Structural basis of the rind disorder oleocellosis in Washington navel orange (Citrus sinensis L. Osbeck)

Oleocellosis, a physiological rind disorder of citrus fruit, is an unattractive surface blemish caused by phytotoxic effects of released rind oils. The development of oleocellosis in Washington navel orange (Citrus sinensis L. Osbeck) was examined by following a time sequence of surface symptoms and microscopic rind changes. The two natural causes of oleocellosis were simulated: mechanical damage to the fruit and transfer of rind oil between fruit. Mechanical fruit injury resulted in rupture of the epidermis above oil glands. Released surface oil appeared to infiltrate the rind via the ruptured epidermis resulting in rapid degeneration of cortical, but not epidermal, cell contents. Oil application to the rind surface produced a more severe blemish than did mechanical damage. The oil appeared to diffuse through the cuticle causing degeneration of the contents of all cell layers, including the epidermis. Loss of membrane integrity was detected within 30 min, followed by cell content degeneration and cell collapse. The resulting blemish, characterized by rind collapse and darkening, developed substantially within 3 d and was attributed to the cellular damage.     

Cellulase Activity, Endogenous Abscisic Acid, and Ethylene in Four Citrus Cultivars during Maturation

At maturity, the fruit of two early maturing orange cultivars, Hamlin and Pineapple (Citrus sinensis [L.] Osbeck), contained more ethylene and abscisic acid than the late maturing Valencia and Lamb Summer (C. sinensis [L.] Osbeck) cultivars. Ethylene (up to 95 nl/l in internal atmosphere) and abscisic (50 μg/kg dry weight flavedo) increased most rapidly in Pineapple, leading to increased cellulase activity and loosening of the fruit. Fruit of the two late maturing cultivars contained less than 25 nl/1 ethylene and 40 μg abscisic acid/kg dry weight of flavedo at peak maturity. Cellulase activity and loosening of the fruit of these late maturing cultivars was slight.     

Growth Rate and Water Relations of Citrus Leaf Flushes

Growth rates of seasonal leaf flushes of ‘Valencia’orange [Citrus sinensis (L.) Osbeck] were measured and waterrelations characteristics of young (new) and over-wintered (old)citrus leaves were compared. New flush leaves had lower specificleaf weights and lower midday leaf water potentials than comparablyexposed old leaves. Spring and summer flush new leaves had higherosmotic potentials than old leaves. These differences becamenon-significant as the new leaves matured. During summer conditions,water-stressed new leaves reached zero turgor and stomatal conductancealso began to decrease in them at higher leaf water potentialsthan in old leaves. Old leaves were capable of maintaining openstomata at lower leaf water potentials. Opened flowers and newflush leaves lost more water, on a dry weight basis, than flowerbuds, fruit or mature leaves. The results illustrate differencesin leaf water potential and stomatal conductance which can beattributed to the maintenance of leaf turgor by decreases inleaf osmotic potentials as leaves mature. These changes in citrusleaf water relations are especially important since water stressresulting from high water loss rates of new tissues could reduceflowering and fruit set. Citrus sinensis (L.) Osbeck, orange, Citrus paradisi Macf., grapefruit, growth rate, leaf water relations, osmotic potential, water potential, stomatal conductance     

Morphology and Anatomy of Stems and Pedicels of Spring Flush Shoots Associated with Citrus Fruit Set

Flowering and vegetative shoots of ‘Shamouti’ orange[Citrus sinensis (L.) Osbeck] and ‘Marsh’ seedlessgrapefruit (Citrus paradisi Macf.) were examined for correlationof their morphology and anatomy with fruit set. Fruit set isfavoured on leafy inflorescences whereas abortion is nearlycomplete on leafless inflorescences. Leafless inflorescencesof ‘Shamouti’ with one flower were found to havea very thin stem which contained few vascular bundles, whereasthose with three flowers had better-developed vascular systems.The vascular system of leafy inflorescences is significantlydifferent from that of leafless ones and contains a distinctcentral xylem cylinder. The vascular area of leafless inflorescencesis only about one-quarter of that of the leafy ones. The vascularsystem of grapefruit resembles that of the ‘Shamouti’orange. This study emphasizes the importance of the dimensionof the vascular system for fruit set and provides a possibleexplanation for the better fruit set on both leafy and leaflessinflorescences with several flowers compared with single-floweredinflorescences. Anatomy; citrus; fruit set; leafless inflorescence; leafy inflorescence; pedicel; vascular system; vegetative shoot     

