty acids cause the release of intracellular ions and
proteins when in contact with sensitive fungi (16), suggesting that they disrupt properties and
functions of the cytoplasmic membrane. Benyagoub et al. (5) hypothesized that this fimgal
sensitivity was related to a low sterol content and to a high degree of unsahiration in
phospholipid fatty acids in fungal membranes, factors which increase membrane fluidity.
Indeed, they showed that the antifungal fatty acids caused a dose-dependent elevation in
fluidity in artificial membranes constnicted fiom the total lipids of the sensitive fungus
Cludosporium cucumerinunr Ellis et Arth, whereas artificial membranes made with lipids of P.
flocculosa demonstrated no changes in fluidity (5).
In general, elevated fluidity is known to cause disorder, i.e., a higher degree of
mobility of phospholipid acyl chains in the membrane bilayer. This alteration in acyl chah
packing can result in changes in membrane dynamics which would affect the activity of
membrane-bound proteins (12). Since toxic fatty acids, in general, seem to interfere with
multiple, apparently unrelated membrane enzymes (13), it has been proposed that the
interaction between the fatty acids and cellular enzymes in sensitive fungi is indirect and non-
specific (19). However, to our knowledge, there are no documented cases which propose a
specific mode of action of unusual fatty acids in living cells and explicitly discuss the
differential response of cells to these compounds.
It has been suggested that free fatty acids alter membrane fluidity either by (i)
partitioning into the lipid bilayer of cells (27) or by (ii) inclusion into fatty acyl chains of
membrane phospholipids (1 3); toxic molecules containhg double bonds, such as P. ficculosa
antifungal fatty acids, may also act by causing changes in permeability towards low-
molecular-weight substances (1 8) by (iii) binding to or altering membrane sterols (10,26). In
this study, these three hypotheses were tested using the free fatty acid cis-9-heptadecenoic acid
(CHDA) produced by P. flocculoscl to treat a range of fiingi. To this encl, our objectives were
(i) to localize CHDA in the membranes of growing h g i exposed to a sublethal dose of this
compound; (ii) to analyze its comsponding efTect on fimgal growth; (iii) to quanti@ cellular
sterols and phospholipid fatty acid unsamation in a number of fungi; and (iv) to determine a
possible link between intrinsic membrane components in fungi and smsitivity to CHDA.
3.2. MATERIALS AND METHODS
3.2.1. Fungal matenal.
Botvtis cinerea Pers. :Fr., Cladosporium cucumerinum, Idriella bolleyi (S prague) Arx
(=Micodochiurn bolleyi [Sprague] de Hoog et Hermanides-Nijhof), Phytophthora infstans
(Mont.) de Bary, Pseudozymu rugulosa (Traquair, Shaw, et Jarvis) Boekhout et Traquair
(=Sporothrix rugulosu Traquair, Shaw, et Jarvis), and Pythium uphonidermatum (Edson)
Fitzp. were maintained on potato dextrose agar (PDA). Sphaerotheca fuliginea
(Schlechtend.:Fr.) Pollacci, a biotrophic fungus unable to grow on artificial media, was
maintained on long English cucumber plants (Cucumis sativus L. cv. Corona).
3.2.2. Synthesis of CHDA.
CHDA was synthesized as previously described (1) and stored at
-24OC in crystalline form.
3.2 -3. Effects of CHDA on fimgal growth and spore germination.
For assessrnent of fimgai growth inhibition, two agar disks (1 5 mm) of the fur@ under
study were suspended in 100 ml of potato dextrose broth (PDB). CHDA dissolved in N,N-
dimethylfomiamide @MF) was added to the broth to give a final concentration of O. 15 mg/ml,
a concentration s h o w previously to reduce growth of C. cucumerinum by approximately 50%
on the basis of dry weight (1). An equivalent concentration of DMF (0.5%) was added to PDB
to serve as a control. Growth was quantified by dry weight measurement following
lyophilization after three day s of culture (25T) on a rotary shaker (1 50 rpm).
To assess spore germination, fûngal spores were suspended in 50 ml of PDB. CHDA
was added as a DMF solution at a concentration of 0.15 mg/ml. DMF alone (0.5%) serveà as a
control. Percent spore germination was detemined after a 24-h incubation period at 25OC. One
hundred spores were assayed for germination. Spores were considered to have germinated
when the length of the g e m tube equaled or exceeded that of the spore itself.
3.2.4. Extraction of lipids.
Total lipid extraction was carried out via a modified Bligh and Dyer method (6).
Lyophilized fimgal cells fiom the above-described expriment were suspended in chloroform-
methanol-water (100:100:50 mllg of dry weight) and homogenized with a Polytron
(Kinematica; Brinkrnan Instruments, Ontario, Canada) for 1 min on ice. The homogenized
solution was protected from light and extnicted for 3 h on a rotary shaker (150 rpm) at 2S°C.
Solvents were separated fiom fùngal biomass by filtration (Whatrnan paper no. 1), and the
extraction procedure was repeated with chlorofonn-methanol-water (200: 10050 mVg of dry
weight) overnight. The combined extracts were diluted with chlorofom and water (1: 1 by
volume), thus partitioning the water-soluble contaminants into the aqueous phase. Trace
amounts of water were removed by dilution with benzene. The organic phase was evaporated
on a rotary evaporator and taken to dryness under a strrarn of nitrogen. The resulting residue
constituted total lipids.
3.2.5. Anaiysis of lipids.
Neutral and polar lipids were separated by acetone precipitation as described by Kates
(2 1). Lipid fractions were separated by thin-layer chromatography on Silica Gel 60 (0.25 mm)
using hexane-diethyl ether-acetic acid (78:20:4 by volume) for neutral lipids and chlorofom-
acetone-methanol-acetic acid-water (10:4:2:2:1 by volume) for polar lipids. Neutral lipid
classes and individuai polar lipids were visualized by iodine staining and were identified by
comparing Rfvalues with authentic standards (Sigma, St. Louis, Mo.).
Fatty acid methyl esten (FAME) were prepared directly from phospholipids by
transesterification using BFj-methanol (14%) for 60 min at 70°C. Individuai FAME were
quantified by a gas chromatograph (GC) (Mode1 5890 series 11; Hewlett-Packard, Mississauga,
Ontario, Canada) coupled to a flame-ionisation detector (FID), using a 30-m DB-225 capillary
column (J&W Scientific, Rancho Cordova, Calif.). Peaks were integrated with a HP
integrator, mode1 3392A. The oven temperature program was as follows: 100°C, 20°C/Mn to
2OO0C, held 5 min, 10°C/rnin to 240°C, held 5 min. H2 injector and FID temperatures were
230 and 250°C, respectively. FAME were identified by comparing retention times with those
of authentic standards (Chromatographie Specialties, Brockville, Ontario, Canada) and
quantified by calibration c w e s of individual compounds. Methylheptadecanoate (1 7:O) was
used as an intemal standard. The degree of unsaturation (Nmol) was determined as previously
described (3 1). where Almol= [% 1 8: 1 + 2(% 1 8:2) + 3(% 1 8:3)]/100.
Sterols were obtained by alkaline hydrolysis of neutral lipids using 1 ml of KOH (33%
wt./vol.) in 10 ml ethanol (95%) for 2 h at 90°C (5). The unsaponifiable fraction containing
sterols was obtained by washing the hydrolysate with hexane. The hydrolysate was then
brought to pH 1 to 2 with HCl (6M), and the saponifiable fraction, containing the fiee fatty
acids, was obtained by washing with hexane.
Total sterols were quantified by gas chromatography using a method proposed by
Rangel et al. (28), with the following modifications. The oven temperature program was
100°C, held 2 min, 20°C/min to 200°C, held 1 min, 10°C/min to 300°C, held 5 min. He
injector and detector temperatures were 290 and 300°C, respectively. Peaks were integrated
with ChemStation software (Hewlett-Packard). Using this method, it was possible to separate
C27, C28, and C29 sterols. C27 and C28 stemls were identified by analyzing mass spectrum
data and comparing retention times with cholesterol and ergosterol, respectively. Sterol classes
were quantified using calibration curves of cholesterol and ergosterol. 5,î4[28]-stigmastadien-
38-01 (C29) was used as an intemal standard and was not present in any of the fbngi. The
steroWphospholipid (S/P) molar ratio was determined using an average molecular weight of
725 for phospholipids (P), 386 for cholestane (C27) derivatives, and 396 for ergostane (C28)
derivatives as follows: S/P molar ratio = (C271386 + C28/396)/(P/725).
3.2.6. Evolution of phospholipid unsaturation.
I. bolleyi and P. aphunidermutum were subjected to m e r analyses of phospholipid
unsaturation in the presence and absence of CHDA. Two agar disks (15 mm each) of fimgal
mycelia were suspended in 100 ml of PDB. CHDA was amended to the broth at a final
concentration of 0.15 mg/ml in 0.5 % DMF. DMF alone (0.5 %) was added to the culture
media to serve as a control. Growth was quaatified daily over a 7&y culture period (2S°C) on
a rotary shaker (150 rpm) by dry-weight measurement following lyophilization. Growth
curves were plotted and phospholipid maturation of some samples was analyzed based on
their growth stage (early and mid-logarithmic, early stationary, and stationary growth phase).
Nmol of b g i were calculated as describcd above.
3.2.7. Analysis of CHDA.
Following incubation of fûngi with CHDA and extraction of lipids from both the
fimgal mass and the culture medium, free fatty acids and phospholipid fatty acids were
analyzed for the presence of CHDA. FAME were prepared from both fatty acid fiactions with
BF3-methanol(14%) and were gas chromatographed as described above. CHDA methyl ester
was identified by comparing retention time with the synthetic standard and quantified by
calibration curve.
3.2.8. Statistical analysis.
Al1 experiments consisted of two replicates of each fungus and were repeated three
times. Analysis of variance (ANOVA) was performed, and Fisher's protected least significant
difference (LSD) was used as a mean sepmation test.
3.3. RESULTS
3.3.1. General effects of CHDA on fiuigi.
CHDA exhibited activity against al1 tested fungi, although the degree of sensitivity
varied considerably. For the concentration used in this study (0.15 mg/ml), growth inhibition
was significantly higher in P. infistans and P. aphanidermatum than in the other fungi (Table
1). Mycelial growth was significantly more hhibited for B. cinerea and C. cucumerinum then
for 1. bolleyi. P. rugulosa had only minimal growth inhibition. Inhibition of conidial
germination was nearly complete for S. fuliginea, elevated in B. cinerea and C. cucumerinum,
and minimal for I. bolleyi and P. rugulosa (Table 1 ) .
