Les champignons endophytes sont des microorganismes non-pathogènes impliqués dans des associations mutualistes avec les plantes. Les endophytes foliaires, en particulier, représentent un groupe très divers, mais leurs interactions avec la plante hôte et ses micro-organismes associés sont peu connues. Des travaux préliminaires initiés par notre équipe, explorant la diversité microbienne foliaire du conifère Cephalotaxus harringtonia, ont permis d’isoler la souche fongique Paraconiothyrium variabile (Ascomycota), un antagoniste du phytopathogène Fusarium oxysporum. Au cours de leur interaction, on détecte des quantités moindres de beauvéricine, une mycotoxine synthétisée par F. oxysporum. En parallèle, on observe une augmentation de la synthèse de deux oxylipines, l’acide 13-hydroperoxyoctadécadiénoïque (13-HPODE) et l’acide 13-oxo-octadécadiénoïque (13-oxo-ODE), dans la zone de confrontation. L'objectif de ce travail était de comprendre les mécanismes conduisant à la diminution de la beauvéricine au cours de l'interaction et d'explorer le rôle des oxylipines dans la régulation de cette dernière. Dans mon travail de thèse, je montre la présence de deux gènes lox chez P. variabile (pvlox1 et pvlox2) codant tous deux pour des manganèse lipoxygénases, potentiellement à l'origine des acides 13-HPODE et 13-oxo-ODE. Pvlox2 est spécifiquement induit pendant l'interaction, et ces résultats sont corroborés par une synthèse accrue de 13-HPODE chez P. variabile. Par ailleurs, l'interaction avec l’endophyte, ainsi que l'ajout de l’oxylipine 13-HPODE, régulent positivement la voie de biosynthèse de la beauvéricine, comme l’indiquent les teneurs plus élevées en mycotoxines observées chez F. oxysporum. Enfin, nous avons montré que la beauvéricine inhibait la croissance de l’endophyte, mais que ce dernier était capable de dégrader la mycotoxine, expliquant ainsi les faibles quantités de beauvericine observées initialement dans la zone de compétition. Ce travail contribue à la compréhension du rôle des oxylipines dans la communication inter-microbienne. / Endophytic fungi are non-pathogenic microorganisms involved in mutualistic associations with their host. Foliar endophytes, in particular, represent a very diverse group but little is known about their interactions with the host and its associated micro-organisms. In preliminary work, exploring the leaf microbial diversity of the conifer Cephalotaxus harringtonia, our team isolated the fungal strain Paraconiothyrium variabile (Ascomycota), an antagonist of the phytopathogen Fusarium oxysporum. During their interaction, decreased amounts of the F. oxysporum mycotoxin beauvericin, and higher amounts of the two oxylipins, 13-hydroperoxyoctadecadienoic acid (13-HPODE) and 13-oxo-octadecadienoic acid (13-oxo-ODE), were observed in the confrontation zone. The objective of the present work was to understand the mechanisms leading to beauvericin decrease during the interaction and to explore the role of oxylipins in beauvericin regulation. In my thesis work I show the presence of two lox genes in P. variabile (pvlox1 and pvlox2) coding both for manganese lipoxygenases, potentially at the origin of 13-HPODE and 13-oxo-ODE. Pvlox2 is specifically induced during the interaction, which lead to an increased synthesis of 13-HPODE in P. variabile. The endophyte itself, as well as the oxylipin 13-HPODE, up-regulated the beauvericin biosynthesis gene beas, which was paralleled by higher mycotoxin content in the mycelium of F. oxysporum. Finally, we showed that beauvericin inhibited the endophyte’s growth, but the latter was capable to degrade the mycotoxin, which explains the lower amounts of beauvericin found in the competition zone. This work presents pioneer undertaking to elucidate the role of oxylipins in inter-microbial crosstalk.

The effect of different blanching treatments and packaging materials on the enzymatic (lipoxygenase and peroxidase) activity and fatty acid stability of two different varieties of sweet corn on the cob (Jubilee and GH 2684) was evaluated during nine months of frozen storage at -23.3°C. The initial moisture content in the kernels of the two sweet corn varieties averaged 72.5%. After nine months of frozen storage the moisture content in the kernels of corn depended greatly on the packaging material used. The ears stored in Cryovac B and E bags showed the best moisture retention (72.2% final moisture content), followed by the polyethylene bags (71.4%) while the ears stored without packaging material showed severe dehydration (70.1%). The peroxidase and lipoxygenase activities were determined using spectrophotometric assays on a crude extract obtained from liquid nitrogen powdered corn. Both unblanched varieties of sweet corn showed similar initial peroxidase specific activity and general behavior during the nine months of frozen storage. The presence of lipoxygenase isozymes with different thermal stabilities in both varieties was suggested by the higher lipoxygenase specific activity found in Jubilee after freezing and nine months of frozen storage (0.135 units/mg protein) compared with the GH 2684 variety (0.115 units/mg protein). Complete inactivation of lipoxygenase was obtained after 9 minutes steam blanching at 100°C. Peroxidase was more heat resistant showing some remaining specific activity after 9 minutes steam blanching with a complete inactivation after 15 minutes steam blanching. No regeneration of either enzyme was observed during the nine months of frozen storage suggesting a permanent disruption of the active site of both enzymes. Relative fatty acid content was determined by gas chromatographic analysis of fatty acids methyl esters. The major fatty acids present in both varieties were palmitic (14.93%), stearic (2.79%), oleic (31.54%), linoleic (46.87%) and linolenic (1.89%) acids. Good stability of the polyunsaturated fatty acids was observed during the nine months storage at -23.3°C, with autoxidation as the main mechanism responsible for the decrease in the relative percent of polyunsaturated fatty acids. Some enzymatic oxidation also occurred, decreasing the linolenic acid content. The control of the degradation of polyunsaturated fatty acids depended mostly on the frozen storage temperature (-23.3°C) and not on the oxygen permeability of the different packaging materials. The results obtained in our study suggested that blanching of the ears of sweet corn had an important effect on reducing the enzyme activity but little effect on the polyunsaturated fatty acid degradation after 9 months of storage at -23.3°C. / Graduation date: 1994