Comparison Between the Effect of the Fruit and of Exogenous GA3-Applications on Source-Sink Relationships in Citrus Sprouts

Inflorescence leaves of citrus (Citrus sinensis L. Osbeck) accumulatecarbohydrate reserves at the beginning of the fruit set period.This effect is mimicked by exogenous GA₃ applications in deflorateinflorescences. The effect of the hormone on two processes thatcould result in carbohydrate accumulation, i.e. increased ribulosebisphosphate carboxylase (RuBPCase) activity and changes inthe import/export balance of assimilates in the sprouts, wasstudied. Neither RuBPCase activity nor respiration rates were affectedby the hormonal treatment. On the other hand, blocking of thephloem connections with the rest of the plant induced an increasein carbohydrate contents in the leaves that was proportionallylower in GA₃-treated sprouts than in control deflorate inflorescences.This increase was minimal in leaves from inflorescences at laterstages of fruit development. This was in spite of a lower fruitgrowth rate that, in turn, brought about a decrease in totaldry weight of the sprout. In contrast, total dry weight washigher in girdled deflorate inflorescences, whether or not theywere treated with the hormone. The presence of the fruit induceda thickening of the stems of intact inflorescences that wasalso seen in ungirdled GA₂-treated sprouts. This increase indry weight per unit length, however, had a different chemicalbasis depending upon whether it was caused by the fruit or thehormone. From these data, the possible causes for the enhancement ofcarbohydrate accumulation in inflorescence leaves, and the involvementof gibberellin in the effect of fruit on the sprouts is discussed. Carbohydrates, Citrus sinensis L. Osbeck, gibberellic acid, girdling, orange, ribulose bisphosphate carboxylase, source-sink     

Histological and Physiological Characterization of Rind Breakdown of 'Navelate' Sweet Orange

Rind breakdown of ‘Navelate’ sweet orange is characterizedby sunken colourless areas of the peel which develop into reddish-brown,dry areas partially covering the exposed portion of the maturefruit. Sudden changes in relative humidity at fruit colour breakseem to be responsible for the natural development of this disorder,which begins at the transitional zone of the flavedo-albedoand advances across the flavedo reaching the epidermis. Affectedcells have reduced amounts of cytoplasm located in a centralposition and have twisted and squashed walls, forming areasof collapsed cells amongst the healthy cells of the flavedoand albedo. Comparisons of healthy and damaged areas of affectedfruits showed no significant differences in wax morphology andcuticular thickness or permeability. Our results suggest thatan excessive loss of water from hypodermal and albedo cellsis responsible for the disorder. Copyright 2001 Annals of BotanyCompany Citrus, physiological disorder, cell collapse, rind blemish, fruit quality     

Photosynthesis and Diffusion Conductance of the Valencia Orange Fruit under Field Conditions

CO₂ uptake and diffusion conductance of Valencia orange fruits(Citrus sinensis L. Osbeck) were measured in the field duringthe growing season of 1977/78 to ascertain if, as in the leaf,stomata control photosynthesis and transpiration under changingenvironmental conditions. Measurements were made on 15 yearold trees grown in a sandy loam soil and receiving either adry or a wet treatment. Fruit diffusive conductance was measuredwith a modified water vapour diffusion conductance meter andgross photosynthesis was measured with a ¹⁴CO₂ uptake meter.Photosynthetically active radiation (PAR) was measured witha quantum sensor. Fruits exposed to light assimilated CO₂ ata rate which was 25–50% of that assimilated by leaves.The uptake was dependent on fruit size, PAR, chlorophyll content,and on diffusive conductance of the fruit epidermis. Epidermalconductance showed a diurnal trend which was similar in shapeto that of the leaf except in the late afternoon. Cuticularconductance of the fruit was calculated and ranged between 0.22and 0.30 mm s^–1. It was speculated that the CO₂ uptakeby the fruit could support the growth of flavedo cell layerswhen exposed to light. Dry soil caused an increase in the ¹⁴CO₂uptake by fruit possibly caused by the increased potential areaof the stomatal opening per unit of fruit surface area.     