3.3.2. Total lipid composition.
Quantitative analysis of total lipids indicated that I. bolleyi and P. rugulosa had
significantly higher intrinsic lipid content than did the other five fùngi (Table 1). P. infistans,
P. aphanidermutum, and S. fuliginea had comparable lipid content whereas C. cucumerinum
had lower levels of total lipids. B. cinerea had the lowest lipid content of al1 fûngi tested. I.
bolleyi and P. rugulosa also had the highest proportion of total sterols, whereas B. cinerea, C.
cucumerinum, and S. fuliginea had statistically similar content (Table 1). P. infestons and P.
aphidermatum contained no sterols. Thin-layer chromatography analysis revealed that
phospholipids were the most abundant polar lipids in ail b g i . Phospholipid contents varied
twofold between the highest (S. filigineu) and lowest (C. cucumerinum) values of tested fungi.
S R molar ratio was significantly higher in I. bolleyi and P. rugulosa than in B. cinerea, C.
cucumerinum and S. fitliginea. Because sterols were not detected in P. infestans and P.
uphanidermatum, SIP molar ratio was 0.0 (Table 1).
3.3.3. Phospholipid fatty acid composition.
For al1 tested fungal controls, palmitic acid (16:O) was the most prevalent saturated
phospholipid fatty acid. The most quantitatively important utl~aturated fatty acids were oleic
acid (1 8: 1) in P. infisruns and P. rugulosa, linoleic acid (1 8:2) in B. cinerea, C. cucumerinum,
1. bolleyi, and P. aphanidermatum, and linolenic acid (18:3) in S. fuliginea. Overall, B.
cinerea, i. bolleyi, and S. filiginea had a significantly higher degree of faiîy acid unsaturation
due to their high proportions of linoleic or linolenic acids. C. cucumerinum had an
intermediately high Nmol value, whereas P. infistans, P. rugulosa, and P. aphonidermatum
had lower values (Table 1). Among h g i treated with CHDA, the A/mol value remained
unchanged for I. bolleyi and P. rugulosa but was significantly higher than controls (P = 0.05)
for B. cinereu (1.9 versus 1.6), C. cucumerinum ( 1.5 versus 1.1 ), P. infistans (1.2 versus 0.9),
and P. aphanidermu?um (0.9 versus 0.8). Only in S. fuliginea was Nmol value significantly
lower (1.3 versus 1.7) than in controls (P = 0.05).
3.3.4. Evolution of phospholipid fatty acid unsaturation.
For 2. bolleyi and P. uphanidermatum, the degree of fany acid unsaturation evolved as
a function of growth stage (Table 2). For I. bolleyi, both mycelial controls and mycelia treated
with CHDA entered different growth phases afier equivalent culture periods. For 1. bolleyi,
there was no difference in Nmol between treatments at any growth phase. However, in the
presence of CHDA, growth of P. aphunidermutum lagged behind that of the control. Thus,
after 3 days of incubation, the P. uphanidermatum control had entered early stationary growth
phase, whereas the CHDA-treated mycelia was in the mid-logarithmic phase. At day 4, the
CHDA-treated fungus was in early stationary phase when the control was well into its
stationary growth phase. The Ah01 value varied accordingly with the growth phase of P.
aphanidermatum. Ah01 was higher in mid-logarithmic phase than in early stationary growth
phases at day 3. Also, the stationary growth phase was associated with a lower Nmol value
than the early stationary phase at ûay 4.
CHDA was not detected in the culture medium or in the phospholipids of any fungus.
However, CHDA was found in the fiee fatty acids in quantities consistent with the dose of
application (P = 0.05) for al1 fûngi.
3.4. DISCUSSION
P. jlocculosa produces antifungal fatty acids known to affect the mycelial growth and
conidial germination of h g i (2, 5, 17). Thus, understanding the mode and site of action of
these antifwgal fatty acids is of major importance in the development of P. flocculosa as a
biocontrol agent. The sensitivity of fungi to these antifhgal fatty acids was recently
hypothesized to be linked to low sterol content and a high degree of phospholipid fatty acid
unsaturation, factors which contribute to an elevated membrane fluidity (5). Here, we have
tested a range of fûngi differing in their specific lipid composition and their sensitivity to the
antifungal fatty acids and have monitored CHDA, one of the antifungal fatty acids, in fùngal
membranes. This has allowed us to present new evidence on the relative sensitivities of h g i
to this compound and provide insight into its specific mode of action.
1. bolleyi and P. ruguiosa, the fhg i most resistant to CHDA, had markedly higher
sterol content than al1 the other fungi, while both P. infetans and P. aphanidermatum, the
most sensitive fungi, lacked sterols altogether. These data support the concept (5) that a low
proportion of sterols is linked to higher sensitivity to P. jlocculosa antifungal fatty acids.
These results were expected because sterols are known to buffer stress-induced modifications
in membrane fluidity (7), thus protecting the fungus in the presence of potentially disniptive
toxic fatty acids. This is of particular importance in the context of the use of P. flocculosa as a
biocontrol agent of S. fuliginea, the pathogen that causes powdery rnildew of cucumber.
Indeed, the low sterol content in S. fuliginea relates well not only to its CHDA sensitivity but
also to the effective biocontrol of cucumber powdery mildew by P. flocculosci.
The degree of phospholipid unsaturation did not correlate with the sensitivity of the
h g i to CHDA. These results suggest that an elevated degree of phospholipid unsaturation is
not as closely involved in sensitivity as was suggested previously (5). To address more deeply
the involvement of phospholipid fatty acid unsaturation in CHDA-mediated events, untreated
h g i were compared with those cultured in the presence of CHDA. In the most CHDA-
resistant fungi, P. rugulosa and I. bolleyi, then was no change in the degree of unsaturation
between treated and control mycelia, but sensitive fungi, with the exception of S. fulginea,
demonstrated a surprising elevation in the Mm01 value. When a stress-induced elevation in
membrane fluidity occurs, cells are expected to compensate by IoweMg their phospholipid
unsaturation to maintain optimal membrane fluidity for growth (25). Results fiom I. bolleyi
and P. aphunidermatum revealed that this elevation in the degree of phospholipid fatty acid
unsaturation does not seem to be an adaptive response to the toxic effect of CHDA but rather a
consequence of growth lag in sensitive fungi. Indeed, slower growth of sensitive fbngi in the
CHDA treatment led to sampling of these fungi in their most active phase (log phase), where a
higher degree of fluidity is necessary. Other results (23, 30, 31) have demonstrated
conclusively that cells need a higher degree of fluidity for such biological processes as
germination and growth. This also explains why S. fuliginea spores treated with CHDA, which
did not geminate, had a lower phospholipid fatty acid unsaturation than the germinated spores
in the untreated controls.
Our results clearly indicate that sterols are not the targets of CHDA, since P. infistans
and P. aphanidermatum, which do not contain or produce sterols, were very sensitive to
CHDA. If sterols were the active sites of CHDA, these fungi would be unaffected by the
treatment .
At the dose used in this study, CHDA was not retrieved fiom the media in which fùngi
had been cultwed, indicating its uptake or insertion into fùngal cells. When fimgal lipid
fractions were analyzed, CHDA was never present in the fatty acyl chains of phospholipids.
This indicates that modification or utilization of CHDA in fungi is not involved in its toxicity.
Moreover, CHDA was completely recovered from the fiee fatty acid fraction of fungi,
indicating that CHDA is present in fiee form in fûngal membranes. As described previously
(22,27), fatty acids, in general, are known to fmly partition Uito membranes. Previous reports
indicate that cis-unsaturated fatty acids induce disorder in neighboring acyl chains due to their
bulkiness caused by the high motional freedom at a certain distance fiom the carboxyl group
(24,29). The cis double bond produces a fhed kink or bend in the fatty acid. Rotation of the
molecule causes disorder in the neighboring membrane acyl chains, thus causing an elevation
in membrane fluidity (9). Nonspecific changes in physical characteristics of the bilayer could
therefore induce conformational changes in membrane proteins and thereby aiter their normal
function (12, 13). These results also explain the higher activity of other P. flocculosa
antifùngal fatty acids (4) which possess additional methyl branch structures capable of
disrupting membrane chain packing by occupying a larger cross-sectional area in the
membrane bilayer (24).
Overall, our data and that of previous reports (5, 17) are best explained by a model in
which the lipid bilayer of h g a l membranes are the primary target of the action of CHDA and
the other closely related fatty acids produced by P. jlocculosa (Fig. 1). At the molecular level,
current data suggest that a sublethal dose of CHDA would readily partition into fimgal
membranes. It is not modified or otherwise utilized but causes an elevation in membrane
fluidity in its free form. Fungi with a high sterol content can buffer the stress-induced
elevation in fluidity and can grow at their maximal rate (7). By contrast, fungi containhg little
or no sterol cannot deal as well with the presence of CHDA, and the ensuing loss of membrane
integrity retards their growth rate. At higher doses of CHDA, the greater elevation in fluidity
(higher disorganization) (5) would cause changes in membrane penneability. This would
cause the release of intracellular electrolytes and of proteins (1 7) and, eventuaîly, cytoplasmic
disintegration (1 6) of mycelia and spores.
This study, in which living fûngal cells were used as model membranes, is the fust, to
our knowledge, to propose a specific mode of action for unusual fatty acids and the molecular
events involved in their toxicity. This is of great relevance in understanding the bais of the
antagonistic effect of P. jlocculosa on S. filiginea, one of the targeted pathogens for use of the
biocontrol agent. This study gives greater insight into the means by which P. flocculosa
protects its habitat on the leaf surface and thus how it is able to exert its biocontrol activity
against powdery mildew h g i , such as S. fitligineu, with which it shares its ecological niche.
ACKNOWLEDGMENTS
This work was supported by a grant fiom the Natural Sciences and Engineering
Research Council of Canada and Plant Roducts Co. Ltd. We thank R. Boulanger, S. Caron,
and S. Couture for technical assistance, C. Labbe for graphic work, T. Carver for proof idhg
the manuscript, and C. Willemot and M. Paquot for critical nview of the manuscript and the
model.
