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Exposure of harvested grapefruit to UV-C (254 nm) irradiation was previously found to induce resistance against the green mold decay caused by Penicillium digitatum. In order to gain insight into the mechanism of this UV-induced resistance we initiated a study for isolation of genes induced during this process. Using the differential display method we cloned cDNA representing an mRNA which is accumulated in grapefruit peel upon UV irradiation. Sequence analysis revealed that this cDNA represents a gene encoding for an isoflavone reductase-like protein and was termed IRL (isoflavone reductase-like). The grapefruit IRL protein sequence has high homology also to a novel family of other isoflavone reductase-like proteins present in few non-legume plants and whose function is not clear yet. The UV dose, and time following it, which lead to maximal accumulation of the IRL transcript were found to be similar to those leading to maximal induced resistance. The expression of the IRL gene was demonstrated to be induced also by wounding and pathogen infection.

ABSTRACT Interactions between CaCl(2), grapefruit peel tissue, Penicillium digitatum, and the yeast antagonist Pichia guilliermondii strain US-7 were investigated. Application of 68 or 136 mM CaCl(2) to grapefruit surface wounds reduced the incidence of green mold caused by Penicillium digitatum by 43 to 52%. In laboratory tests, a cell suspension (10(7) cells/ml) of Pichia guilliermondii containing either 68 or 136 mM CaCl(2) reduced the incidence of green mold from 27 to 3%. In large scale tests, dip application of 136 mM CaCl(2) with US-7 (10(7) cells/ml) significantly decreased the number of wounds infected by Penicillium digitatum. CaCl(2), with or without yeast cells, stimulated ethylene production in grapefruit tissue. Increasing concentrations of CaCl(2) resulted in decreased spore germination and germ tube elongation of Penicillium digitatum. Pectinolytic activity of crude enzyme preparations of Penicillium digitatum was also inhibited by the presence of increasing concentrations of CaCl(2). US-7 exhibited a strong ability to maintain cytosolic Ca(2+) homeostasis at levels that did not exceed 1.4 muM when exposed to 150 mM CaCl(2). On the other hand, strain 114 of Debaryomyces hansenii, which failed to give any protection against infection by Penicillium digitatum, showed reduced capacity to maintain Ca(2+) homeostasis. The effect of calcium in reducing infection of grapefruit wounds by Penicillium digitatum could be due to direct effects on host tissue (making cell walls more resistant to enzymatic degradation) or the pathogen (interfering with spore germination, growth, and inhibition of fungal pectinolytic enzymes). Alternatively, the ability of US-7 to maintain calcium homeostasis may allow it to grow or assist in its competitive ability in a microenvironment that, because of high levels of calcium ions, is inhibitory to growth of the green mold pathogen.

In recent years, both the public and health authorities have become increasingly concerned about the presence of pesticides in our food supply and the environment. As a direct result of this mounting concern, research efforts for the development of alternative methods for the control of postharvest diseases of fruits and vegetables have been intensified. Considerable attention has been placed on assessing the potential of the use of biological antagonists as a viable alternative to the use of synthetic fungicides. Naturally occurring microbial antagonists have been shown to control several rot pathogens on diverse commodities. Such antagonists have various modes of action: antibiosis or competition for nutrients and space or both, induction of resistance in the host tissue, and direct interaction with the pathogen. The commercialization of certain antagonists to control postharvest decay of fruits and vegetables appears to be feasible and may present an alternative to synthetic pesticides.

The application of ultraviolet light (UV-C, 254 nm) hormesis on fruits and vegetables to stimulate beneficial responses is a new method for controlling storage rots and extending the shelf-life of fruits and vegetables. The present study was aimed at treating tomatoes ( Lycopersicon esculentum ) with different UV-C dosages (1.3 to 40 KJ/m 2 ) to induce resistance to black mold ( Alternaria alternata ), gray mold ( Botrytis cinerea ), and Rhizopus soft rot ( Rhizopus stolonifer ). These diseases were effectively reduced when tomatoes were inoculated following UV-C irradiation. UV-C treated tomatoes were firmer in texture and less red in color than the control tomatoes, indicating a delay in ripening. Slower ripening and resistance to storage rots of tomatoes are probably related. The positive effect of UV-C on tomatoes decreased as treatments were performed at stages of increased ripeness.