The Dormancy of Buds of Citrus sinensis (L.) Osbeck Inserted into Rootstock Stems: Factors Intrinsic to the Inserted Bud

Buds of sweet orange, harvested from shoots of different timeof flushing and from different positions along the shoot, wereused to examine whether lack of burst of inserted buds was acharacteristic of the bud. Bursting of inserted buds was significantlyslower in buds taken from (a) older branches (b) shoots producedunder winter conditions, and (c) basal rather than apical budson the same shoot. The slowness to burst when transferred matched a tendency todormancy in buds on shoot segments grown in vitro, suggestingthat the variation in budburst was intrinsic to the bud. Budburstwas correlated with the extent of secondary bud development;the majority of buds from apical regions of the shoot had developeda secondary bud by the time of implantation, but basal budshad not. Adequate vascular connections with the host tissueswere found in both burst and unburst buds. Citrus sinensis (L.) Osbeck, sweet orange, buds, endodormancy, budding     

Carbohydrate Availability in Relation to Fruitlet Abscission in Citrus

Abscission of flowers and fruitlets in the Washington navelorange (Citrus sinensis [L.] Osbeck) has been characterizedin relation to carbohydrate availability. A main wave of flowerabscission occurs shortly after anthesis while the carbohydratereserves in the tree are high. Fruitlet abscission starts approx.30 d after the commencement of flowering, while carbohydrates(mainly starch) are being accumulated in the leaves. Flowerand early fruitlet abscission are not caused by carbohydrateshortage. During late fruitlet abscission sucrose concentrationin the leaves falls to a low value demonstrating a limitationin supply and competition among the developing fruitlets forcarbohydrates. Concentrations of sucrose and reducing sugarsin the peel of the fruitlets also fall to low values, and arelationship could be demonstrated between these free sugarlevels and abscission. Ringing increases carbohydrate supplyto fruit and reduces late fruitlet abscission, but only hasa marginal effect on the growth of the fruitlets, which seemsless sensitive than abscission to carbohydrate shortage. Thelimitation of carbohydrate supply to the fruitlets occurs whilestarch levels in the leaves remain high. Slow mobilization ofstarch reserves may be one factor limiting set in Citrus. Copyright2001 Annals of Botany Company Carbohydrate supply, citrus, fruit growth and abscission, ringing, navel orange, starch, sugar metabolism     

Effects of Volatiles Evolved from Excised Plant Tissues and Lemon Oil on the Growth of Mung Bean Seedlings

Volatiles evolved from excised segments of leaves of oleander(Nerium oleander) and cedar (Cedrus deodara), peels of lemon(Citrus limon), orange (Citrus sinensis) and lime (Citrus aurantifolia),and roots of wasabi (Wasabia japonica) inhibited growth of mungbean seedlings. The volatiles evolved from lemon oil also markedlyinhibited elongation of mung bean seedlings. The active constituent(s)in the volatiles were distinct from ethylene. The mode of inhibitionby the volatiles from lemon oil was different from that of ethylene.The volatiles from lemon oil alleviated the inhibitory effectof ethylene on the growth of seedlings. The volatiles from lemonoil supressed the formation of lamellar structure in chloroplasts,thus resulting in etiolation of seedlings. (Received October 25, 1982; Accepted December 29, 1982)     

Symptomatic expression of tristeza-infected citrus plants in Pakistan

Variable symptoms were recorded during a survey in the citrus trees infected or suspected to be infected with citrus tristeza virus based on ELISA tests. Sweet orange manifested main symptoms like bud union crease, pin holing, bark cracking, incompatibility, yellowing and dropping of leaves. Pin holing was particularly prevalent in Sweet orange (Citrus sinensis Osbeck), Kinnow (Citrus reticulata Blanco) and Grapefruit (Citrus paradisii Mcaf.), while incompatibility was only in Sweet orange and Grapefruit.     

Changes in cellulase and pectinase activities in fruit tissues and separation zones of citrus treated with cycloheximide

Cellulase activity increased in separation-zone tissues 1 day after “Valencia” orange (Citrus sinensis [L.] Osbeck) was treated with 20 micrograms per milliliter cycloheximide. Exocellulase was detected only in the separation zones of treated fruit, whereas endocellulase was present in zones from both treated and control fruit. Endocellulase activity in separation-zone tissue of treated fruit was nearly three times as great as that in control tissues. Cellulase activity was restricted to separation-zone tissue. Pectinase and an albedo-macerating factor activity were very low and were not influenced by the treatment. The cycloheximide effect in these experiments was apparently caused by ethylene produced by wound tissue.     