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TABLE 2. Evolution of growth phase and phospholipid fany acid unsaturation of selected
h g i in absence (control) and presence (treated) of a sub-lethal dose of CHDA.
L bolleyi 2
3
4
6
P. aphanidermutum 2
3
4
7
Control
Treated
Control
Treated
Control
Treated
Controt
Treated
Control
Treated
Control
Treated
Control
Treated
Control
Treated
"EL, early logarithmic phase; ML, mid-logarithrnic phase; ES, early stationary phase; S, stationary phise.
Ydlmol = [b/. 18: 1 + 2(% l8:2) + 3(% 18:3)]1100.
=Within control and matcd fungi at the samc gowth period, = significantly differcnt at P = 0.05; ** =
significantiy ciiffernt at P = 0.01.
Figure 1. Proposed model of activity of CHDA, an antifungal compound produced by P.
/locculosa.
(A) Susceptible fungi. CHDA partitions into the hydrophobic region of fungal membranes,
producing significant changes in packing of lipid molecules by inducing disorder in
neighboring acyl chains due to its high motional fieedom. The resulting change in membrane
dynamics would affect the activity of membrane-bound proteins. These effects result in
alteration of membrane potentials which leads to its collapse, as reported in susceptible fùngi
exposed to P. /locculosa (1 7). (B) Resistant h g i . Sterols b a e r stress-induced fluctuations in
membrane fluidity by ordering fatty acyl chains. This would maintain more optimal activity of
membrane-bound enzymes and proteins by minimizing the impact of the CHDA-induced
disordering effect . Minimal membrane alterations are induced, and resistant fungi can
overcome this stress, as observed with 1. bolleyi (2). This model is consistent with the activity
of the other related antifungal fatty acids produced by P. jlocculosa, namely 6-methyi-9-
heptadecenoic acid, whereby its methyl branch wodd cause an even greater disturbance in the
lipid environment which would explain its greater activity in vitro (l,5).
CHAPITRE IV
MOLECULAR AND PHYSIOLOGICAL ANALYSIS OF THE POWDERY MILDEW
ANTAGONIST PSEUDOZM FLOCCULOSA AND RELATED FUNGI
T. J. AVIS', S. J. CARON', T. BOEKHOUT*, R. C. HAMELIN', and R. R. BÉLANGER'
'~é~artement de phytologie, Centre de recherche en horticulture, Universitt Laval.
un entra al bureau voor Schimmelcultures, Yeast Division, Delft, The Netherlands.
'centre de Foresterie des Laurentides, Natural Resources Canada, Sainte-Foy, Quebec.
Ce chapitre a dté publit dans la revue Phyto@oIogy (2001) 91 :249-254
Lors du développement du biofongicide Sporodexm par le laboratoire de Bio-contrôle
de l'université Laval, seule la souche de Pseudozyma jlocculosa (Traquair, L. A. Shaw &
Jarvis) Boekhout & Traquair (Boekhout, 1995), isolat A22, a été dtudiee quant A son potentiel
d'utilisation comme agent de lutte biologique contre le blanc. Cependant, d'autres isolats de
Pseudozyma ont Cté identifies depuis ce temps, et n'ont jamais Cté caractérises pour leur
potentiel antagoniste au blanc. Il a dté montre précédemment que la selection de l'agent
antagoniste le plus approprié dans un programme de lutte biologique pouvait dépendre de
l'évolution du contexte agronomique d'utilisation (Kiss et Nakasone, 1998; Grondona et al.,
1997). Par exemple, un agent antagoniste effcace dans des conditions agronomiques précises
peut être moins performant lorsque certains facteurs changent tels l'agent pathogène ciblé, les
conditions culturales et les régions géographiques d'utilisation, d'où l'importance de possdder
plusieurs candidats potentiels différents.
A h d'identifier et de selectiomer le(s) individu(s) le(s) plus efficace(s) et approprid(s)
dans le cadre du développement d'un produit de lutte biologique il faut avoir des outils de
sélection précis.
Cette recherche s'intéressait donc a l'étude de la variabilité des isolats connus de
Pseudorynuj7occulosa en ce qui a trait à leur potentiel antagoniste ainsi qu'a leur pertinence
pour leur développement dans le cadre d'un programme de lutte biologique contre le blanc.
Ainsi, des caractéristiques phénotypiques et génotypiques ont été utilisées dans le but d'établir
l'identité et la diversité de cet organisme.
Dans une première partie, différents isolats de P. jlocculosa et d'autres especes très
voisines, ont et6 analyses. L' Ctude de séquences d'ADN ribosomique a permis de discriminer
P. jlocculosa d'autres espèces du même genre, et a permis d'identifier deux isolats de
Pseudozyma non identifies comme appartenant B l'espéce flocculoso. Les empreintes
génétiques basées sur les microsatellites ont t6vdlé trois souches distinctes de P. jlocculm
panni les isolats testés.
Dans un dewieme temps, les propriCtés biologiques et la production de métabolites
antifongiques ont et6 Cgaiement Ctudiées. De ces travaux, seuls les différents isolats de P.
flocculosa ont montré une activitb antagoniste et &galement, la production d'au moins un des
deux acides gras antifongiques préalablement Ctudids (voir chapitres précedents).
Cette étude a finalement permis la production de marqueurs mol~culaires utiles pour
distinguer P. jlocculosa d'autres espèces voisines et pour identifier différentes souches au sein
de cette espèce. Suite ces travaux, il a Ctk possible d'identifier de nouveaux candidats
potentiels, bien qu'ils soient gendtiquement diffbrents, comme agents de lutte biologique
contre le blanc. Enfin, la production de marqueurs génétiques permettra Cventuellement de
construire des outils moleculaires spécifiques aux isolats afin de faciliter la recherche et le
développement de P. jlocculosa comme agent de lutte biologique contre les champignons du
blanc.
ABSTRACT
A nurnber of phenotypic and genotypic characteristics wen used to ascertain the
identity and diversity of Pseudoryntajlocculosa, a natural antagonist of powdery mildews that
has received littie attention in tems of taxonomy. To this end, several putative isolates of P.
jZocculosa as well as several closely related species were analyzed. Ribosornal DNA
sequences distinguished P. flocculosa from other Pseudozyma spp. and identi fied two
previously unknown Pseudozyma isolates as P. flocculosa. Random amplified microsatellites
revealed three distinct P. flocculoso strains arnong the tested isolates. Biocontrol propenies
and antifungal metabolite production were limited to the P. j?occulosa spp. Results produced
useful molecular markers to (i) distinguish P. jlocculosa fiom other related h g i , (ii) identiQ
different strains within this species and, (iii) aid in the construction of isolate-specific
molecular tools that will assist in research and development of P. jlocculosa as a biocontrol
agent of powdery d d e w fimgi.
4.1. INTRODUCTION
Pseudorymaflocculosa (Traquair, L. A. Shaw & Jarvis) Boekhout & Traquair (syn.:
Sporothrix jlocculosa Traquair, L. A. Shaw & Jarvis) (6) is a yeastlike fungus with strong
antagonistic activity against powdery mildew fungi (3, 1 1, 12, 1 6). Cytochemical observations
revealed that the antagonist induces rapid collapse of powdery mildew conidial c h a h and
cytoplasmic disintegration of fimgal cells (13). The biocontrol agent produces extracellular
fatty acids with antifungal properties (4,7). Bioassay of these fatty acids confmed that they
induce the same toxic effect in fungal cells as P. flocculosa itself (1, 14), confirming their
importance in the biocontrol potential of the antagonist.
Current development of this fungus as a biocontrol agent has highlighted the
importance of assessing the identity and diversity of P. flocculosa isolates. First and foremost,
taxonomie confusions have hampered basic research and development of the fungal antagonist
as a biocontrol agent. The fungus was initially classified in the Sporothrix genus (20), though
recent studies have suggested that it is more closely related to the genus Pseudo~yma (2, 5).
Assessing identity and diversity is also of great importance in the selection of suitable
biocontrol candidates as well as in authentication of P. flocculosa isolates for industrial
procedures and registration purposes (9). Indeed, the efficacy of different isolates may Vary
with changes in crops and geographic regions of intended use. This may require the re-
evaluation of the most effective isolates of the fungus as these conditions evolve in agronomic
practices (1 7). Furtheme, authentication of fungai isolates is critical for consistent results in
eficacy in research and commercial trials as well as in the process of mass production in an
industrial setting (19, 23). In al1 of these processes, it is essential to have means for the
discrimination of P. flocculosa fiom other closely related organisms and to assess variability h
within the P. jlocculoso spp. In this context, DNA fingerprinting seems to offer a sensitive and
reliable approach to genetically characterize the biocontml agent.
Our objectives were, thenfoie, (i) to assess ribosomal DNA (rDNA) sequences and
randorn amplified microsatellite (RAMS) fingerprints as tools in identityldiversity studies of
P. flocculosu and related fungi, and (ii) to correlate this information with biological activity in
an attempt to provide a basis of selection of pruper candidates for biocontrol programs.
4.2. MATERLALS AND METHODS
4.2.1. Biological Materials.
Fungal isolates listed in Table 1 were derived fkom single-spore cultures and were
maintained on slants of potato dextrose agar (PDA) at 4OC. Cells were cultured in potato
dextrose broth (PDB) on a rotary shaker (150 rpm) at 25OC. The fimgal biomass was
centrifuged at 10,000 rpm for 20 min, and the culture medium was discarded. Fungal cells
were washed with sterile distilled water and centrifbged for an additional 20 min. The water
was discarded, and the fimgal biomass was transfemd to sterile 1.5-ml microtubes. The cells
were lyophilized and stored at -20°C until use.