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published_or_final_version / abstract / toc / Pharmacology / Doctoral / Doctor of Philosophy

Biomasses of Penicillium camemberti, Penicillium roqueforti and Geotrichum candidum strains were grown and harvested after a culture incubation period that corresponded to the maximal dry weight of mycelium as well as to lipoxygenase (LOX) activity. The crude enzymatic extracts were recovered from the homogenized mycelium cells and the partially purified LOX extracts were obtained by ammonium sulfate precipitation. Using linoleic acid as substrate, the partially purified LOX extracts from P. camemberti, P. roqueforti and G. candidum exhibited a major activity, at pH 6.50, 5.50 and 3.75, respectively, and a minor one at pH 8.00. The partially purified LOX extracts exhibited an overall preferential specificity towards free fatty acids, including linoleic, linolenic and arachidonic acids, than that for fatty acid acylglycerols, including mono-, di- and tri-linolein. The Km and Vmax values for partially purified LOXs were investigated. Normal phase high-performance liquid chromatography (NP-HPLC) and gas-liquid chromatography/mass spectrometry (GC/MS) analyses showed that the LOX activity of the partially purified LOX extracts converted mainly linoleic acid into the corresponding 9- and 13-hydroperoxides (HPODs); however, the production of a relatively important proportion of 10-HPOD, ranging from 4 to 9% of the total HPODs, was also demonstrated with the partially purified LOX extracts from Penicillium sp. The chiral phase HPLC analysis demonstrated the production, by the partially purified LOX extracts from Penicillium sp., of both (R)- and (S)-enantiomers of HPODs, with a slightly higher proportion of the (S)-enantiomers. The crude enzymatic extracts from Penicillium sp. were incubated, at pH 6.50, with individual racemic mixture of 9(R,S)-, 10(R,S)-, 12( R,S)- and 13(R,S)-HPODs, prepared by photooxidation and separated by NP-HPLC. The experimental results indicated that only 10( S)-HPOD isomer was cleaved by a hydroperoxide lyase activity that resulted by the for

It is widely accepted that prostaglandins (PGs), produced via the cyclooxygenase pathway from arachidonic acid, are essential to the ovulatory process in the pig. In support of this, ovulation is preceded by an increase in follicular fluid (FF) PG concentration, indomethacin (INDO) suppresses both the PG increase and ovulation, and ovulation can be restored by administration of exogenous PGs (Downey and Ainsworth, 1980; Prostaglandins 19: 17-22). Recent studies in the rat have shown that ovulation is also preceded by a rise in ovarian concentrations of 15-hydroxyeicosatetraenoic acid (15-HETE), a product of the lipoxygenase pathway (Tanaka et al., 1989; Endocrinology 15: 1373-1377) and inhibition of this pathway suppresses ovulation (Reich et al., 1983; Prostaglandins 26: 1011-1020). Furthermore, INDO, a cyclooxygenase inhibitor, inhibits 15-lipoxygenase as well as PG synthesis (Tanaka et al., 1989 Endocrinology 15: 1373-1377). The PMSG/hCG prepuberal gilt model was used to investigate the involvement of 15-HETE in the procine ovulatory process, and the effect of INDO on the 15-lipoxygenase pathway. Follicular fluid concentrations of 15-HETE were elevated 40 h post hCG (p $<$ 0.01). The effects of INDO and nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase activity, on ovulation rate, FF 15-HETE and FF PGF$ rm sb{2a}$ were investigated by intraovarian administration of INDO or NDGA. INDO inhibited ovulation rate (p $<$ 0.01) and PGF$ rm sb{2a}$ (p $<$ 0.01) as well as 15-HETE (p $<$ 0.01). NDGA also suppressed ovulation rate (p $<$ 0.01) but did not inhibit 15-HETE or PGF$ rm sb{2a}$ production. In in vitro experiments, 15-HETE production by both granulosa cell (GC) and theca interna cell (TIC) cultures 40 h post hCG was greater (p $<$ 0.01) than at 0 h post hCG. INDO inhibited 15-HETE production in 40 h post hCG TIC cultures (p $<$ 0.01) but not GC cultures, while NDGA inhibited 15-HETE production by both cell types (p $<$ 0.01). These results sugges

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 165-181). Also available via the Internet.

Thesis (doctoral)--Rijksuniversiteit te Utrecht, 1983. / Summary also in Dutch. Vita and acknowledgements in Dutch. Additional references and errata slip inserted. Includes bibliographical references (p. 75-79).

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