Oviposition behaviour and larval development of Anastrepha fraterculus from Argentina in citrus

Citrus peel physicochemical attributes are considered the main components conferring partial or even total resistance to fruit fly (Diptera: Tephritidae) infestation. Fruit fly females adapt their ovipositional strategies to overcome such resistance. Here, we explored the effects of citrus species (Rutaceae) on the ovipositional behaviour of the South American fruit fly, Anastrepha fraterculus (Wiedemann), and on its immature development. Particularly, we investigated the effects of (1) citrus species on oviposition behaviour and immature development, (2) citrus species on oviposition preference and on the location of the eggs at different depth in the citrus peel, and (3) harvest season and post‐harvest storage time on oviposition behaviour and immature development in lemon. Citrus species influenced ovipositional behaviour and affected survival of immature stages. Females laid eggs in lemon [Citrus limon (L.) Burm.], orange [Citrus sinensis (L.) Osbeck], and grapefruit (Citrus paradisi Macfadyen). In orange and lemon, larvae were found dead close to the oviposition areas, suggesting chemically mediated resistance mechanisms. Under choice conditions, females preferred grapefruit over lemon and bigger clutches were found in the layers where embryonic development is favoured. Unsuitability of lemon as a medium to complete development was neither affected by harvest season nor by storage time of the fruit after harvest. The physical and chemical characteristics of the peel were distinctive to each citrus species and may have affected the specific levels of resistance of these citrus species to infestation by A. fraterculus.     

Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage

Regeneration and transformation systems from mature plant material of woody fruit species have to be achieved as a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of resulting horticultural traits. We report here, for the first time, a procedure for genetic transformation and regeneration of mature tissues of woody plants that overcomes the long juvenile periods and high heterozygosity that are characteristic of most of these species. An improved regeneration frequency from mature explants was obtained by invigoration of the plant material through grafting of mature buds on juvenile seedlings. Co-cultivation of the explants in feederplates after inoculation with Agrobacterium tumefaciens resulted in enhanced transformation frequencies. Furthermore, in vitro shoot-tip grafting of the regenerated mature shoots on seedling rootstocks provided a rapid and efficient system for plant production. Citrus is the most extensivel y grown fruit crop worldwide and sweet orange (Citrus sinensis L. Osbeck) accounts for approximately 70% of the Citrus total production. Mature transgenic sweet orange plants have been obtained, which flowered and bore fruit in 14 months     

The Occurrence of Abscisic Acid and Abscisyl-beta-d-Glucopyranoside in Developing and Mature Citrus Fruit as Determined by Enzyme Immunoassay

The contents of (+)-cis-abscisic acid (ABA) and alkaline-hydrolyzable ABA-conjugate(s) were analyzed by means of enzyme immunoassay in partially purified extracts of developing and mature sweet orange fruit (Citrus sinensis [L.] Osbeck cv Washington navel). A relatively small increase in ABA was observed in the fruit exocarp during the natural color transition from green to orange. At the same time, the ABA-conjugate level increased approximately 12-fold in this tissue. The contents of ABA and ABA-conjugate equaled 15.0 ± 0.7 and 107.8 ± 2.1 nanomoles per gram fresh weight, respectively, in the exocarp at harvest. Other tissues also contained considerable quantities of these compounds. Whereas the highest ABA content was observed in the exocarp, the highest ABA-conjugate content was observed in the central vascular axis of the fruit and equaled 187.0 ± 10.3 nanomoles per gram fresh weight. The only immunoreactive conjugate found in significant quantity in mature fruit was identified as abscisyl-β-d-glucopyranoside (ABA-GE) based on (a) immunological cross-reactivity, (b) thin layer chromatography co-chromatography with authentic standards in two solvent systems, (c) susceptibility to both chemical and enzymic degradation, and (d) mass spectroscopy.     

Chlorophyllase activity in chlorophyll-free citrus chromoplasts

Chromoplast fractions from mature, chlorophyll-less ‘Valencia’ orange (Citrus sinensis L. Osbeck) flavedo (= the outer coloured layer of citrus peel) showed considerable chlorophyllase activity. Acetone powders prepared from chromoplast fractions had 2.5× higher specific activity than those prepared from whole flavedo. Exposure of mature, chlorophyll-less fruit to ethylene caused a 2.5 to 4.0 fold increase in chlorophyllase activity. Juice chromoplasts showed negligible chlorophyllase activity. The results suggest that chlorophyllase activity as well as its induction by ethylene are not dependent upon the presence of chlorophyll in the tissue.     

