A. Fuchs

A comparison of methods for the preparation of 14C-labelled plant tissues for liquid scintillation counting

Abstract Four methods for the preparation of 14 C-labelled plant tissues for liquid scintillation counting have been compared for accuracy and precision. The wet oxidation method of Mahin and Lofberg, although easy and rapid, proved to be unsuitable for obtaining a reliable estimate of 14 C present in lyophilized plant material, showing non-quantitative recovery and poor reproducibility. Solubilization in Hyamine hydroxide provided accurate and reproducible results, if experimental conditions were strictly adhered to; however, especially when larger samples were employed, severe—and variable—color quenching resulted in very low counting efficiency, which necessitated construction of quench correction curves specific for each source of plant material. Van Slyke wet combustion, in a device slightly modified after Jeffay and Alvarez, on the other hand, always resulted in reproducible and reliable data, irrespective of the carbon compound administered and the nature of the plant material. With samples of up to about 10 mg, Schoniger dry combustion in the Micro-Mat BF 5010 oxidation system even outrivalled Van Slyke wet combustion, because of speed and ease of manipulation. The latter methods are, therefore, evidently superior to the wet oxidation method of Mahin and Lofberg and solubilization in Hyamine hydroxide in preparing plant tissue samples for liquid scintillation counting.

Resistance to triforine: a nonexistent problem?

Permanent resistance to triforine in Cladosporium cucumerinum was obtained after UV irradiation of spore suspensions and selection of resistant strains in the presence of triarimol or triforine. About 50 strains were examined for mycelial growth on malt agar, viability upon routine subculturing, growth and inhibition by triforine in thin-layer chromatographic bioassays, and virulence towards cucumber seedlings. In addition, sporulation and spore germination, as well as effects of ergosterol were investigated in a restricted number of strains. Although all strains differed considerably from each other in these characteristics, they were quite similar in a reduced overall-fitness. These triforine-resistant mutants of C. cucumerinum might, therefore, have a greatly reduced chance of survival in competition with the wild-type strain, as was actually demonstrated for two strains in greenhouse experiments with mixed-inoculations of cucumber seedlings. Such a reduced chance of survival might also explain why under practical conditions development of resistance to triforine has not been observed yet.The triforine-resistant C. cucumerinum strains showed cross-resistance to a number of systemic fungicides known or supposed to inhibit ergosterol biosynthesis, viz. Denmert, fenarimol, imazalil, triadimefon and triarimol. The practical implications and potential hazards of such cross-resistance for the use of already established fungicides are discussed.

PHOTO ACTIVATION OF ISOFLAVONOID PHYTOALEXINS: INVOLVEMENT OF FREE RADICALS

Abstract— Upon irradiation with ultraviolet light the isoflavonoid phytoalexins phaseollin, 3,6a, 9‐trihydroxypterocarpan, glyceollin, tuberosin and pisatin, but not medicarpin, brought about inactivation ofglucose–6‐phosphate dehydrogenase in an in vitro assay system. Photoinactivation of the enzyme by photoactivated pisatin in air‐saturated solutions was hardly affected by singlet oxygen quenchers such as NaN3, bovine serum albumin, histidine or methionine. Neither addition of the hydroxyl radical scavengers mannitol, Na‐benzoate and ethanol nor the presence of catalase or supcroxide dismutase protected the enzyme against photoinactivation, suggesting that OH, H2O2 and O2 are not the reactive oxygen species involved. However, the free radical scavenger S‐(2‐amino‐ethyl)isothiouronium bromide hydrobromide (AET) protected the enzyme against inactivation by photoactivated pisatin. Direct evidence for the generation of free radicals was obtained by ESR measurements of solutions of phaseollin, pisatin and medicarpin in hexane irradiated with ultraviolet light in the presence or absence of O2. Phaseollin produced the most stable free radicals, whereas medicarpin hardly gave rise to free radical formation; pisatin took a somewhat intermediate position by producing a strong ESR signal which, however, decayed rather quickly. Photodegradation of all phytoalexins, except for medicarpin, was accompanied with loss of fungitoxicity, as shown in thin‐layer chromatographic bioassays, and formation of new products.