In previous studies workers have shown that three yeast strains (strains US-7, 82, and 101) have biological control activity against various postharvest fungal pathogens of fruits and vegetables, including Penicillium rots of apples and citrus and Botrytis rot of apples. In these reports the researchers have described these strains as Debaryomyces hansenii (anamorph, Candida famata) or Candida sp. strains. In this study we performed additional physiological, DNA reassociation, and mannan characterization tests that clearly established a new taxonomic classification for these strains, Candida guilliermondii. We also propose amendment of the physiological test profile in the taxonomic description of C. guilliermondii.

The analogs of rhizobitoxine, aminoethoxyvinylglycine (AVG) (l-2-amino-4-2'-aminoethoxy-trans-3 butenoic acid) and methoxyvinylglycine (MVG) (l-2-amino-4-methoxy-trans-3-butenoic acid), that are potent inhibitors of ethylene biosynthesis at 0.1 millimolar also inhibited protein synthesis and charging of tRNA especially at 1 millimolar and higher concentrations. The saturated analog of MVG inhibited ethylene synthesis while the saturated analog of AVG did not. Both saturated AVG and MVG inhibit methionyl- and leucyl-amino acyl-tRNA synthetase. Because of the inhibition of amino acid metabolism in plant tissues by these rhizobitoxine analogs caution is advised in interpreting the results obtained with concentrations of compounds above 0.1 millimolar.

Ethylene production by detached cotyledons of cotton (Gossypium hirsutum L.) seedlings grown under cycles of 12 h darkness and 12 h light has been shown to be rhythmic, with a minimum and maximum 4 and 16 h, respectively after the start of the cycle (Rikin, Chalutz and Anderson, 1984, Plant Physiol. 75, 493-495). Treatment with silver ions stimulated the rhythmic ethylene production in both regular and "inverted" cycles (i.e. dark period changed to light period, and vice versa). The rate of the conversion of [3,4-(14)C]methionine into ethylene also followed the stimulation of rhythmic ethylene evolution by silver ions in both regular and inverted cycles, while treatment with aminoethoxyvinylglycine (AVG) decreased this stimulation. Conversion of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) into ethylene was not affected by silver ions, but was dependent upon the immediate light conditions, regardless of the time in the light-dark cycle, light decreasing and darkness increasing this process. It is concluded that silver ions stimulate the normal rhythmic ethylene production, and this stimulation is regulated at a step prior to the conversion of ACC into ethylene. The rhythmicity in other processes (cotyledon movement, phenylalanine ammonia-lyase activity, resistance to the herbicide 3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide [bentazon]) was not affected by a decrease in the rhythmic changes in ethylene production by AVG or interference in ethylene action by silver ions. Thus, these rhythmic changes were not regulated by the rhythmic changes in ethylene production.

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 [(14)C]methionine.Following induction by the fungus, filtrates of induced shake cultures also evolved ethylene in increasing amounts by both enzymic and monenzymic reactions. Tracer experiments indicated that the ethylene released by the filtrate was derived from a fungal metabolite of methionine and not directly from methionine.

Characterization of the phosphate effect on ethylene production by Penicillium digitatum is reported. A low level of phosphate (0.001 millimolar) was about 200 to 500 times as effective as a high phosphate level (100 millimolar) in stimulating ethylene production and the stimulation was readily reversed by addition of phosphate. This phosphate effect did not operate in static cultures. The precursor of ethylene in the stimulated low phosphate system was glutamate but not alpha-ketoglutarate, which is a precursor in static systems. Actinomycin D and cycloheximide effectively inhibited the low phosphate/high ethylene-producing system. Alkaline phosphatase and protein kinase activities were higher in low than in high phosphate systems. We suggest that phosphate level regulates ethylene production by P. digitatum and that the regulation involves a phosphorylation or dephosphorylation reaction of some enzyme system associated with ethylene production. Phosphate-mediated control of ethylene production may also involve the transcriptional and translational machinery of the fungal cell. P. digitatum apparently can produce widely different levels of ethylene by different pathways, depending on culture conditions under which it is grown.

