Giczey, Gábor

Hornok, László

Kerényi, Zoltám

Érudit

Érudit est un consortium interuniversitaire sans but lucratif composé de l'Université de Montréal, l'Université Laval et l'Université du Québec àMontréal. Il a pour mission la promotion et la valorisation de la recherche. Érudit offre des services d'édition numérique de documentsscientifiques depuis 1998.Pour communiquer avec les responsables d'Érudit : info@erudit.org Article "Strain Improvement in Biocontrol Fungi" László Hornok, Gábor Giczey et Zoltám KerényiPhytoprotection, vol. 79, n° 4, 1998, p. 136-138.   Pour citer cet article, utiliser l'information suivante : URI: http://id.erudit.org/iderudit/706172arDOI: 10.7202/706172arNote : les règles d'écriture des références bibliographiques peuvent varier selon les différents domaines du savoir.Ce document est protégé par la loi sur le droit d'auteur. L'utilisation des services d'Érudit (y compris la reproduction) est assujettie à sa politiqued'utilisation que vous pouvez consulter à l'URI https://apropos.erudit.org/fr/usagers/politique-dutilisation/Document téléchargé le 13 février 2017 04:57Research Challenges and Needs for Safe Use of Transgenic Organisms Strain Improvement in Biocontrol Fungi Lâszlô Hornok, Gâbor Giczey and Zoltân Kerényi Agricultural University and Agricultural Biotechnology Center, Gôdôllô, Hungary INTRODUCTION Antagonistic and hyperparasitic fungi could be our useful allies in integrated pest management technologies allow-ing us to reduce the amount of pesti-cides released into the environment. Ampelomyces quisqualis, Coniothyrium minitans, Peniophora gigantea, Pythi-um oligandrum, as well as several spe-cies of Gliocladium and Trichoderma are the most potent biocontrol agents efficient against either leaf or soil in-habiting plant pathogens. The antago-nistic activity of thèse fungi is attribut-ed to various mechanisms including antibiosis, parasitism, compétition and induced résistance (Goldman et al., 1994). There are many examples of the successful expérimental use of hyper-parasitic fungi, their application on a commercial scale is, however, limited to a few cases. than the original strain. Protoplast fu-sion was also attempted to produce superior biocontrol strains (Harman and Hayes, 1993). However, the efficiency of this procédure is hampered by the genetic incompatibility existing between the parental strains; postfusional un-stability was also found both in intraspe-cific and interspecific hybrids (Stasz et al., 1989). Transformation seems to be the most powerful method to achieve an outstanding combination of the dé-sirable traits in a single strain, because this technique precludes the introduc-tion of unfavorable properties into the récipients. IMPROVEMENT OF BIOCONTROL EFFICACY BY USING BIOTECHNOLOGICAL METHODS Q. Q. 3 w Ci r*« Z o H O UJ H O ce û. O AIMS AND METHODS OF STRAIN IMPROVEMENT The main reason of the scarcity of the commercial use of biocontrol fungi is their lower efficiency and reliability compared to chemical fungicides. One of the means by which the evectiveness of biological control could be increased is the use of genetically improved an-tagonistic (hyperparasitic) fungi. The biocontrol value of a wild-type isolate can be enhanced by différent methods like mutagenesis, protoplast fusion and transformation. Papavizas and Lewis (1983) selected UV-induced mutants from Trichoderma viride and some of the mutants proved to be more efficient Species of the genus Trichoderma are by far the most intensely studied or-ganisms in this field of research. Thèse fungi produce extracellular cell-wall degrading enzymes, including chitinas-es, cellulases, /3-1,3-glucanases and proteases. The mycolytic activity of thèse enzymes has long been recog-nized as a major factor in the hyperpar-asitic mechanism exerted by Trichoder-ma (Elad et al., 1982). Différent stratégies hâve been tried to increase the chitinase activity