Regulation of ethylene production by phosphate in Penicillium digitatum

Inorganic phosphate regulated ethylene production in shake culturesof Penicillium digitatum. Decreasing the phosphate level ofthe medium from 100 to 0.01 mM markedly increased, about 100-fold,the rate of ethylene production, in 96 hr, which was confinedentirely to the fungal mycelium. Exogenous addition of between0.01 to 100 mM phosphate, to high ethylene producing, low-phosphatecultures strongly inhibited their ethylene production and increasedthe ATP content of the mycelium. Phosphate also inhibited ethyleneproduction in apple slices. Addition of calcium ions to theincubation medium stimulated the production of ethylene in appleslices, subhook epicotyl segments of pea and shake culturesof P. digitatum. We suggest that this stimulatory effect wascaused by the reduction of inhibitory levels of phosphate, whichcomplexed with calcium. Thus, phosphate in conjunction withcalcium may play an important role in regulating ethylene productionnot only in P. digitatum but also in higher plants. ¹ On leave from the Agricultural Research Organization, TheVolcani Center, Israel. ² On leave from the M.S. University of Baroda, India. (Received September 7, 1977; )     

The biogenesis of ethylene in Penicillium digitatum

The origin of ethylene in Penicillium digitatum has been shown to be intimately associated with the Krebs cycle. 2-Ketoglutaric acid and glutamic acid are the most efficient precursors of ethylene, which is derived from carbons 3 and 4 of these substrates as a unit. However, which of these is the immediate precursor has not been established. Since 2-ketoglutaric acid is a very efficient precursor and succinic acid is an inefficient one, 2-ketoglutaric acid must be the branching point at which the pathway of ethylene biosynthesis leads away from the Krebs cycle. This conclusion is in full agreement with the following observations: Three of the four hydrogen atoms of the ethylene molecule were derived from protons of the medium; C-2 but not C-1 of acetate was incorporated into ethylene; and [2,3-¹⁴C]succinic acid but not [2,3-³H]succinic acid was incorporated.     

Factors affecting ethylene production by some plant pathogenic bacteria

Summary Methionine, up to 10^–3 M, added to a basal medium enhanced bacterial ethylene production in 14 of the 20 bacteria tested. The effects of substrate, cofactors, light, and temperature on ethylene production byPseudomonas solanacearum #25 revealed that the greatest effect occurred when 10^–5 M methionine and 10^–4 M FMN were combined, from which 4.10l/l of ethylene were produced. Higher levels of methionine resulted in production of high levels of non-enzymically produced ethylene and death of the bacteria. This non-enzymic production of ethylene was eliminated in the dark. Copper had no effect upon ethylene production. Twenty-nine and 35°C were inhibitory, whereas 19°C appeared to be near optimum for ethylene production.Pseudomonas solanacaerum #25 and some other bacteria are capable of ethylene production and methionine and FMN enhance this production.This work was supported by the Fred C. Gloeckner Foundation and the University of Minnesota Graduate School Grant in Aid #496-0307-4909-02.     

Methionine-induced Ethylene Production by Penicillium digitatum

Shake cultures, in contrast to static cultures of Penicillium digitatum grown in liquid medium, were induced by methionine to produce ethylene. The induction was concentration-dependent, and 7 mM was optimum for the methionine effect. In the presence of methionine, glucose (7 mM) enhanced ethylene production but did not itself induce ethylene production. The induction process lasted several hours, required the presence of viable mycelium, exhibited a lag period for ethylene production, and was effectively inhibited by cycloheximide and actinomycin D. Thus, the methionine-induced ethylene production appeared to involve induction of an enzyme system(s). Methionine not only induced ethylene production but was also utilized as a substrate since labeled ethylene was produced from [¹⁴C]methionine.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Effects of lipophilic and water-soluble membrane probes on ethylene synthesis in apple and Penicillium digitatum

Several chemicals were used to probe the in situ ethylene formingenzyme systems in apple tissue and Penicillium digilatum. 2,4-Dinitrofluorobenzene,a membrane permeant probe, inhibited ethylene production effectivelyin apples but far less effectively in P. digitatum. In contrast,salicylaldehyde, another membrane permeant probe, effectivelyinhibited the P. digitatum system but, except at 0.1 mM concentration,little influenced the apple system. l,5-Difluoro-2,4-dinitrobenzene(DFDNB), a membrane permeant probe which cross-links proteinswith proteins and with phospholipids, strongly inhibited ethylenebiosynthesis in both apple and P. digitatum, whereas dimethylsuberimidate, the protein cross-linking reagent, inhibited slightlythe apple system but not P. digitatum system. Picrylsulfonate(TNBS), a non-permeant membrane probe, up to 0.1 mM, did notinhibit any of the two systems studied. However, in the presenceof exogenous methionine in the apple system and glutamate inP. digitatum, TNBS at 0.1 and 1 mM caused inhibition of ethylenesynthesis. These probes did not affect respiration of appleslices under similar incubating conditions, excepting for DFDNBwhich on longer incubation did inhibit respiration, but theeffect on ethylene synthesis was 15 times greater. Divalentcation ionophores, A23187 [GenBank] and X537 A, had no effect on ethylenesynthesis in both the systems. The water soluble iron chelatingagent, o-phenanthroline, was a more potent inhibitor of theapple system but minimally affected P. digitatum. In contrast,the lipophilic chelator, bathophenanthroline, was a more potentinhibitor of the P. digitatum system. Assay of the fatty acidcomposition of polar lipids from crude membrane fractions showedconsiderably greater linoleic to linolenic ratio in P. digitatumthan in apple. We suggest that the ethylene formations in appleand P. digitatum are sensitive to a modification of membranestructure and that specific chelator-sensitive metals (perhapsiron and copper) are involved in ethylene synthesis in boththese systems. ¹ On leave from the M.S. University of Baroda (India); presentaddress: Department of Plant Genetics, The Weizmann Instituteof Science, Rehovot, Israel. ²Present address: Agricultural Research Organization, The VolcaniCenter, Bet-Dagan, Israel. (Received February 23, 1979; )     