4.2.2. DNA extraction.
Genomic DNA was prepared as follows. Lyophilized h g i (= 10 mg) were mixed with
an equal amount of diatomaceous earth (Sigma Chemical Co., St. Louis) and ground with a
pestle. Six hundred microlitres of extraction buffer (100 mM Tris-HCl at pH 9.5, 2%
cetryltrimethylammonium bromide, 1.4 M NaCI, 1% polyethylene glycol 8000, 20 mM
EDTA, and 1% D-mercaptoethanol) was added to the macerated cells and incubated at 65°C
for 1 h. The mixture was extracted with 600 uL of phenol/chloroform/isoarnyl alcohol
(25:24:1) and centrifuged at 10,000 X g for 5 min. The supernatant was transferred to a new
microtube and re-extracted with 400 uL of phenol/chloroform/isoamyl alcohol. The
supernatant was precipitated with 1 vol of cold isopropanol and centrifuged et 10,000 X g for
5 min. The precipitate was washed with 70% ethanol, vacuum-dried and resuspended in 50 uL
of Tris-EDTA b d e r ( 1 W Tris-HCI and 1 mM EDTA, pH 8). Al1 DNA was diluted (1 : 100)
to a dilution detemined to yield the most reproducible polymerase chain reaction (PCR)
amplification
4.2.3. PCR amplification conditions.
The regions of rDNA were arnplified (Table 2): the entire region containhg both
intemal transcribed spacers (ITS), partial sequences of the large rnitochondrial subunit
(mtLSU), and partial sequences of the small nuclear subunit (nSSU). PCR reactions were
carried out by the Taq DNA polymerase system (Boehringer Mannheim Biochemica,
Mannheim, Germany) in volumes of 25 uL containing 1 X the supplied reaction bufier
(including 1.5 mM MgC12), 100 uM each deoxynucleoside triphosphate, 1 uM each primer, 1
unit of Taq DNA polymerase, and 1 uL of the template DNA. Amplifications were performed
in a thermal cycler (MJ Research Inc., Watertown, MA) programmed for an initial
denaturation step at 95OC for 3 min, 35 cycles of 92°C for 30 s, 58OC (ITS and mtLSU) or
52'C (nSSU) for 30 s, and 72OC for 1 min. The amplifications were completed with a IO-min
final extension at 72°C.
RAMS fmgerprints were produced using single-primer amplification with GT and
CCA primers (Table 2). PCR was carried out (Expand Long Template PCR; Boehringer
Mannheim Biochemica) in 25 uL volume containing 1 X the supplied buffer (including 1.5
m M MgC12), 100 uM each deoxynucleoside triphosphate, 0.4 uM primer, 2% dimethyl
sulfoxide, 1 unit of Taq Expand DNA polymerase, and 1 uL of the template DNA. The
thermal cycler was programmed for an initial denaturation step at 95°C for 10 min, 34 cycles
of 94°C for 30 s, 58°C for 1 min, and 68OC for 2 min. The amplifications were completed with
an &min final extension at 68'C. Amplicons were visualized on 1.5% agorose gels in Tris-
acetate-EDTA buffet at 3 Vkm for 1.5 h. Gels were stained with ethidium bromide and
photographed under W light.
4.2.4. Nucleotide sequencing determination.
rDNA PCR products (ITS, partial mtLSU, and partial nSSU) were purified on minispin
columns (QIAquick; Qiagen, Hilden, Germany) and directly sequenced using an aflatoxin
biosynthesis inhibitor automated sequencer (373A Strech; Applied Biosystems, Mississauga,
Ontario, Canada). Multiple sequence alignmeut was performed with Clustal W, available on-
line fiom the Baylor College of Medicine. Following visual inspection, final alignment of the
sequences was performed by hand. Nucleotide sequences are available in the GenBank
database as Accession Nos. AF294690 to AF294724.
4.2.5. Phy logenetic analy sis.
Phylogenetic analyses were performed by phylogenetic analysis using parsirnony
(PAUP 4.0bl; Sinauer Associates, Sunderland, MA) on rDNA sequences of the entire ITS
region, partial mtLSU, and partial nSSU, both as individual and combined data sets. Indels
were coded as single events. Unweighted parsimony analyses were performed on the
individual data sets, excluding uninformative characters, using the heuristic search option with
1,000 random addition sequences with MULTREES on and tree bisection-recomection branch
swapping. Maximum parsimony analysis of the combined data set was by the branch-and-
bound option in PAUP for exact solutions. The nSSU data set was comprised of a 14-taxon
matrix, including sequences fiom Sporothrix schenckii (GenBank Accession No. M85053).
The combined rDNA data set was comprised of an Il-taxon matrix, fiom which the three
Sporothru spp. were excluded. Saccharomyces sp. (GenBank Accession No. AB040998) and
Tilletiopsis washingtonensis were selected as outgroups for rooting the nSSU and combined
rDNA trees, respectively. Clade stability was assessed by 1,000 parsirnony bootstrap
replications. Neighbor-joining trees were also infened with uncorrected "P" and maximum-
likelihood distance methods. Concordance of the three rDNA datasets was evaluated with the
partition-homogeneity test implemented with PAUP, using 1,000 random repartitions. The
Kishino-Hasegawa likelihood test implemented in PAUP was used to compare various
constrained and unconstrained topologies (Table 3).
4.2.6. Production of antifûngal fatty acids.
lsolates were tested for the production of 9-heptadecenoic and 6-methyl-9-
heptadecenoic acids, two antifungal fatty acids produced by P. jlocculosa (1). Fungi were
cultured in PDB for three days on a rotary shaker (150 rpm) at 2S°C, followed by a 28-day
still culture, protected fiom light. Culture media was separated fiom fimgal biomass by
centrifugation at 10,000 rpm for 20 minutes. Culture media was extracted (3 X 50 mi) with
chlorofom (4). The combined organic phases were roto-evaporated and taken to dryness with
a Stream of nitrogen. The oily midue, containing both antifùngal fatty acids, was derived with
Phenacyl-8 (Pierce Chernical Company, Rockford, XL) (8). Phenacyl ester fatty acids were
analyzed by reverse-phase high pressure liquid chromatopphy (Nova-Pak C-18 column, 60
A, 4 um, 3.9 x 300 mm) coupled to a photodiode array detector (Waters Limited, Mississauga,
Ontario, Canada). The eluent was a gradient of 80 to 100% acetonitile, acidified with 0.1 %
H3P04, at a 1 .O d m i n flow rate, as follows: 80 to 100% over 15 min, 100% held foi 5 min,
and 100% to 80% over 2 min. 9-heptadecenoic and 6-methyl-9-heptadecenoic acids were
identified in fungal culture medium extracts by comparing retention times and W spectra
with authentic standards (1). The experiment was repeated three times for each fungus.
4.2.7. Biocontrol activity of h g a l isolates.
Fungal isolates were assayed for their ability to antagonize Sphaerothecu fuliginea
(Schlechtend.:Fr.) Pollacci, causal agent of cucurnber powdery mildew. Foliar disks (5 cm)
were cut from S. fuliginea-infected cucumber leaves and placed on 20-20-20 agar containhg 2
gniter of 20-20-20 'al1 purpose' soluble fertilizer and 8 g/liter bacto-agar. Disks were cut from
the leaf portions covered by at least 95% with colonies of S. fuliginea. Spores of tested isolates
(1 x 106 spores/ml) were placed in water with 0.02% Aqua-Aid (Ken Crowe Inc., Montréal,
Qudbec, Canada). Spore were sprayed directly on the infccted cucumber disks and monitored
daily over a 7-day period for antifûngal activity evaluated as collapse of S. fulginea conidial
chains. Aqua- Aid alone (0.02%) served as a control. The experiment was repeated three times
for each fungus.
43. RESULTS
nie partial nSSU of rDNA data set consisted of 812 nucleotide characters, 65 of which
were parsimony idormative. These data yielded a single most parsimonious tree (MPT) (Fig.
1). Neighbor-joining tzees were found using the unconected P and maximum-likelihood
distance options and were topologically concordant with the MPT. Clade stability, as assessed
by 1,000 bootstrap replications, identified strong support of two independent groups: the
Pseudozymu clade that included the four P. flocculosa isolates, PBC, and PH, and the
Sporothrîx clade.
When the Sporothrix spp. were excluded, there was extensive variability within the
three rDNA sequences in al1 tested Pseudoryna spp. 'the ITS rDNA data set rrvealed that PF-
A22, PF-1, PF-RM, PF-IS, PBC, and PH had identical sequences, with the exception of a 2-bp
insertion at positions 1 30 (T) and 13 1 (T) for PH. Sequences comprishg the 1,183-bp partial
mtLSU of rDNA data set contained a large intron of 733 bp for the four P. flocculoscl isolates,
PBC, and PH at the 3' end of these sequences. Partial sequences of the nSSU of rDNA
revealed that P. prolifca contained a large intron between position 64 and 480. Results of the
partition-homogeneity test (P = 0.072) indicated that the three rDNA trees reflected the same
underlying phylogeny . The three data sets were thus combined and analyzed by several tree
building prognuns.
Maximum parsimony yielded five equally parsimonious trees for the combined data set
(Fig. 2). Neighbor-joining trees were topologically concordant with the MPTs. Clade stability,
as assessed by 1,000 bootstrap replications, identified strong support of multiple independent
grouping of al1 Pseudozyma spp. as well as grouping the two unidentified Pseudozyma spp.
(PBC and PH) in the P. flocculosa clade in a polytomy set of relationships. Topological
constraints forcing the monophyly of different P. flocculosa groups were not significantly
different than the unconstrained MPT when subjected to the Kishino-Hasegawa likelihood test
(P = 0.05) (Table 3).
RAMS analysis revealed extensive differences in microsatellite fmgerprints between a
group consisting of the four P. jlocculosa isolates, PBC, and PH and a second group
comprised of the other Pseudozyma spp. GT primer yielded differences in banding patterns
between the four P. fIocculosa isolates fiom Ontario and Québec (PF-A22, PF-1, PF-RM, and
PF-1s) and the isolates fiom British Columbia (PBC) and the Netherlands (PH) (Fig. 3). With
al1 other major fragments the sarne, a PCR product of approximately 620 bp was present in the
four isolates from Ontario and Québec, whereas in PH and PBC, the "equivalent" fragment
was of approximately 600 bp. Moreover, CCA primer provided an amplicon positioned at
1,500 bp in PH and PBC that was absent in the four P. flocculoso isolates (Fig. 4). With CCA
primer it was also possible to differentiate the PH and PBC isolates fiom each other. Indeed,
the PBC isolate yielded a fragment of approximately 600 bp which was absent in the PH
isolate. Neither RAMS primer distinguished differences in banding patterns between the four
P. flocculosa isolates from Ontario and QuCbec.
Analysis of both antifbngal fatty acids revealed that PF-A22, PF-1, PF-RM, PF-IS,
PBC and PH produced 9-heptadecenoic acid (Fig. 2). 6-methyl-9-heptadecenoic acid was
produced by PF-A22, PF-1, PF-RM, PF-IS, and PBC. PH did not produce 6-methyl-9-
heptadecenoic acid. None of the other tested fungi produced either of the antifungal fatty
acids.