of one of the most potent biocontrol spe-cies, T. harzianum. Haran et al. (1993) introduced the chitinase gène of Serra-tia marcescens into the génome of T. harzianum under the control of the 136 constitutive 35S promoter and obtained two transformants which expressed higher levels of chitinase activity in the présence of glucose. However, when thèse transformants were grown on chitin, the transgenic strains expressed lower chitinase activity than the paren-tal wild-type strain. The authors ex-plained this phenomenon by cleavage of the heterologous enzyme by host proteases and the interférence of the resulting protein fragments with host chitinases during the secretory process. Margolles-Clark et al. (1996) used a transformation construct that contained the coding région of the 42 kDa endo-chitinase gène (ThEn-42) of T. harzianum under the control of the cellulase pro-moter, cbhl from Trichoderma reesei and could achieve a ten-fold increase of the chitinase activity in most of the 20 transformants tested. This was an élégant approach to the chitinase over-production under in vitro conditions, butthe behaviorof thèse transformants under natural conditions is difficult to predict, as their increased chitinase production was elicited by cellulase-inducing components (owing to the chbl promoter) and no spécifie induc-tion occurred in the présence of chitin, the real substrate of the overproduced enzyme. In our laboratory, another evolution-ary variant of the 42 kDa endochitinase encoding gènes, named Tham-ch was cloned by screening the genomic li-brary of Trichoderma hamatum, strain Tam-61 with a PCR-amplified chitinase séquence from the same fungus (Gic-zey et ah, 1998). A 3.5 kb genomic DNA fragment containing the coding région, as well as the 5' and 3' regulatory sé-quences was reintroduced into the host strain by PEG-mediated homologous transformation under sélection pressure provided by hygromycin B. The inté-gration and the stability of the trans-forming construct was demonstrated by Southern blotting in 10 transformants. The intégration of the transforming vector was stable only in one copy and occurred through homologous recom-bination in 9 of 10 transformants, while random intégration was detected in one transformant. Ail but one transformant expressed higher levels of chitinase activity in comparison to the wild type récipient strain; the maximum level of increase rose 5-fold. Duplicating the copy number of the entire endochiti-nase gène under its own regulatory séquences is especially suitable for improving the biocontrol capability of Trichoderma as the highly conserved 42-kDa endochitinase-encoding gène, présent in ail mycophagous species of Trichoderma (Fekete et ai, 1996) has been shown to be specifically triggered in mycoparasitic interactions (Carsolio et al., 1994). Triggering occurs when a spécifie "mycoparasitic" protein com-plex binds to the promoter séquences of the gène and displaces the binding of a catabolite repressor protein (Lorito et al., 1996). The transformation strate-gy we used was thus based on the molecular régulation of the Trichoder-ma endochitinase gène and RNA-blot analysisconfirmedthe rationalityofthis décision: when fungal cell wall prépa-ration was added as a sole carbon source, significantly higher levels of Tham-ch mRNA were detected in ail the transformants compared to the wild-type récipient. Another advantage of the 42-kDa endochitinase gène is that its overexpression causes no adverse effects on the transformants as de-scribed for the first time by Margolles-Clark et al. (1996) in T. harzianum. The disadvantage of our method is, howev-er, that a bacterial antibiotic résistance gène was introduced into the transfor-mants, precluding thus their outdoor testing. Gènes encoding lytic enzymes others than chitinases has also been used for the genetic improvement of Trichoder-ma strains. Co-transformation of