The stimulatory effect of catalase on the formation in vitro of ethylene by an ethylene-forming enzyme purified from Penicillium digitatum

The dependence of the rate of formation of ethylene on the concentration of an ethylene-forming enzyme was determined with a purified preparation of the ethylene-forming enzyme from Penicillium digitatum IFO 9372. The relationship was n ot linear. When catalase and bovine serum albumin were added to the reaction mixture, the rate of formation of ethylene was directly proportional to the concentration of the enzyme. The non-linearity of the reaction, in the absence of these additives, is probably due to the hydroxyl radical ions (OH) produced by the Fenton reaction which occurs in the reaction mixture when ferrous ions and oxygen are present together under reducing conditions.     

Geraniol biotransformation-pathway in spores of Penicillium digitatum

Spores of Penicillium digitatum ATCC 201167 transform geraniol, nerol, citral, and geranic acid into methylheptenone. Spore extracts of P. digitatum convert geraniol and nerol NAD+-dependently into citral. Spore extract also converts citral NAD+-dependently into geranic acid. Furthermore, a novel enzymatic activity, citral lyase, which cofactor-independently converts citral into methylheptenone and acetaldehyde, was detected. These result show that spores of P. digitatum convert geraniol via a novel biotransformation pathway. This is the first time a biotransformation pathway in fungal spores has been substantiated by biochemical studies. Geraniol and nerol are converted into citral by citrol dehydrogenase activity. The citral formed is subsequently deacetylated by citral lyase activity, forming methylheptenone. Moreover, citral is converted reversibly into geranic acid by citral dehydrogenase activity.     

Inhibition of Ethylene Production in Penicillium digitatum

Production of ethylene by static cultures of Penicillium digitatum, which utilize glutamate and α-ketoglutarate as ethylene precursors, was inhibited by methionine, methionine sulfoxide, methionine sulfone, and methionine sulfoximine. Rhizobitoxine did not affect ethylene production but its ethoxy and methoxy analogues were effective inhibitors of ethylene production; its saturated methoxy analogue and kainic acid stimulated ethylene production. Tracer studies showed that the inhibitors blocked the conversion of [³H]glutamate into [³H]ethylene.

In shake cultures of this fungus, which utilize methionine as the ethylene precursor, rhizobitoxine and its unsaturated analogues all inhibited, while the saturated methoxy analogue stimulated ethylene production. In both types of cultures inhibition was irreversible and was diminished by increasing concentrations of the ethylene precursor. The inhibitory activity or lack of it by rhizobitoxine and its analogues appears to be a function of their structural resemblance to glutamate and methionine as well as of their size and stereoconfiguration. These data suggest similarities between the ethylene-forming system in the fungus and in higher plants despite differences in precursors under some cultural conditions.

     

指状青霉Penicillium digitatum侵染柑橘果实的细胞学研究

采用电子显微镜技术系统研究了指状青霉Penicillium digitatum对柑橘果实的侵染过程及超微结构特征。结果表明室温条件下,接种12h后,伤口附近的分生孢子开始萌发产生芽管;然后从伤口处直接侵入果实表皮细胞内;接种24h后,受侵染果实细胞中的菌丝向相邻细胞扩展蔓延,寄主细胞壁开始消解,质壁分离,细胞内含物及各类细胞器凝集,颜色加深,最后完全消解,伤口部位的果皮开始变软,伤口处的菌丝向外生长;84h后伤口处病斑软化,部分长出白色的霉层;96h后病斑软化面积直径达3cm,白色霉层面积逐渐扩大;120h后白色霉层中间伤口处霉层颜色加深变为灰绿色;144h后整个果实变软腐烂。果胶质标记结果表明,菌丝侵入果实后产生果胶酶并降解柑橘细胞壁中的果胶,使得细胞壁松弛,软化腐烂。     

Factors affecting growth and urease production by Trichophyton spp.

Among Trichophyton spp. examined for urease production, T. rubrum was negative, whereas T. mentagrophytes appeared to be the most active species. Urease was not detected in cell-free culture fluids of the tested fungi. The endocellular urease of the test fungi was essentially constitutive. Moreover, addition of urea to the growth medium of these organisms markedly inhibited their mycelial biomass and ureolytic yield. Environmental factors showed variable effects on the test fungi and there was no correlation between mycelial growth and urease activity of these fungi.     

The role of wounds in the infection of oranges by Penicillium digitatum Sacc

Inocula of spores of Penicillium digitatum in water applied to apparently uninjured skin of oranges do not cause lesions to develop. Addition of citric acid, orange juice, or various extracts of rind had little effect on susceptibility to infection. When spores in water are applied to wounds made between oil vesicles, lesions develop only from wounds that penetrate deeply into the albedo. The flavedo of most oranges seems to be resistant to infection even when damaged, but in a few consignments it showed much less resistance. Increasing the number of conidia in the inoculum caused more lesions to develop but some fruits developed lesions from inocula containing very few spores. The method and timing of spore application to wounds had a considerable effect on the incidence of lesions; emanations from infected fruit had no effect. Lesions developed more rapidly and readily when suspensions of spores in water were applied to wounds in the skin that damaged oil vesicles; wounds as shallow as 0–25 mm allowed lesions to develop.