Effects of ethylene on the susceptibility to Botrytis cinerea infection of different tomato genotypes

Ethylene at 10 and 100 μl/litre stimulated germ-tube elongation of Botrytis cinerea spores incubated within normal and non-ripening nor tomato fruits, but had little influence on the total percent of germination. Values of germ-tube length within the mature-green normal fruits and the mature-green or mature nor fruits were similar to those recorded within the normal mature fruits when held in air. Exposure of the normal and the mutant fruits to 100 μl/litre ethylene immediately after inoculation with B. cinerea insignificantly increased lesion development, but resulted in increased sporulation. When tomato fruits were exposed to ethylene for 3 days before inoculation a marked stimulatory effect on rot development was exhibited on the mature-green normal fruits but not on the nor mutant fruits. The results indicate that exogenous ethylene may directly stimulate germ tube growth of B. cinerea in both normal and mutant fruit, but that it may affect subsequent fungal growth indirectly, via stimulation of the ripening process, only in preclimacteric normal tomato fruit.     

Initiation at the flowering stage of postharvest Botrytis stem-end rot in normal and non-ripening tomato fruits

Flowers of a normal tomato cultivar and of the two non-ripening mutants rin and nor, were sprayed with a Botrytis cinerea spore suspension. Stem-end infection developed in 84–100% of the harvested fruits of these sprayed flowers when held in high r.h. In both nor and rin fruits, rot development remained restricted to the stem-end area and fruit shoulders, whereas in the normal fruit decay spread rapidly from the stem-end over the entire fruit. Spraying the flowers with iprodione prior to spore inoculation resulted in decreased incidence of decay and suppressed hyphal growth and sporulation in all types of fruits. The results indicate that floral organs serve as a pathway for B. cinerea stem-end initiation in both the normal and the mutant fruit and suggest that this mode of penetration is not related to the marked resistance to infection attributed to the mutant fruits.     

Stock-Scion Interactions of Normal and Fruit Ripening Mutants rin and nor in Tomato

Scions of the non-ripening rin and nor tomato strains (Lycopersicum esculentum Mill.) were grafted on normal understock plants (cv. Rutgers) in an effort to study the influence of roots and vegetative tissue on the ripening behavior of the tomato fruit. Receiprocal grafts of ‘Rutgers’ scions on rin and nor understocks as well as grafted and ungrafted controls were also established. No alteration in the ethylene, and CO₂ evolution and color development of either mutant fruits on normal understock or of normal fruits on mutant understock occurred. We suggest that the inability of rin and nor mutant fruits to ripen normally stems either from the presence in mutant fruit of a non-translocatable ripening inhibitor, or from the absence of a non-translocatable ripening factor.     

Resistance of rin and nor tomato mutants to postharvest Rhizopus infection

Fruits of the two non-ripening mutants of tomato, rin and especially nor, were markedly more resistant to Rhizopus stolonifer infection than the normal Rutgers fruit. Following artificial inoculations by contact with a diseased normal tomato covered with mycelium and sporangia, no infection of unwounded nor fruit occurred at its mature-green stage. At the mature stage the resistance of nor mutant fruit was manifested by a prolongation of the incubation period of the disease as well as by a markedly reduced incidence of rotted fruits. Chilling injury of fruit, prior to spore inoculation, was found to be a good means for indicating the relative resistance of the mutants as compared with the normal tomato. The relationship between the resistance of the mutant tomatoes to Rhizopus infection and their response to induced peel damage as a result of the contact or the chilling procedure, led to the assumption that fruit resistance is associated with the inability of the fungus to penetrate the periderm, rather than with fungal development within the fruit.     

Pectolytic and Cellulolytic Activity of Botrytis cinerea Pers. Related to Infection of Non-ripening Tomato Mutants

Enzymes of Botrytis cinerea were detected in vitro using various carbon sources. Pectin-pectate as a sole carbon source induced both polygalacturonase (PG) and pectin lyase (PL) activity, whereas carboxymethylcellulose served as an inducer for cellulase (C_x) activity. PG activity appeared earlier than C_x activity when induced by their respective sources. Both PG and PL activities were detected earlier and their level was higher on cell walls of the normal tomato fruit, than of the nor mutant, and in each case activity was higher on cell walls of the mature fruits than of the mature-green ones. Whereas relatively high rates of PG and PL activity were recorded on autoclaved tomato homogenate (TH) of both the normal and the nor fruits, only trace levels of PG activity were recorded on unautoclaved media, except for those prepared from ripe normal fruits, and no PL activity was detected on either of the unsterilized media. Botrytis-infection resulted in PG activity in the enzyme-less rin and nor mutant fruits at both stages of maturity and in the normal and hybrid fruits at their mature-green stage. In the ripe normal and hybrid fruits, infection increased the level of PG activity recorded prior to inoculation. An association was drawn between the low PG activity recorded in the nor mutant and its hybrid at initial stages of invasion and their resistance to infection. Following infection an increase in the level of C_x activity over that recorded in healthy fruits was found in all the tomato genotypes, whereas no PL was recorded in either healthy or infected fruits.     