When selected fbngi were bioassayed against S. fuliginea, only the four P. flocculosa
isolates, PBC, and PH induced collapse of S. fulginea conidial chains (Fig. 2). The remaining
h g i showed no evidence of biocontrol activity against S. fuliginea under our experimental
conditions.
4.4. DISCUSSION
P. jlocculosa is a recently characterized yeastlike fungus with the potential for
biological control of powdery mildew disease. When selecting h g i for a specific purpose or
function it is essential to correctly classify and identifi isolates for the selection of appropriate
candidates in given situations. This precise identification is also necessary to ensure
consistency in experimental, industrial, and commercial processes. In this study, a mixture of
phenotypic and genotypic characteristics were studied to ascertain the identity and diversity of
the known P. flocculosa isolates in order to gain insight into the strains that are of interest in
biocontrol prograrns.
P. jlocculosa was initially classified in the genus Sporothrix based on classical
mycology techniques (20). Recent studies have suggested that the fungus is in fact fiom the
genus Pseudoryma, based, in part, on sequences of the large nuclear subunit (nLSU) of rDNA
(2, 5). In this work, the nSSU of rDNA was used to directly compare Pseudotyma isolates,
including P. flocculosa, with three Spororhrix spp. These results conclusively demonstrated
that P. flocculosa is indeed distant from Sprothrix spp. and is more closely related to the
genus Pseudoryma.
Cladistic analysis of the combined data set of three rDNA sequences clearly
demonstrated that the tested Pseudoqma fungi are distributed into five distinct species based
on their high bootstrap values (95% or greater). This is consistent with previous reports that
have used the nLSU of rDNA (2,5). Moreowr, the Kishino-Hasegawa likelihood test forcing
the monophyly of the unknown Pseudotymu isolates fiom British Columbia (PBC) and the
Netherlands (PH) with the four P. jZocculoso isolates indicated that both PBC and PH should
be considered as belonging to the P. jlocculosu species.
Microsatellite fmgerprinting of P. jlocculosa isolates suggested that the four isolates
fiom Ontario and Quebec are very closely related. These results were expected beuiuse these
four isolates were initially derived fiom a single strain. Also, microsatellite fmgerprints of
PBC and PH indicated that they are both distinct strains of P. flocculosa. Previous results with
random arnplified polymorphic DNA analysis also nvealed that PH was distinct fiom PBC
and PF-A22, although this analysis could not distinguish the latter two isolates bctween each
other (2 1 ).
Under the test conditions used in this study, biocontrol activity against S. fuliginea,
causal agent of cucumber powdery mildew, was limited to the six P. /locculosa isolates,
including PH and PBC, indicating that biological control properties are not a general
characteristic of Pseudoryna spp. but are specific to certain species. Although P. rugulosa has
been reported to antagonize S. fuliginea (1 1). this was not demonstrated in our experiments.
Two of the antifhgal fatty acids that mediate the biocontrol properties of P. flocculosa
were produced in a11 P. flocculosa isolates, with the exception of PH, which only produce 9-
heptadecenoic acid. This may play an important role in the selection of the most prolific P.
flocculosa isolate in biocontrol programs. P. rugulosa produces a related antifbngal fatty acid,
4-methyl-7,ll -heptadecadienoic acid, but did not produce either of the two antifungal fatty
acids tested in this sndy. This may well effect the general biocontrol property of P. rugulosa,
which has been known to be less effective than P. jlocculosa in controlling powdery mildew
(1 1)-
Overall, this study indicates that P. jlocculosa can be genetically and biochemically
discriminated fiom other related fungi. C m n t results demonstrated that there are three
distinguishable P. flocculosa strains capable of biocontrol activity against S. fuliginea.
Although this biocontrol fimgus has a very limited genetic base, i.e., there are few known
isolates, these findings provide a basis to ideatm, authenticate, and rnonitor these isolates, and
possibly others yet to k discovered. This is of paramount importance in the production and
release of P. jlocculosa in a biocontrol program. For example, as experimental and
commercial-sale testing of these isolates against other powdery mildew h g i as well as in
different cultural and geographical conditions continues, these molecular markers will be
useful in the selection of the most effective isolates to use in a given situation. Moreover,
should more than one isolate of P. jlocculosa be mas-produced in an industrial setting, these
molecular markers would be instrumental in the detection of cross contamination between
isolates in a quality control test (18). These findings could permit the construction of
molecular tools based on isolate-specific sequences. This may allow the possible detection of
mutations which could, if followed by selection or genetic drift, alter the genetic integrity and,
potentially, the biocontrol property of the fungus. The discovery of specific and usefbl genetic
markers in this study will, thus, be instrumental in gaining insight into the diable use of a P.
flocculosa-based biological fungicide against powdery rnildew diseases.
ACKNOWLEDGMENTS
This work was supported by a gant fiom the Natural Sciences and Engineering Research
Council of Canada and Plant Products Co. Ltd. We thank D. Auclair, H. Germain, and N.
Lecours for technical assistance, and K. O'Donnell for assistance with phylogenetic analysis.
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fungus Sporothrix flocculosa against Erysiphe graminis var. tritici. Biocontrol Sci.
Technol. 3:427-434.
1 3. Hajlaoui, M. R., Benhamou, N., and Bélanger, R. R. 1992. Cytochemical study of the
antagonistic activity of Sporothrix jlocculosa on rose powdery mildew, Sphaerotheca
pannosa var. rosue. Phytopaîhology 82583489.
14. Hajlaoui, M. R., Traquair, J. A., Jarvis, W. R., and Bdlanger, R. R. 1994. Antifungal
activity of extracellular metabolites produced by Sprothrix flocculosa. Biocontrol Sci.
Tecbnol. 4:229-237.
1 5 .Hantula, J., Dusabenyagasani, M., and Hamelin, R. C. 1 996. Random arnplified
microsatellites (RAMS): A novel method for characterizhg genetic variation within fungi.
Eur. J. Plant Pathol. 26: 1 59- 166.
16. Jarvis, W. R., Shaw, L. A., and Traquait, J. A. 1989. Factors af5ecting antagonism of
cucumber powdery mildew by Stephanouscus flocculosus and S. rugulosus. Mycol. Res.
92: 162-165.
1 i.Kiss, L., and Nakasone, K. K. 1998. Ribosomal DNA intenal transcribed spacer
sequences do not support the species statu of Ampelomyces quisqualis, a hyperparasite of
powdery mildew h g i . Curr. Genet. 33:362-367.
18. Markovic, O., and Markovic, N. 1998. Ce11 cross-contamination in ce11 cultures: the silent
and neglected danger. In Vitro Cell. Dev. Biol. 34: 1-8.
19. Stacey, G.N., Bolton, B. J., and Doyle, A. 1991. The quality control of ce11 banks using
DNA fingerprinting. Pages 361 -370 in: DNA Fingerprinting: Approaches and
Applications. T. Burke, G. Dolf, A.J. Jeffreys and R Wolff, eds. Birkhauser Verlag, Basel,
S witzerland.
20. Traquair, J.A., Shaw, L. A., and Jarvis, W. R. 1988 New species of Stephanoascw with
Sporothrix anamorphs. Can. J . Bot. 66:926-93 3.
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using random arnplified polymorphic DNA analysis. Can. J. Plant Pathol. 19:380-389.
22. White, T.J., Burns, T., Lee, S., and Taylor, J. 1990. Amplification and direct seqwncing of
fimgal ribosomal RNA genes for phylogenetics. Pages 3 15-322 in: PCR Protocols: a Guide
to Methods and Applications. M.A. Innis, D.H. Gelfand, J.J. Sninsky, and T.J. White, eds.
Academic Press, New York.
23.Woodhead, S.H., O'Leary, A. L., O'Leary, D. J., and Rabatin, S. L. 1990. Discovery,
development, and registration of a biocontrol agent fiom an industrial perspective. Can. J.
Plant Paîhol. l2:328-33 1.
Table 1 . Fungal isolates and species used in this study.
Species Designation Isolatea Host of origin Geographic origin
Pseudozyrna/locctllosa
P. /Iocculosa
P. floccu losa
P. /Ioccufosa
Psdozyma sp.
Pseudozymu sp.
P. mgdosa
P. antariica
P. aphidis
P. prolijka
Spïmîrhrir infita
S. nivea
Tilletiopsis washingtonensis
PF-A22
PF- 1
PF-RM
PF-IS
PH
PBC
PR
PAN
PAP
PP
SI
SN
TW
CBS 167.88
This study
ATCC 74320
This study
This study
This study
CBS 170.88
CBS 5 16.83
CBS 517.83
CBS 3 19.87
ATCC 24422
ATCC 76232
ATCC 96 1 56
T w u m paterne
Rose
Not applicable
Not applicable
unknown
Cucumis sativus
Zea mays
Sediment
Aphid secretion
Scirpus microcarpus
Soi1
Wastewater
Cucumis sativus
Ontario, Canada
QuCbec, Canada
Québec, Canada
Québec, Cana&
Netherlands
British Columbia, Canada
Ontario, Canada
Antarctica
Gemany
Canada
Gemany
Saskatchewan, Canada
British Columbia, Canada
%BS = Centraalbureau voor Schimmelcultures; ATTC = American Type Culture Collection.
Table 2. Polyrnerase chah reaction-amplified regions and primers.
Genomic regiona Primer sequenceb ~ e f ?
ITS 1 f 5'-C'M'GGTCATITAGAGGAAGTAA-3' (10)
Entire ITS
Partial mtLSU ML6 5'-CAGTAGAAGCTGCATAGGGTC-3' (22)
Partial nSSU
RAMS
GT repeat GT SV-VWGTGTGTGTGTGTGTG-3' (15)
CCA rcpeat CCA 5'-DDBCCACCACCACCACCA-3' (15)
'ITS = intemal transcribed spacer, mtLSU = mitochondial large subunit, nSSU = small nuclear subunit, and RAMS = random
amplified microsatellites.
bnie following desigat ions are used for degenerate si tes: V (G, A, or C), H (A, T, or C), D (G, A, or T), and B (O, T, or C).