T. longibrachiatum with the hygromycin résistance gène and the egll gène, en-coding j8-(1,4)-endoglucanase resulted in a significant increase of the extracel-lular endoglucanase activity (Sànchez-Torres et al. 1994). In a more récent study (Migheli et al., 1998) thèse trans-formants were found to show enhanced biocontrol activity against Pythium ul-timum on cucumber. The biocontrol ability of T. harzianum has successfully been improved by increasing the copy number of a basic proteinase gène (Flores et al. 1997); in greenhouse tests 137 thèse transformants proved to be supe-rior in reducing Rhizoctonia infection of cotton. Should we encourage research to un-coverthe secondary metabolite profiles of lesser known biocontroi fungus spe-cies? o. a 3 PERSPECTIVES OF THE GENETICALLY MODIFIED HYPERPARASITIC FUNGI In this review examples were listed to illustrate research focussed on engineer-ing more efficient biocontroi fungi. Many genetically improved strains were produced bu t - to the bestof our knowl-edge - none of them hâve yet been tested under natural conditions. The efforts to construct genetically modi-fied biocontroi fungi seem to be mono-centric, in fact they are restricted to membersofthegenus Trichodermaand gènes encoding (myco)lytic enzymes hâve been the only targets of genetic manipulation. When this is considered the follow-ing questions (and appropriate answers) could promote the matter of strain improvement in biocontroi fungi. What biocontroi species, other than Trichoderma could be recommended for genetic engineering? What could bethe main points of the sélection; usefulness in biocontroi, easiness in laboratory handling or environmental safety? What traits, others than mycolytic ability are worth of improving; second-ary metabolites, adaptation ability, com-petitiveness, tolérance against biotic and abiotic stress factors? The majority of biocontroi fungi pro-duces secondary metabolites, that could account for their biocontroi activity, but may adversely affect non-target organ-isms in nature. Can we advise molecu-lar biologists to improve secondary metabolite production in thèse fungi? Alternatively, can we advise them to produce disarmed strains, deprived of secondary metabolite production and improved in other biocontroi traits? ACKNOWLEDGEMENT Some experiments described in this paperwassupported bya research grant from OTKA (F 023692). REFERENCES Carsolio C, Gutiérrez A, Jiménez B, Van Montagu M, Herrera-Estrella A (1994) Proc Natl Acad Sci USA 91: 10903-10907. Elad Y, Chet I, Henis Y (1982) Can J Micro-biol 28: 719-725. Fekete C, Weszely T, Hornok L (1996) FEMS Microbiol Lett 145: 385-391. Flores A, Chet I, Herrera-Estrella A (1997) Curr Gen 31: 30-37. Giczey G, Kerényi Z, Dallmann G, Hornok L (1998) FEMS Microbiol Lett (in press) Goldman GH, Hayes C, Harman GE (1994) Trends Biotechnol 12: 478-482. Haran S, Schickler H, Pe'er S, Logemann S, Oppenheim A, Chet I (1993) Biol Control 3: 101-108. Harman GE, Hayes CK (1993). The genetic nature and biocontroi ability of progeny from protoplast fusion in Trichoderma. In: Biotechnology in Plant Disease Con-trol (Chet I, éd.), pp. 237-255. Wiley-Liss, New York. Lorito M, Mach RL, Sposato P, Strauss J, Peterbauer CK, Kubicek CP (1996) Proc Natl Acad Sci USA 93: 14868-14872. Margolles-Clark E, Harman GE, Pentilla M (1996) Appl Environ Microbiol 62: 2152-2155. Migheli Q, Gonzâles-Candelas L, Dealessi L, Camponogara A, Ramôn-Vidal D (1998) Phytopathology 88: 673-677. Papavizas GC, Lewis JA (1983) Phytopathol-ogy 73: 407-411. Sanchez-Torres P, Gonzalez R, Pérez-Gonzalez JA, Gonzalez-Gandelas, Ramôn D (1994) Appl Microbiol Biotechnol 41: 440-446 Stasz TE, Harman GE, Gullino ML, (1989) Exp Mycol 13: 364-371. 138 

Strain Improvement in Biocontrol Fungi

http://www.erudit.org/revue/phyto/1998/v79/n4/706172ar.pdf

Strain Improvement in Biocontrol Fungi

Abstract

Similar works

Full text

Available Versions

Érudit