Comparison of Propylene-induced Responses of Immature Fruit of Normal and rin Mutant Tomatoes

Continuous application of propylene to 40 to 80% mature fruits of normal tomato strains (Lycopersicon esculentum Mill.) advanced ripening in fruits of all ages by at least 50%. Although preclimacteric respiration was stimulated by propylene treatment, there was no concomitant increase in ethylene production. Once ripening commenced, the rates of endogenous ethylene production were similar in both propylene-treated and untreated fruits. Continuous exposure to propylene also stimulated respiration in immature fruits of rin, a nonripening mutant. Although respiration reached rates similar to those during the climacteric of comparable normal fruits there was no change in endogenous ethylene production which remained at a low level. Internal ethylene concentrations in attached 45 to 75% mature fruits of rin and a normal strain were similar. It is suggested that the onset of ripening in normal tomato fruit is not controlled by endogenous ethylene, although increased ethylene production is probably an integral part of the ripening processes.     

Effect of Sodium Chloride on Fruit Ripening of the Nonripening Tomato Mutants nor and rin

Tomato (Lycopersicon esculentum Mill) plants of the nonripening mutant nor, the ripening-inhibited mutant rin, and the normal cultivar `Rutgers' were grown in nutrient solution supplemented with 3 grams per liter NaCl from the time of anthesis. In plants treated with NaCl, all the ripening parameters of the fruits of the nor mutant increased, but those of the rin mutant did not. The ripening of the fruits of the NaCl-treated nor plants was characterized by the development of a red color and taste, increased pectolytic activity, and increased evolution of CO₂ and ethylene. These changes do not normally take place in nor under control conditions. The values of these ripening parameters in nor were lower than those of the normal Rutgers fruits. In addition, both in nor and rin and in the normal variety, exposure of the plants to NaCl shortened the developmental period of the fruit, decreased the fruit size, and increased the concentrations of total soluble solids, Na⁺, Cl⁻, reducing sugars, and titratable acids in the fruit. The role of NaCl in overcoming the inability of nor to ripen is discussed.     

Promotion by Ethylene of the Capability to Convert 1-Aminocyclopropane-1-carboxylic Acid to Ethylene in Preclimacteric Tomato and Cantaloupe Fruits

The intact fruits of preclimacteric tomato (Lycopersicon esculentum Mill) or cantaloupe (Cucumis melo L.) produced very little ethylene and had low capability of converting 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. When these unripe tomato or cantaloupe fruits were treated with ethylene for 16 hours there was no increase in ACC content or in ethylene production rate, but the tissue's capability to convert ACC to ethylene increased markedly. Such an effect was also observed in fruits of tomato mutants rin and nor, which do not undergo ripening and the climacteric increase in ethylene production during the senescence. The development of this ethylene-forming capability induced by ethylene increased with increasing ethylene concentration (from 0.1 to 100 microliters per liter) and duration (1 to 24 hours); when ethylene was removed this capability remained high for sometime (more than 24 hours). Norbornadiene, a competitive inhibitor of ethylene action, effectively eliminated the promotive effect of ethylene in tomato fruit. These data indicate that the development of the capability to convert ACC to ethylene in preclimacteric tomato and cantaloupe fruits are sensitive to ethylene treatment and that when these fruits are exposed to exogenous ethylene, the increase in ethylene-forming enzyme precedes the increase in ACC synthase.     

Physiology of the tomato mutant alcobaca

Alcobaca is commonly regarded as an abnormally ripening mutant of the tomato (Lycopersicon esculentum Mill.). Alcobaca fruits were found to be similar to cv. Rutgers fruits in the following characteristics: time between full anthesis and the onset of ripening, response to ethephon, flavor, pH and concentrations of titratable acids, total soluble solids and reducing sugars. The pattern of CO₂ and ethylene climacteric are similar in the two plant types, but the peak levels were lower and occurred later in alcobaca than in ‘Rutgers’. The mutant fruits differed from fruits of normal varieties in their greatly prolonged shelf life, their relatively low activity of polygalacturonase (PG) and polymethylgalacturonase (PMG), and their low level of endogenous ethylene. Fruits of the mutant harvested before the onset of ripening failed to reach normal pigmentation and remained yellow. Fruits harvested at the onset of ripening reached an orange color, while fruits ripened while attached to the plant reached almost normal pigmentation. These results suggest that alcobaca is a slow ripening mutant and does not belong to the category of non-ripening mutants.     