'TS = this study.
Figure 1 . Single most parsimonious tree inferred fiom partial sequences of the small nuclear
subunit (nSSU) of rDNA. Bootstrap replication frequencies > 50% are indicated above nodes.
GT CCA
100 P. flctc~laro isolrtc A22
Production of 9-hcpfadcccnoic
acid
Ycs
Ycs
Ycs
Roduction of Coll~pse of 6-Wyl -9 - S. filigintu
heptsdccaroic conidia acid
Ycs Yes
Yes Ycs
Figure 2. Strict consensus of the five most parsimonious trees based on the combined dataset of three rDNA sequences. Bootstrap
replication frequencies > 50% are indicated above nodes. GT = GT-primed microsatellite fingerprint (Fig. 3); CCA = CCA-primed
microsatellite fingerprint (Fig. 4).
CHAPITRE V
DISCUSSION G~NÉRALE ET CONCLUSIONS
L'objectif global de cette recherche visait l'étude des propriétés et de la spécificité des
mecariiismes de l'activite antagoniste de l'agent Pseudozymo jlocculosa, afin de produire des
outils de sélection d'isolats les plus appropriés à introduire dans le cadre d'un programme de
lutte biologique contre le blanc des cultures serricoles.
Pour aborder cet objectif, nous nous sommes d'abord intéressés à la caracttrisation de
l'activite biologique des acides gras antifongiques produits par P. flocculosa. Lors de travaux
précédents, les acides gras antifongiques n'avaient pas 6tC spécifiquement testés quant a leur
activité biologique contre des champignons de blanc mais plutôt contre des champignons
modèles, ce qui avait permis alors de simplifier leur détection (Benyagoub et al., 1996a). Il
s'est ensuite avéré necessaire de déterminer si les acides gras étaient des principes actifs dans
l'activité antagoniste de P. flocculosa contre les agents pathogènes du blanc, maladie contre
laquelle l'agent antagoniste est développe comme biofongicide.
La réalisation de la synthèse chimique de deux acides gras antifongiques produits par
P. jbcculosa, i'acide 9-heptadécenoïque et l'acide 6-méthyle-9-heptadécenoïque, a dté un atout
majeur dans notre systéme puisqu'elle a permis de mener des expériences sur leur activite sans
les d i~cul tes liées B leur extraction et lt leur purification dans les filtrats de culture.
L'utilisation de ces acides gras a permis de montrer qu'ils avaient une forte activite
inhibitrice contre Sphaerotheca fuliginea, agent causal du blanc du concombre. En fait, ces
acides gras induisaient le même effet inhibiteur que P. flocculosa lui-même i.e. l'effondrement
des chaînes des conidies du blanc (Hajlaoui et Bélanger, 1991)' ce qui a confirmb leur rôle
dans l'activité biologique de P. flocculosa.
De plus, ces expériences ont permis de mesurer l'effet inhibiteur propre a chacun des
deux acides gras Ctudibs. Ainsi, une diffdrence structurale entre les deux acides gras, en
l'occurrence un groupement méthyle supplémentaire chez l'acide 6-méthyle-9-
heptadkenoïque, a r6v61é que ce groupement fonctionnel induisait une activité inhibitrice plus
tlevde que l'acide 9-heptaddcenoïque. A la lumiére de ces rdsultats, on pourrait sélectionner
des souches de P. jlocculosa ayant une production supCrieure en acides gras antifongiques
méthyles comme moyen de sdlection de candidats ayant un meilleur potentiel de lune
biologique contre le blanc. Cependant, la production Nt vitro de ces acides gras est
présentement longue et hasardeuse. En effet, il est fastidieux de détecter ces moldcules dans
les filtrats de cul- de P. flocculosa, puisque la technique actuelle demande au moins 30
jours de croissance ou l'tpuisement du milieu. Une nouvelle mdthode de culture est
prdsentement l'essai au laboratoire de Bio-contrôle pour permettre la production de
métabolites toxiques en deçh d'une semaine, ce qui reprtsenterait un facteur non ndgligeable
dans le cadre d'un programme de criblage de souches potentiellement efficaces.
Afin d'etablir dbfmitivement le rôle de ces métabolites dans l'eficacite antagoniste de
P. jlocculosa dans la phyllosph&e, il serait révélateur de connaître l'importance de la
production de ces acides gras in situ. Cependant, la degradation rapide de ces produits en
conditions naturelles ne nous a pas permis de les detecter 4 la surface de feuilles de concombre
infectees et traitées avec P. jZocculosa. Une solution envisagée serait l'utilisation d'anticorps
spécifiques aux acides gras antifongiques préalablement complexés à une protéine (haptène).
Cependant, il semble tr5s difficile de créer un anticorps vraiment spécifique suite une
construction acide gras-haptène vue la taille trks petite des acides gras antifongiques.
L'utilisation de P. flocculosa dans le cadre d'un programme de lutte biologique
necessitait une connaissance approfondie de son mode d'action, ceci dans l'objectif de tenter
de prddire son efficacité a court et B long terme (Bélanger et Deacon, 1996). Ainsi. nous avons
entrepris une étude sur les mécanismes d'action des acides gras antifongiques de l'agent
antagoniste, et sur les propriétés intrinsèques des champignons ayant différents niveaux de
sensibilitd face à ces acides gras.
Cette dtude a permis de confirmer l'hypothbse Crnise par Benyagoub et al. (1996b) il
l'effet que la sensibilité relative de différents champignons face aux acides gras antifongiques
etait lide principalement il leur contenu en stdrols membranaires. Nos r6sultats ont montré
qu'un contenu faible ou nul en stdrols chez les champignons causait une sensibilitd accrue aux
acides gras antifongiques. De ce fait, le spectre d'activitt des acides gras antifongiques &tait liC
h une propridtd intrinséque des champignons ciblés. Ceci est similaire aux résultats obtenus
avec la gliotoxine, produite par Gliocladium virens, dont le spectre d'activitt etait lui aussi lie
A une composante intrins@ue des champignons ciblés, en l'occurrence, les groupements thiol
de la membrane cytoplasmique (Jones et Hancock, 1988). Ainsi, le faible contenu en stérols
membranaires du champignon de blanc, S. filiginea, semble dtterminer non seulement sa
grande sensibilité aux acides gras antifongiques, mais aussi sa susceptibilitt face A P.
jlocculosu. Dans ce contexte, il serait de mise de tester si d'autres champignons de blanc sont
plus ou moins sensibles aux acides gras produits par l'agent antagoniste. Par exemple,
Sphaerothecapannosa var rosae, agent causal du blanc du rosier, semble plus susceptible il P.
jlocculosa (Bureau, 1999) que son homologue chez le concombre. Il serait donc intéressant
d'étudier sa sensibilitb face aux acides gras et d'ttablir le lien entre sa sensibilité supérieure et
son contenu en stérols membranaires.
Lors de ces travaux, il a aussi ét6 montré que les acides gras fongitoxiques s'insdraient
naturellement dans les membranes cellulaires des champignons, observation ayant été déjà
faite avec d'autres types d'acides gras (Pjura et al., 1984), et que leur activité biologique se
manifestait sans utilisation, ou autre modification, par les champignons ciblés. Les acides gras,
sous leur forme native, agissaient donc en provoquant une désorganisation générale, ou
augmentation de la fluidité, dans les membranes cellulaires (Benyagoub et al., 1996b). Ces
résultats ont permis de proposer un modèle d'activité des acides gras. Cette activité biologique
serait due à une perturbation des lipides membranaires causée par la rotation des acides gras
autour du double lien en conformation cis. Lon de leur rotation, les acides gras repousseraient
ou désorganiseraient les chaînes acyles des phospholipides qui sont, entre autres composantes
membranaires, responsables de l'intdgrité des cellules fongiques ( D e W et al., 1973; Burt et
al., 1991). Ce modèle est aussi en accord avec l'activité biologique supérieure des acides gras
méthylés. Ainsi, le groupement methyle occuperait un espace physique supplémentaire dans
les membranes fongiques (Macdonald et al., 1984), ce qui aurait pour effet de dbsorganiser
plus efficacement la structure membranaire.
La derniére partie de ces travaux consistait à dtablir l'identité et illustrer la diversité des
isolats connus de P. flocculosa et autres Pseudozyrna spp. Pour ce faire, un nombre de
caract6ristiques gdnotypiques et phhotypiques a Ctb choisi pour des fins analytiques. Au
niveau gdnétiqw, des séquences d'ADN ribosomique ont Cte caractérisées, et des empreintes
génbtiques basées sur les microsatellites ont bté produites. Grâce & ces travaux, il a ttk
possible de distinguer P. flocculosa d'autres champignons du même genre, en plus d'identifier
comme appartenant B I'espéce~occtllosa, deux isolats du genre Pseudozyma jusqu'alors non
identifiés. Le test des microsatellites a également pennis de mettre en fidence l'existence de
trois souches distinctes B l'int&ieur de l'espécejlocculosa. Ces dsultats sont d'une importance
capitale dans le dtveloppement de P. jlocculosu comme agent de lutte biologique. En effet, ils
permettront de développer des tests de contrôle de qualitd afin de vdrifier l'identité du
champignon, la presence de contaminations et l'intégrité du produit h a 1 dans un contexte de
production industrielle (Avis et al., 200 1).
En sus des tests génétiques, l'activité biologique et la production des acides gras
antifongiques ont et6 égaiement suivies chez les mêmes isolats afin de relier les résultats
génétiques aux propriétés antagonistes du genre Pseudozyma. Suite à ces travaux, on a pu
montré que l'activité antagoniste contre le blanc et la production des acides gras etaient
limitées à l'espèce flocculosa. De plus, les trois différentes souches de P. flocculosa,
préalablement identifiées, avaient des activités biologiques distinctes qui pouvaient
potentiellement affecter, de façon positive ou négative, leur efficacité il r6primer le blanc dans
différentes situations d'utilisation puisque d'origines geographiques différentes (Kiss et
Nakasone, 1998, Grondona et al., 1997). Cette étude sur la caracterisation ghotypique et
phénotypique a aussi montrd qu'une des trois souches de P. flocculosa ne produisait pas l'acide
6-méthyle-9-heptadécenoïque contrairement aux deux autres. Toutefois, cette souche avait
montré le plus haut niveau d'activité biologique contre le blanc lors d'essais en laboratoire. Ce
résultat nous laisse croire qu'il existe peut-être d'autres molécules antifongiques qui n'ont
toujours pas été identifides. À la lumière de ce résultat, la création de mutants dbficients dans
la production des acides gras antifongiques serait une option pour déterminer l'importance du
rôle des acides gras dans l'interaction P. jlocculosa-champignons de blanc. Ainsi, il serait
possible de ddterminer si les acides gras sont les seuls principes actifs contre le blanc ou s'il
existe d'autres métabolites qui peuvent agir de façon additive ou en synergie avec les acides
gras antifongiques.