Cellulysin-induced ethylene production in tobacco (Nicotiana tabacum L.) leaf discs was enhanced several-fold by prior exposure of the leaf tissue to ethylene. This enhancement in the response of the tissue to Cellulysin increased rapidly during 4 and 8 hours of pretreatment with ethylene and resulted from greater conversion of methionine to ethylene. On treatment with Cellulysin, the content of 1-aminocyclopropane-1-carboxylic acid (ACC) in leaf discs not pretreated with ethylene markedly increased while that of the ethylene-pretreated tissue was only slightly higher than in the tissue incubated in the absence of Cellulysin. Ethylene-treated tissue, however, converted ACC to ethylene at a faster rate than air controls. These data indicate that ethylene stimulates Cellulysin-induced ethylene production by stimulating the conversion of ACC to ethylene. Data are also presented on a possible relation of this phenomenon to ethylene produced by the tobacco leaf upon interaction with its pathogen, Alternaria alternata.

Cotyledons of cotton (Gossypium hirsutum L.) seedlings grown under a photoperiod of 12 hour darkness and 12 hour light showed daily oscillations in ethylene evolution. The rate of ethylene evolution began to increase toward the end of the dark period and reached a maximum rate during the first third of the light period, then it declined and remained low until shortly before the end of the dark period. The oscillations in ethylene evolution occurred in young, mature, and old cotyledons (7 to 21 day old). These oscillations in ethylene evolution seemed to be endogenously controlled since they continued even when the photoperiod was inverted. Moreover, in continuous light the oscillations in ethylene evolution persisted, but with shorter intervals between the maximal points of ethylene evolution. In continuous darkness the oscillations in ethylene evolution disappeared. The conversion of [3,4-(14)C]methionine into [(14)C] ethylene followed the oscillations in ethylene evolution in the regular as well as the inverted photoperiod. On the other hand, the conversion of applied 1-aminocyclopropane-1-carboxylic acid into ethylene did not follow the oscillations in ethylene evolution, but was affected directly by the light conditions. Always, light decreased and darkness increased the conversion of applied 1-aminocyclopropane-1-carboxylic acid into ethylene. It is concluded that in the biosynthetic pathway of ethylene the conversion of 1-aminocyclopropane-1-carboxylic acid into ethylene is directly affected by light while an earlier step is controlled by an endogenous rhythm.

Production of ethylene by static cultures of Penicillium digitatum, which utilize glutamate and alpha-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 [(3)H]glutamate into [(3)H]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.

The concentrations of 3-methyl-6-methoxy-8-hydroxy-3,4-dihydroisocoumarin (MMHD) formed in carrot roots inoculated with certain fungi or treated with indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), were related to the amount of ethylene produced by the root tissue. Ethylene applied exogenously in concentrations above 0.3 ppm induced the formation of MMHD in carrot root discs. Continued production of MMHD required the continued presence of ethylene. The amounts of MMHD in the discs were reduced by CO(2), an inhibitor of ethylene action, and by reduction of the partial pressure of ethylene in fungus-inoculated or 2,4,5-T-treated carrot root discs. The results indicate that ethylene is required for the induction of MMHD formation by carrot root tissue.

Ethylene enhanced the activity of phenylalanine ammonialyase in carrot (Daucus carota L., var. "Nauty") root tissue. Slight increase in enzyme activity was exhibited by root discs incubated in ethylene-free air. It was probably due to the ethylene formed within the sliced tissue. Addition of ethylene to the air stream increased phenylalanine ammonia-lyase activity and the total protein content of the discs until maximum activity was reached after 36 to 48 hours of incubation. The continuous presence of ethylene was required to maintain high level of activity. Ethylene, at a concentration of 10 microliter per liter induced higher activity than at lower or higher concentrations. CO(2) partially inhibited the ethylene-induced activity. Cycloheximide or actinomycin D effectively inhibited the ethylene-induced activity in discs that had not previously been exposed to ethylene. The results appear to support the hypothesis that the mode of action of ethylene may involve both de novo synthesis of the enzyme protein and protection or regulation of activity of the induced enzyme.