Fruit cuticle lipid composition during development in tomato ripening mutants

Recent studies suggest that fruit cuticle is an important contributing factor to tomato (Solanum lycopersicum) fruit shelf life and storability. Moreover, it has been hypothesized that variation in fruit cuticle composition may underlie differences in traits such as fruit resistance to desiccation and microbial infection. To gain a better understanding of cuticle lipid composition diversity during fruit ontogeny and to assess if there are common features that correlate with ripening, we examined developmental changes in fruit cuticle wax and cutin monomer composition of delayed‐ripening tomato fruit mutants, ripening inhibitor (rin) and non‐ripening (nor) and delayed‐ripening landrace Alcobaça. Previous reports show that fruit ripening processes such as climacteric ethylene production, cell wall degradation and color change are significantly delayed, or do not occur, in these lines. In the study presented here, however, we show that fruits from rin, nor and Alcobaça have cuticle lipid compositions that differ significantly from normal fruits of Ailsa Craig (AC) even at very early stages in fruit development, with continuing impacts throughout ripening. Moreover, rin, nor and the Alcobaça lines show quite different wax profiles from AC and each other throughout fruit development. Although cutin monomer composition differed much less than wax composition among the genotypes, all delayed‐ripening lines possessed higher relative amounts of C₁₈ monomers than AC. Together, these results reveal new genetic associations between cuticle and fruit development processes and define valuable genetic resources to further explore the importance of cuticle in fruit shelf life.     

Altered Tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.) Fruit Cuticle Biomechanics of a Pleiotropic Non Ripening Mutant

Tomato (Lycopersicon esculentum Mill.) fruit ripening involves multiple metabolic changes resulting in softening and pigmentation. We investigated the mechanics and morphology of the enzymatically isolated cuticular membrane (CM) of cv. Ailsa Craig wild-type (wt) and nonripening mutant (nor) at three developmental stages. Cuticle thickness and degree of cutinization increased significantly from immature to fully ripe fruits for both wt and nor without differences between them. Mechanical characterization was carried out on dry and fully hydrated samples in uni-axial tension to determine their modulus of elasticity, stress, and strain at failure. Corresponding stress-strain diagrams were biphasic and showed yield for virtually all dry CM samples, while that of hydrated CM displayed considerable differences between wt and nor fruits. Concerning the mechanical properties, the CM of wt fruits was characterized by increasing stiffness and strength during fruit growth and maturation in both dry and hydrated states, whereas the CM of nor fruits was significantly less stiff and weaker at full maturity. Hydration generally caused lower moduli of elasticity and strength, while breaking strain was significantly affected only for the CM of ripe nor fruits. This plasticizing effect of water increased towards full maturity for both wt and nor, and may be related to fiber content in the CM matrix and hydration state of the cuticle. Comparative analysis of two additional wild-type tomato cultivars supported the ripening-related stiffening of the CM of Ailsa Craig wt and the altered mechanical properties of the nor mutant, as well as the plasticizing effect of water.     

Biocontrol of Postharvest Grey Mould on Tomato by Yeasts

The fungal pathogen Botrytis cinerea causes severe rots on tomato fruit during storage and shelf life. Biological control of postharvest diseases of fruit may be an effective alternative to chemical control. Yeasts are particularly suitable for postharvest use, proving to be highly effective in reducing the incidence of fungal pathogens. Yeast fungi isolated from the surface of solanaceous plants were evaluated for their activity in reducing the postharvest decay of tomato caused by B. cinerea. Of 300 isolates, 14 strains of Rhodotorula rubra and Candida pelliculosa were found to be strongly antagonistic to the pathogen in vitro and were selected for further storage experiment. The antagonists were evaluated for their effect on the biological control of postharvest grey mould. Artificially wounded fruits were treated by means of a novel technique: small sterile discs of filter paper imbibed separately in suspensions of each yeast and the pathogen were superposed onto each wound. After 1‐week, 11 isolates were significantly effective in reducing the diameter of lesions by more than 60% compared to the control treated with B. cinerea alone. Total protection was obtained with the strain 231 of R. rubra on fruits challenged with pathogen spores. To our knowledge, R. rubra and C. pelliculosa have not been described as biocontrol agents against grey mould caused by B. cinerea. Our data demonstrate that the application of antagonistic yeasts represents a promising and environmentally friendly alternative to fungicide treatments to control postharvest grey mould of tomato.     