En rdswnd, l'ensemble des rdsultats fait ressortir les découvertes suivantes en ce qui
concerne les propridtds de P. flocculosa: (i) les acides gras antifongiques produits sont des
principes actifs dans le pouvoir antagoniste de P. flocculosa qui pourraient smir d'outils de
sélection des isolats les plus appropries et prolifipues dans le cadre &un programme de lutte
biologique, (ii) les acides gras antifongiques agissent sans autre modification dans les
membranes cellulaires et causent une perturbation physique (ddsorganisation géntrale) des
membranes, (iii) les champignons, avec un faible contenu en stCrols membranaires, sont plus
sensible aux acides gras; cela donne un outil potentiel de prkdiction de l'efficacit6 de P.
jlomlosa bas6 sur cette proptittt! intrinséque de l'agent pathogéne cible en l'occumnce
différentes especes de blanc, (iv) il existe trois souches de P. flocculosa connues qui sont
identifiables sur une base génotypique et phenotypique, ce qui d o ~ e des outils d'identification
et de sdlection importants dans le développement de ces souches comme agents de lutte
biologique contre les champignons de blanc des cultures serricoles.
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Thrane, C., D. F. Jensen, and A. Tronsmo. 2000. Substrate colonization, strain cornpetition,
enzyme production in vitro, and biocontrol of Pythium ultimum by Trichoderma spp.
Isolates P 1 and T3. Eut. J. Plant Pathol. 1 O6:2 15-225.
Traquair, J. A., L. A. Shaw, and W. R. Jarvis. 1988 New species of Stephanoascus with
Spro thrn anamorphs. Can. J . Bot. 66:926-933.
Whipps, J. M. 1997. Developments in the biological control of soil-borne plant pathogens.
Adv. Bot. Res.: Incorporating Adv. Plant Pathol. 26: 1-1 34.
Woodhead, S. H., A. L. O'Leary, D. J. O'Leary, and S. L. Rabatin. 1990. Discovery,
development, and registration of a biocontrol agent from an industrial perspective. Cm. J.
Plant Pathol. 12:328-33 1 .
APPROACHES TO MOLECULAR CHARACTERIZATION OF FUNGAL BIOCONTROL
AGENTS: SOME CASE STUDIES
T. J. AVIS', R. C. HAMELIN*, and R. R. BÉLANGER'
1 Département de phytologie, Centre de recherche en horticulture, Université Laval.
*centre de Foresterie des Laurentides, Natural Resources Canada, Sainte-Foy, Québec.
Ce chapitre a &te publie dans la revue Canadian Journal of Plant Pathology (2001) 23:8-12
En agriculture comme en foresterie, un nombre sans cesse croissant de champignons
sont développés comme agents de lutte biologique contre les ravageurs. Ainsi, l'emploi
d'organismes vivants en phytoprotection a géniré de nouveaux dCfis pour les secteurs de la
recherche et du ddveloppement de l'industrie des produits de lutte biologique. Les marqueurs
moléculaires procurent d'immenses sources de dom6es pouvant assister les chercheurs dans le
ddveloppement d'outils pertinents pour assurer le suivi de l'évolution genbtique et
environnementale des agents. La sdlection d'une technique moléculaire appropriée repose,
d'une part, sur les caractéristiques spécifiques de l'organisme dtudié et, d'autre part, sur
l'information nécessaire pour évaluer une étape précise dans le processus de développement
d'un biopesticide. La connaissance de la ghétique moldculaire des agents antagonistes
fongiques est non seulement souhaitable mais essentielle pour assurer la fiabilité et l'usage
sécuritaire des produits phytosanitaires biologiques.
ABSTRACT
An increasing nurnber of b g a l biocontrol agents are being developed for control of
pests in agriculture and forestry. The use of these living organisms has brought new challenges
in research and development of biocontrol products. Molecular markers provide immense
sources of data which can assist scientists in developing tools to monitor the genetic and
environmental fate of these agents. Selection of appropriate molecular techniques should be
based on the specific characteristics of the organism and on the desired type of information
necessary to evaluate a particular step in the developmental process of a biopesticide. Genetic
assessrnent of b g a l antagonists is not only necessary but essential to gain insight into the
safe and reliable use of biologicai control products.
1. INTRODUCTION
The use of fungal biocontrol agents is becoming an increasingly important alternative
to chernicals in crop protection against weeds, insects, and diseases in both agriculture and
forestry. The discovery of fungal antagonists has led to new challenges in research,
development, and registration of biocontrol products in a market where chemical pesticides
dominate. Bringing a biocontrol product to market is a multitiered process which includes
discovery, eflicacy trials, toxicology testing, mass production, formulation, registration, etc.
(Woodhead et al., 1990; Cross and Polonenko, 1996). Through these different steps, there is
an increasing awareness that precise sampling of the (potential) h g a l antagonist has to be
routinely performed. As well, registration of a specific strain of an antagonist requires that the
identity and stability be ascertained with precision. This is of paramount importance to ensure
eficacy and genetic integrity of the product and to protect intellectual property. In this paper,
we outline the most important objectives and developmental processes by which the
phenotypic and genotypic qualities of a fimgal biocontrol agent c m be assessed. Potential
applications of molecular markers are discussed through case studies to highlight the
importance of molecular characterization of fimgal antagonists throughout the developmental
process.
2. OBJECTIVES AND DEVELOPMENTAL PROCESSES
2.1. Discovery (identity)
The first step in developing a fungal biocontrol agent is the discovery, through
empirical or targeted screening. Potential fungal biocontrol agents have been identified in
numerous cropping systems or natural aosystems (Baker and Cook, 1982; Julien, 1992, Lipa
and Smits, 1999). Following isolation of potential biocontrol agents for a particular weed,
insect, or disease, screening of the fûngal isolates for biocontrol activity is generally
performed in vitro, dong with tests to establish identity and relatedness of the newly found
strains. This is most often accomplished using morphology, nutrient assimilation profiles,
enymatic activity, etc. These methds are now fiequently complemented by molecular data to
allow classification of the antagonistic fungi into existing or new taxa (Samson, 1995). This is
important as the isolates may be closely related to other known fungi which may be pathogens
of plants or animals (in particular humans), or that are already present in the ecosystem. This
could eventually influence the applicability, if any, of the antagonist for the desired purpose.
23. Efficacy trials (diversity)
Once the identity of the fungus is established, it is often desirable to screen different
isolates to assess diversity within the species. This will aid in selecting the most efficient and
antagonistic strains for biocontrol ability in vivo. However, isolates with the highest level of
biocontrol in vitro may not perform as well in vivo since environmental conditions and
cornpetition with other microorganisms are much more restrictive. It is, therefore, essential to
select fùngal isolates under a range of conditions. Also, should the selection process be limited
to a narrow genetic basis, it is foreseeable that isolates with unknown potential could be left
out.
23. Mass production and maintenance of Uolates (quality control)
Perhaps the most important role of genetic assessrnent of fûngal biocontrol agents is
during mass production and maintenance of long-tem cultures, which can lead to
contamination (Markovic and Markovic, 1998) or mutation (Becker and Schwim, 1993), and
if followed by genetic drift or selection, would change the genetic integrity and potentially the
biocontrol properties of the organism. Accordingly, it is essential to develop a quality control
system that allows monitoring of genetic stability over time (Stacey et al., 1991).
2.4. Post-nlease (monitoring)
Once the fungal biocontrol agent is released into the environment, one must implement
a system to monitor its impact on the receiving ecosystem and, in particular, on existing
microbial populations. In this case, one may be interested in following population dynamics of
the introduced fungus, especially if other strains of the same species are present. It may be
important to monitor gene flow in existing populations, particularly if the introduced organisrn
has been geneticall y modified.
2.5. Registration (inteiiechul property)
Regulations conceming cornmercialization of biocontrol agents vary fiom country to
country (Fravel et al., 1999). As more fungal biocontrol agents become registered, the
scientific and industrial communities are increasingly aware that strain authentication is
necessary to address concems about intellectual property and commercial protection. Suitable
and reproducible strain authentication methods are therefore desirable and often necessary in
commercial procedures such as filing patents and product licensing.
3. APPLICATIONS OF MOLECULAR MARKERS IN CHARACTERIZING
BIOCONTROL FUNGI
3.1. Identity, diversity and relatedness of biocontrol fungi
An essential component for the development of a fimgal antagonist is selection of the
most effective isolate for disease or Pest conuol. For example, pycnidial hyperparasites of
powdery mildew fungi are most often regarded as belonging to one species, Ampelomyces
quisqualis Ces., although morphological and cultural differences exist among isolates. One of
the A. quisqualis isolates is now commercially available as AQ-10 Biofungicide (Ecogen hc.,
Langhome, PA). Restriction fragment length polymorphism (Kiss, 1997) and sequence
adysis (Kiss and Nakasone, 1998) of the ribosomal DNA (rDNA) intenial transcribed spacer
(ITS) revealed high genetic diversity within A. quisqualis isolates which would not support
grouping of al1 isolates into a single species. These results indicate (i) that the applicability of
A. quisqualis isolates may be compromised without identification and selection of proper
candidates for use in biological contml and (ii) that only one or few Ampelonyces isolates
have been used while others have been neglected in biocontrol experiments. Future molecular
and field studies on these pycnidial hyperparasites may realize the biocontrol potential of this
still misunderstood fungal antagonist or even highlight the
hyperparasites of powdery mildews.
existence
90
of yet unknown
Other uses in study of diversity in fungal antagon ists include assessrnent of
applicability of candidate f h g i to different hosts or ecological zones. This is important not
only to ensure efficacy of the biocontrol agent under varying conditions, but also to minimize
the risks of large-scale introduction of biocontrol agents, in particular weed pathogens, which
may have undesirable effects such as spread of the fungus and unwanted disease epidemics.