The function of an MBC-releasing deposit of benomyl and thiophanates in plant roots and soil

A comparative study has been made on the effects of short-term treatment versus long-term treatment with benomyl and three thiophanates with regard to distribution of fungitoxicant (MBC, or EBC) within plants and their protection against fungal diseases. In both treatments, plants were exposed to the fungicides for two days and then transplanted to garden soil; long-term treated plants received an additional supply of fungicide as a daily drench for one week.Results of short-term treatment of cucumber seedlings and tomato plants provided additional proof for the hypothesis, that MBC derivatives, substituted at N-1 in the benzimidazole nucleus, are retained to some extent on or in the roots, and gradually converted to MBC which then moves into the aerial parts of the plant. Correlation of infection with concentration of fungitoxicant within leaves showed 0.35 μg/g fresh weight to be the limiting concentration for symptom expression of cucumber powdery mildew. Only with benomyl was MBC to be released long enough into the aerial parts of the plant to protect leaves, unfolding after termination of the treatment, for some weeks.With long-term treatment, a concentration of MBC (or EBC) sufficient for protection was found with all fungicides in all above-ground parts of the plant until the end of the experiment. Thus, a reservoir of fungicide in the planting medium guarantees a continuous supply of fungicide only then may new growth be efficaciously protected.SamenvattingEen vergelijkend onderzoek werd ingesteld naar de effecten van kortdurende behandedeling tegenover voortgezette behandeling met benomyl en drie thiophanaten wat betreft de verdeling van de eigenlijke fungitoxische verbinding (MBC, respectievelijk EBC) in planten en hun bescherming tegen schimmelziekten. Bij beide behandelingen werden de planten gedurende twee dagen aan de fungiciden blootgesteld en daarna in tuingrond geplant; in het geval van de voortgezette behandeling werd dagelijks gedurende één week een hoeveelheid fungicide in het gietwater toegevoegd.De resultaten van de kortdurende behandeling van komkommerzaailingen en tomateplanten ondersteunden de hypothese, dat MBC-derivaten, die op de N-1 plaats in de benzimidazoolring gesubstitueerd zijn, tot op zekere hoogte in de wortels worden ‘vastgelegd’, waar ze geleidelijk in MBC worden omgezet en vandaar als zodanig naar de bovengrondse delen worden getransporteerd. Correlering van infectie met concentratie van het MBC in de bladeren liet zien, dat 0,35 μg/g vers gewicht de grensconcentratie is, waarboven geen symptomen van komkommermeeldauw meer optreden. Alleen in het geval van benomylbehandeling werd MBC voldoende lang naar de bovengrondse delen vervoerd om ook die bladeren die zich na beëindiging der behandeling ontplooiden, gedurende enkele weken te beschermen tegen meeldauw.Bij voortgezette behandeling werd de genoemde concentratie met alle fungiciden in alle bovengrondse plantedelen tot het einde van de onderzoeksperiode gehandhaafd. Klaarblijkelijk garandeert een zekere voorraad van het fungicide in de grond een continue toevoer van het fungicide aan de plant; alleen onder die omstandigheden kunnen nieuwgevormde plantedelen doeltreffend worden beschermd.

The mechanism of pisatin degradation by Fusarium oxysporum f. sp. pisi

Abstract Mycelium of the pea pathogen Fusarium oxysporum f. sp. pisi, pregrown in a medium without pisatin, readily degraded [14C]-pisatin. The first degradative step involved demethylation of the methoxyl group at C-3. The metabolite produced, 6a-hydroxy-inermin, was decomposed by reductive opening of the dihydrofuran ring, giving rise to 3-hydroxy-inermin-isoflavan. The isoflavan was broken down further, with a substantial loss of radioactivity, apparently because of production of volatile compounds such as CO2. Experiments with [14C]-6a-hydroxy-inermin substantiated this metabolic pathway. Mycelium of the non-pea pathogen Fusarium oxysporum f. sp. lycopersici, pregrown under similar conditions, accumulated pisatin but not 6a-hydroxy-inermin. Neither of the two compounds was degraded by this fungus.

Bacterial breakdown of benomyl. I. Pure cultures

With different soil and water samples as inoculum and the benzimidazole fungicides benomyl (either as Benlate or as the pure compound) and thiabendazole as selective agents, a large number of, mainly fluorescent, Pseudomonas strains were isolated which nearly all were able to grow in a mineral medium with benomyl as the sole source of carbon. However, no growth occurred with any of a series of other benzimidazole compounds, viz. benzimidazole, 2-aminobenzimidazole (2-AB), thiabendazole and fuberidazole. Although benomyl—or rather its non-enzymatic breakdown product methyl benzimidazol-2-yl carbamate (MBC)—was partially degraded to 2-AB, most probably n-butylamine, which arises after splitting off of the butylcarbamoyl side chain, was the actual carbon source for the Pseudomonas isolates.When incorporated in a lactate medium, 2-AB markedly inhibited the growth of Pseudomonas spp. at a concentration of 250 μg/ml, with complete inhibition being attained at 500 μg/ml. For Bacillus spp. grown in liquid peptone media benzimidazole compounds were inhibitory at concentrations of 500–1000 μg/ml, with a toxicity increasing in the order: benzimidazole

Metabolism of radioactively labeled quinic acid and shikimic acid in healthy and Fusarium-infected tomato plants