Possible Mechanism of the Detached Unripe Green Tomato Fruit Turning Red

In general, a mature green tomato will ripen and turn red on the vine or off the vine. Interestingly, an unripe green (UG) tomato also will turn red after being detached from the plant, but the mechanism behind this is unclear. Our study showed that detached UG fruits were able to become red-ripened at the room temperature (25?°C), although fruit quality was lower than that of the vine-ripened fruit. In addition, detached UG fruits exposed to light accumulated more lycopene and total soluble sugars than those incubated in darkness. When the detached UG fruits were stored at a low temperature (10?°C), the fruit-ripening process was nearly blocked, which displayed a non-ripening phenotype. At a high temperature (35?°C), the detached UG fruit showed a yellow-color on ripening, and fruit qualities such as lycopene and soluble sugars were obviously lower than those stored at 25?°C. Moreover, detaching the UG fruit from the plant evoked a rapid increase in total respiration as well as alternative pathway respiration, but ethylene production was not stimulated during the first 24 h of storage. Importantly, the application of nPG, an inhibitor of alternative pathway respiration, markedly suppressed the wound-induced rise in total respiration and delayed the ripening of detached UG fruit. These findings imply that wound-induced respiration might be a signature for launching the onset of the ripening of detached UG fruit, and the optimal conditions of light and temperature are beneficial for the color change and the ripening processes.     

Effect of suppression of ethylene biosynthesis on flavor products in tomato fruits

To elucidate the role of ethylene in the production of flavor compounds by tomato fruits, wild-type tomato (Lycopersicon esculentum L., cv. Lichun) and its transgenic antisense LeACS2 line with suppressed ethylene biosynthesis were used. The metabolism of individual sugars was ethylene-independent. However, citric acid and malic acid were under ethylene regulation. The content of these acids was higher in transgenic tomato fruits and returned to normal level after transgenic fruits were treated with ethylene. Because most of amino acids, which are important precursors of volatiles, were shown to be correlated with ethylene, we surmise that amino acid-related aroma volatiles were also affected by ethylene. Headspace analysis of volatiles showed a significant accumulation of aldehydes in wild-type tomato fruits during fruit ripening and showed a dramatic decrease in most aroma volatiles in transgenic tomato fruits as compared with wild-type fruits. The production of hexanal, hexanol, trans-2-heptenal, cis-3-hexanol, and carotenoid-related volatiles, except β-damascenone and β-ionone, was inhibited by suppression of ethylene biosynthesis. No remarkable differences were observed in the concentrations of cis-3-hexenal and trans-2-hexenal between transgenic and wild-type tomato fruits, indicating these two volatiles to be independent of ethylene. Thus, there are various regulation patterns of flavor profiles in tomato fruits by ethylene. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 92–101. The text was submitted by the authors in English. Both authors equally contributed to this work.     

Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

Tomato Fruit Development and Ripening Are Altered by the Silencing of LeEIN2 Gene

Loss-of-function ethylene insensitive 2 （EIN2） mutations showed ethylene insensitivity in Arabidopsis, which indicated an essential role of EIN2 in ethylene signaling. However, the function of EIN2 in fruit ripening has not been investigated. To gain a better understanding of EIN2, the temporal regulation of LeEIN2 expres- sion during tomato fruit development was analyzed. The expression of LeEIN2 was constant at different stages of fruit development, and was not regulated by ethylene. Moreover, LeEIN2-silenced tomato fruits were developed using a virus-induced gene silencing fruit system to study the role of LeEIN2 in tomato fruit ripening. Silenced fruits had a delay in fruit development and ripening, related to greatly descended expression of ethylene-related and ripening-related genes in comparison with those of control fruits. These results suggested LeEIN2 positively mediated ethylene signals during tomato development. In addition, there were fewer seeds and Iocules in the silenced fruit than those in the control fruit, like the phenotype of parthenocarpic tomato fruit. The content of auxin and the expression of auxin-regulated gene were declined in silenced fruit, which indicated that EIN2 might be important for crosstalk between ethylene and auxin hormones.     

Stimulation of ethylene production by a cell wall component from mature green tomato fruit

Pectic (carbonate-soluble, covalently-bound pectin, CBP) material stimulated increased ethylene production when vacuum-infiltrated into whole, mature green tomato ( Lycopersicon esculentum Mill. cv. Rutgers) fruit. Activity was greatest if CBP was extracted from mature green tomatoes with jellied locules. CBP extracted from mature green tomatoes with immature seeds had no elicitor activity, while CBP from turning or red ripe tomatoes was only moderately active. Infiltration of CBP from normal mature green fruit into ripening inhibitor ( rin ) mutant tomato fruit stimulated ethylene production and attenuated red pigmentation in these fruits. Partial purification of the active material was accomplished using DEAE-Sephadex and BioGel P-100 chromatography. The most highly purified fraction is comprised of neutral carbohydrate (95%) with a relatively low content of amino acids (1%) and a uronic acid content of less than 5%. This material may be an endogenous trigger of ethylene production and ripening.