Chondrostereum purpureum (Pers.:Fr) Pouzar is an example of an indigenous plant
pathogenic fungus that has exhibited eficacy in biological control of some deciduous forest
weed species in Canada (Wall, 1990). However, C. purpureum has also k e n known to be
pathogenic on desirable tree species, though epidemics have never been reported (Gosselin et
al., 1999). The value of this biocontrol agent also depends on the applicability of selected
isolates to different ecozones. For these reasons, it is valuable to establish patterns of spread
and gene flow, as well as to develop a fingerprint of the potential biocontrol agent for
environmental monitoring (see following section). Diversity studies have recently been
undertaken to assess host or ecological specialization in this fungus. Previous methods, such
as isoenymes and rDNA anaiysis, detected little or no genetic variation among isolates of the
fungus. Therefore, Gosselin et al. (1996) used random amplified polymorphic DNA to screen
multiple regions of anonymous DNA to better assess the diversity within C. purpureum
isolates from Québec. Random amplified polymorphic DNA detected extensive
polymorphism, although there was no evidence of host or ecologicd specialization. Ramsfield
et al. (1996) used restriction fragment length polymorphism analysis of the large
nontranscribed spacer of rDNA repeats of a worldwide collection of C. purpureurn. The
distribution of identified auclear type patterns suggested that gene flow was occurring across
North America and the authors tentatively concluded that the relative nsk of releasing a single
native isolate of this fungus across Canada was likely to be low. A more precise evaluation of
the genetic structure of Canadian populations of C. purpureum was described by Gosselin et
al. (1999) using RAPD analysis, which provides multilocus f ingerp~ts and is more sensitive
to low variation. The analysis revealed that C. purpureum is a highly heterogeneous fungus
and there was no association betweea DNA profiles and ecological or host origin. The authors
concluded that the risk of disease epidemics should be low since the amount of gene diversity
present within subpopulations should negate the risk of introducing new, highly aggressive
genotypes. This factor, and the lack of ecological specialization, has led to the conclusion that
any genotype fiom central or eastem Canada selected for its biocontrol potential should be
considered indigenous regardles of the region where its use is intended.
However, such wide-based applicability of fùngal isolates, as in the case of C.
purpureum, is the exception rather than the nom. For instance, Trichodermu harziunum Rafai
is a fungal antagonist that is registered or currently developed as a biocontrol agent of
numerous plant diseases (Chet and Inbar, 1994). One of the most widely asked questions in
Trichoderma research is whether the ability for biocontrol is a general property or a specific
attribute of a restricted number of isolates. Since T. harzianum is also the causal agent of green
mold disease (Ospina-Giraldo et al., 1999), the understanding of the nature and diversity of
this fimgus is critical for its potentially widespread use for control of phytopathogenic fungi
since there could be a risk of unwanted disease on nontarget hosts. Molecular study of T.
harzianum was undertaken to clarify this relationship (Ospina-Giraldo et al., 1999). The ITS
fiom rDNA of these isolates was sequenced, and although both green mold and biocontrol
isolates shared a ment ancestor, they could be distinguished as different phylogenetic groups.
In the case of biological control of chestnut blight by Trichodermu sp., positive biocontrol
isolates belonged to a few genetically defined groups as revealed by a variety of single primer
amplifications (designated as RAPD by the authors, though the primers were repeated rather
then random sequences; Arisan-Atac, 1995). Grondona et al. (1997) published a
comprehensive study of T. harziunum isolates to determine if there were distinct functional
groups within the species, which were correlated with biological activity, and if this could aid
in the selection of isolates for biological control. A combination of physiological, biochernical
(enzyme production), and molecular (ITS sequences) criteria were used to establish
intraspecific groups, which could be related to different levels and specrnim of plant pathogen
control. They concluded that the efficacy of panicular strains of T. hurziunum depended on the
intended target and the required functions for biocontrol. This again highlights the importance
of selection of the most efficient strains for each intended use. Other studies have used
molecular markers, such as RAPD (Zirnand, 1994) or microsatellites and ITS (Schlick et al.,
1994). as tools for identification of specific, mutant and patent strains of T. hanianum.
Recent work with Pseudozyma jloccuiosa (Traquair, L. A. Shaw & Jarvis) Boekhout &
Traquair, an antagonist cumntly in the process of registration review in Canada and the
United States for control of powdery mildew in greenhouse crops, has highlighted the
importance of establishing correct identity of the biocontrol agent. The fungus was initially
classified in the genus Sporothrix based on classical mycological techniques (Traquair et al.,
1988). This caused some concem fiom governing bodies during the registration process as
some Sporothrix species, such as S. schenckii Hektoen & Perkins, are known human
pathogens (Bennett, 1990). Based on physiological, biochemicd, and molecular studies, in
particular sequence analysis of the large nuclear subunit of rDNA, Boekhout et al. (1995)
reclassified the fungus in the Pseuduzyma genus. Further studies to idenMy molecular markers
of P. jlocculosa (rDNA) and of specific isolates of the fungus which have been selected for
use in biological control (microsatellites) have confirmed that this fungus is genetically distant
fiom Sporothrix (Avis et al., 200 1).
3.2. Quality control and m o n i t o ~ g of biocontrol products
Following identification of a biocontrol agent and evaluation of its antagonistic
potential, maintenance of fungai strains is necessary to have a reliable source of authenticated
fungal strains (Stacey et al., 1991). This should enable consistency of experimental data in
research and the reproducibility required in industrial-scale production of the b g i . For these
and other processes, such as patent and production license applications, a quality control test is
essential to ensure the highest possible biocontrol efficacy. Also, it is important to have this
type of test to allow monitoring of fimgal isolates that will be released into natural or cultural
environments. Most of these quality control procedures are based on the use of DNA markers
that allow authentication of strains and permit monitoring of contamination (Markovic and
Markovic, 1998) and potential mutations (Becker and Schwinn, 1993) of the biocontml agent,
although morphological, physiological, and (or) biochemical testing must complement the
genetic approach.
An important consideration when authenticating strains and monitoring mutations is
the proportion and region' of the genome which are assessed by the quality control test. The
objective is therefore to mess a high number of meaningfûl (useful) loci throughout the
fungal genome. There are two prevalent approaches in a genetic quality control test. One is to
survey a high nmber of hypervariable loci (Stacey et al., 1991) through the use of multilocus
fmgerprints such as mini- and micro-satellites. Another is to look into specific ngions of DNA
that have biological meaning in the system under study, such as genes implicated in relevant
biological hctions (Fratamico and Strobaugh, 1998) or DNA stretches which are specific to a
particular isolate (Hamelin et al., 1996).
In C. purpureum, a quality control system is under development by use of sequence-
characterized amplified regions in improvement of useful markers for the monitoring of
commercial stniins of the weed pathogenic fungus (Becker et al., 1999). These markers an
generated by sequencing the RAPD fragments that are of particular interest in isolates of C.
purpureum. When sequences are known, it is possible to design primers that are longer than
RAPD primers and are exactly complementary to both the 5' and 3' ends of the original RAPD
fragment. Amplification with these primers identifies a single locus corresponding to the
original RAPD fragment. Thus, fiom the original dominant RAPD locus, it is possible to
produce a highly reproducible, codominant marker which is specific to the selected isolates of
C. purpureum. The production of several sequence-characterized amplified regions, when
RAPD fragments are correctly selected, could then allow for the evaluation of C. purpureum
based on more usefid markers for the monitoring of industnally relevant strain of the fungus.
In the case of Trichoderma, the approach towards a quality control test is being
evaluated through production of polymerase chah reaction (PCR) fingerprints by use of semi-
random primers designed to primarily target intergenic, more variable areas in the genome
(Bulat et al., 1998). So called universally primed PCR (UP-PCR) uses single, 15 to 20-base
pair long primers which produce multiple amplification products without prior knowledge of
DNA sequences. When compared to ITS ribotyping (Bulat et al., 1998) and RAPD (Lübeck et
al., 1999), UP-PCR had the ability to better resolve very similar Trichodermu strains because
of the reliable generation of numerous fragments (60-100 per primer), which cover an
important portion of the genome. LQbeck et al. (1999) consider UP-PCR to be a powerf'ul tool
in monitoring strains of interest as well as for identification of industrially important (patent)
strains of Trichoderma. However, M e r refinement of the technique is necessary to identify
the most useful markers for use in a quality control test.
In out laboratory, we are currently working with the multiplex PCR amplification
approach for a genetic quality control test of P. jlocculosa. Multiplex PCR is essentially a
cocktail of different primers which allows the rapid assessment of numerous DNA fragments
in a single amplification (Hamelin et al., 1996). Careful selection of DNA markers fiom both
hypervariable regions (microsatellites), conserved regions (rDNA genes) and, eventually,
genes implicated in the production of metabolites conferring biocontrol ability will aid in the
design of primers that produce specific fmgerprints to the industrial strain of P. jlocculosa.
Through carefùl selection of DNA marken and construction of relevant primers, we expect to
achieve a quality control test which should allow for the assessment of contamination and
mutation in the commercial production of P. j?occulosa. niese markers will eventually be
converted to oligonucleotide probes for use in rnicroarrays that will simultaneously sample a
large portion of the fûngal genome in a single reaction.
4. CONCLUSIONS
As more b g a l biocontrol agents are registered as alternatives to chernical pesticides,
it becomes increasingly important to develop tools that monitor the genetic and environmental
fate of these agents. Indeed, the use of living organisms as pesticides has brought new
challenges in research and development of plant protection products by pathologists,
entomologists, and weed scientists.
Molecular markers provide an immense source of data that can assist scientists in the
study of identity, relatedness, diversity, and selection of proper candidates for biological
control. Furthemore, molecular marken offer a means of constnicting quality control tests
that are essential throughout the developmental processes of these h g i . For such endeavors,
selection of the appropriate molecular techniques should be based on the specific
characteristics of the organism and on the desired type of information necessary for the
evaluation of a particular step in the developmental process.
The increasingly available molecular &ta for fimgal biological control agents has shed
light on the importance of genetic studies in research, development, and ngistration of these
biocontrol products. It is now clear that genetic assessment of huigal antagonisu is not only
necessary but desirable to gain important insight into the safe and reliable use of these
biological control agents.
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