Radioactivity from quinic acid-U-14C was readily incorporated into chlorogenic acid and shikimic acid in healthy andFusarium-infected tomato plants of two varieties, ‘Bonner Beste’ (susceptible) and ‘Moneymaker’ (resistant); radioactively labeled shikimic acid, on the other hand, was converted neither to quinic acid nor to chlorogenic acid.Infection led to increased incorporation of14C inton-butanol extractives, and alcohol-soluble and insoluble esters, except in the resistant variety after feeding of shikimic acid-U-14C. After infection incorporation into the non-hydrolyzable fraction—which a.o. contains lignin—decreased in the susceptible variety, but it increased in the resistant variety, particularly after administration of shikimic acid-U-14C.SamenvattingRadioactief kinazuur werd in gezonde en metFusarium geïnfecteerde tomateplanten behorende tot twee variëteiten, ‘Bonner Beste’ (vatbaar) en ‘Moneymaker’ (resistent), in chlorogeenzuur en shikimizuur omgezet; laatstgenoemde verbinding werd echter noch in kinazuur, noch in chlorogeenzuur omgezet.Infectie leidde tot een toegenomen incorporatie van14C in metn-butanol extraheerbare verbindingen, en in alcohol-oplosbare en onoplosbare esters, behalve na toediening van radioactief shikimizuur in de resistente variëteit. Incorporatie in de zg. niethydrolyseerbare fractie, die o.a. lignine bevat, bleek in de vatbare variëteit na infectie geringer te zijn, maar in de resistente variëteit na infectie toe te nemen, vooral na toediening van shikimizuur.

Simultaneous resistance in fungi to ergosterol biosynthesis inhibitors and dicarboximides

Several strains ofAspergillus nidulans, Cladosporium cucumerinum andPenicillium italicum with known resistance to ergosterol biosynthesis inhibitors were tested for resistance to three dicarboximides. Negligible levels of resistance to iprodione and vinclozolin were observed in one out of three strains ofA. nidulans. Two out of three strains ofC. cucumerinum displayed a low resistance to iprodione, and a high resistance to procymidone and vinclozolin. The latter strains were also moderately resistant to the isoflavonoid phytoalexins medicarpin and pisatin, but sensitive to the antibiotic pimaricin. All sixP. italicum strains examined displayed wild-type sensitivity to all three dicarboximides; the two of these tested in thin-layer chromatographic bioassays proved to be resistant to pimaricin.Iprodione and vinclozolin induced energy-dependent fenarimol efflux inA. nidulans. In line with this observation, in crossed-paper strip assays iprodione and fenarimol antagonized each other in their toxicity towardsA. nidulans; towardsC. cucumerinum, on the other hand, these fungicides behaved independently.The implications and practical consequences of the phenomena observed are briefly discussed.SamenvattingVerscheidene tegen ergosterolbiosyntheseremmers resistente stammen vanAspergillus nidulans, Cladosporium cucumerinum enPenicillium italicum werden getoetst op resistentie tegen dicarboximiden. Eén der drie onderzochte stammen vanA. nidulans bezat enige resistentie tegen iprodione en vinchlozoline. Twee van de drie onderzochte stammen vanC. cucumerinum vertoonden een lage graad van resistentie tegen iprodione en een zeer hoge tegen procymidone en vinchlozoline. Ze waren ook enigermate resistent tegen de fytoalexinen medicarpine en pisatine, maar gevoelig voor het antibioticum pimaricine. Alle onderzochte stammen vanP. italicum waren voor de drie getoetste dicarboximiden even gevoelig als het wild-type; voorzover onderzocht, bleken deze stammen resistent tegen pimaricine.Iprodione en, hoewel in mindere mate, vinchlozoline induceerden energieafhankelijke efflux van fenarimol inA. nidulans. In overeenstemming hiermee antagoneerden iprodione en fenarimol elkander in hun activiteit ten opzicht vanA. nidulans. Ten opzichte vanC. cucumerinum gedroegen deze fungiciden zich onafhankelijk van elkaar.De practische consequenties van de waargenomen verschijnselen worden kort aangeduid.

Odoratol and methylodoratol, two α-hydroxydihydro-chalcone stress metabolites from lathyrus odoratus

Abstract From Lathyrus odoratus treated with mercuric acetate (cotyledons, pods) or with Phytophthora megasperma var. sojae -elicitor (cotyledons) a number of fungitoxic stress metabolises have been isolated, among which are two novel α-hydroxydihydrochalcones (odoratol and methylodoratol). Their structures have been elucidated.

3-Hydroxymaackiainisoflavan, a pisatin metabolite produced by Fusarium oxysporum f.sp. pisi.

Abstract 3,7,2′-Trihydroxy-4′,5′-methylenedioxyisoflavan, a novel fungal metabolite of pisatin, was identified on the basis of NMR and MS data. The isoflavan arises by reductive opening of the dihydrofuran ring in the pterocarpan 6a-hydroxymaackiain, the first breakdown product of pisatin. The trivial name 3-hydroxymaackiainisoflavan is proposed for this substance.