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.     

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; )     

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.

     

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.     

Sulla penetrazione di Penicillium digitatum Sacc. e Penicillium italicum Wehmer nei frutti degli agrumi

Summary

The A., with a series of controls and investigations, accomplished on different groups of Oranges and following various forms of artificial infection with Penicillium digitatum and Penicillium italicum, renders evident the different reactions that these moulds have with regard to the resistance of the cuticle and the epidermic and under-epidermic stratums of the peel of the controlled fruits. Moreover the A. confirms that both the P. digitatum and the P. italicum can act as «parasite of injury» and as «parasite of contact».     

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.     

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.     

Monitoring indole alkaloid production by Penicillium digitatum during infection process in citrus by Mass Spectrometry Imaging and molecular networking

Green mold, caused by Penicillium digitatum, is the most destructive post-harvest disease in citrus. Secondary metabolites produced by fungal phytopathogens have been associated with toxicity to their respective host through the interaction with a wide range of cell targets. Natural products have also been described as important molecules for biocontrol and competition in their respective environment. For P. digitatum, the production of indole alkaloids, tryptoquialanines A and B, have been reported. However, their biological role remains unknown. Mass Spectrometry Imaging (MSI) technique was applied here for the first time to monitor the secondary metabolites produced on the orange surface during infection in order to gain insights about the P. digitatum-citrus interaction mechanisms. Through the combination of MSI and molecular networking it was possible to report, for the first time, the production of tryptoquivalines and fumiquinazolines by P. digitatum and also the accumulation of tryptoquialanines on the fruit surface from 4 to 7 d post inoculation. P. digitatum was also evaluated concerning the ability to sinthesize indole alkaloids in vivo in the different citrus hosts. The biological role of tryptoquialanines was investigated and tryptoquialanine A was submitted to insecticidal bioassays that revealed its high toxicity against Aedes Aegypti, suggesting an important insecticidal action during orange decay.     

The nature of macerating factor of Penicillium digitatum saccardo

The pectic enzymes produced by Penicillium digitatum, which invades oranges, have been compared with those produced by P. notatum which does not attack oranges. The differences found are discussed in relation to the ability of the culture filtrate of P. digitatum to macerate orange-rind tissue. Purification of pectin transeliminase from the culture filtrate of P. digitatum is described and evidence is discussed which suggests that it is this enzyme which is responsible for the maceration of orange-rind tissue.     

Potential for Development of Tolerance by Penicillium digitatum and Penicillium italicum after Repeated Exposure to Potassium Sorbate

Two strains of Penicillium digitatum and one strain of Penicillium italicum were exposed to various levels of sorbic acid and potassium sorbate, and the MICs were determined. Selected strains of the molds were then repeatedly exposed to subinhibitory levels of the compounds to determine whether increased tolerance might develop. The MIC of sorbic acid (pH 4.75) to P. digitatum was between 0.02 and 0.025%. The MIC of sorbate (pH 5.5) to two strains of P. digitatum and P. italicum was found to be between 0.06 and 0.08%. Increasing levels of sorbate resulted in increasing growth suppression of the molds. Populations of P. digitatum were tested for increased tolerance to sorbic acid, and none was found. Individual molds that started from the same parent colony were examined for increased tolerance to potassium sorbate. Two P. digitatum strains developed no observable increased tolerance, but P. italicum developed a slight increase in tolerance to sorbate. When spores of P. italicum and P. digitatum were exposed to higher levels of sorbate for prolonged times, the fungicidal or fungistatic activity of the inhibitor was dependent upon pH, length of exposure time, level of sorbate, and the mold strain.