Phytohormone Changes and Carbohydrate Status in Sweet Orange Fruit from Huanglongbing-infected Trees

Huanglongbing (HLB) infection alters citrus fruit growth and development, resulting in small, misshapen, and poorly colored fruit containing aborted or partially developed seeds. Typically, symptomatic fruit have delayed maturation and abscise prematurely. We studied carbohydrate and phytohormone changes in HLB-affected fruit to explain symptom development because (1) carbohydrate shortage has been linked to fruit growth arrest and eventually abscission and (2) hormonal signals regulate, at least partially, fruit set and development. Symptomatic fruit (S), asymptomatic fruit (AS) from symptomatic trees, and healthy fruit (H) from asymptomatic trees were harvested from ‘Valencia’ sweet orange trees [Citrus sinensis (L.) Osbeck] infected with the HLB pathogen or not, as verified by PCR. Mature S weighed less, had lower °Brix, were smaller, had more aborted seeds, and were greener than AS or H. Starch and sucrose contents were lower in mature S flavedo compared with that of H and AS. S and AS harvested 7 and 12 months after full bloom produced significantly less ethylene than H. Indole-3-acetic acid (IAA) and abscisic acid (ABA) contents in flavedo removed from the stylar end, middle section, or stem end of fruit generally were higher in S flavedo than in AS and H. ABA content was fourfold higher in flavedo from the middle section of S than in AS and H. Flavedo excised from the large shoulder of misshapen S had significantly higher IAA content when compared with the normal-sized area of the same fruit on the opposite side. This increase corresponded to an increase in hypodermal cell area in S flavedo. Overall, these data reveal an imbalance of carbohydrate and phytohormone status in fruit from HLB-infected trees and suggest a role of such changes in fruit symptom development.     

Engineering d‐limonene synthase down‐regulation in orange fruit induces resistance against the fungus Phyllosticta citricarpa through enhanced accumulation of monoterpene alcohols and activation of defence

Terpene volatiles play an important role in the interactions between specialized pathogens and fruits. Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, is associated with crop losses in different citrus‐growing areas worldwide. The pathogen may infect the fruit for 20–24 weeks after petal fall, but the typical hard spot symptoms appear when the fruit have almost reached maturity, caused by fungal colonization and the induction of cell lysis around essential oil cavities. d ‐Limonene represents approximately 95% of the total oil gland content in mature orange fruit. Herein, we investigated whether orange fruit with reduced d ‐limonene content in peel oil glands via an antisense (AS) approach may affect fruit interaction with P. citricarpa relative to empty vector (EV) controls. AS fruit showed enhanced resistance to the fungus relative to EV fruit. Because of the reduced d ‐limonene content, an over‐accumulation of linalool and other monoterpene alcohols was found in AS relative to EV fruit. A global gene expression analysis at 2 h and 8 days after inoculation with P. citricarpa revealed the activation of defence responses in AS fruit via the up‐regulation of different pathogenesis‐related (PR) protein genes, probably as a result of enhanced constitutive accumulation of linalool and other alcohols. When assayed in vitro and in vivo, monoterpene alcohols at the concentrations present in AS fruit showed strong antifungal activity. We show here that terpene engineering in fruit peels could be a promising method for the development of new strategies to obtain resistance to fruit diseases.     

Histological characterization of in vitro adventitious organogenesis in Citrus sinensis

The adventitious bud development was induced in epicotyl segments of Valencia sweet orange (Citrus sinensis L. Osbeck). Seeds were cultured in vitro for three weeks in the dark, followed by one week at a 16-h photoperiod. Epicotyl segments were cultured horizontally for the induction of organogenesis in Murashige and Tucker (1969, MT) culture medium supplemented with 1.0 mg dm⁻³ benzylaminopurine. Samples were observed by light and scanning electron microscopy from day zero to day 25, when buds were well grown. It was shown that the adventitious buds originated directly from the cambial region on the cut ends of the explants.     

Programmed cell death of secretory cavity cells in fruits of Citrus grandis cv. Tomentosa is associated with activation of caspase 3-like protease

Previously, we found that secretory cell degradation typically occurred through programmed cell death during secretory cavity development in Citrus sinensis L. (Osbeck). This finding indicated that secretory cavities could be utilized as a new cell biology model for investigating the regulatory mechanisms of plant programmed cell death. To study further the programmed cell death during secretory cavity development in Citrus fruit, we studied the morphogenetic characteristics of secretory cavities during their development in Citrus grandis cv. Tomentosa. Using light microscope- and electron microscope-TUNEL assays, immunohistochemistry and immunocytochemistry, we described the precise spatial and temporal alterations in caspase 3-like distribution, chromatin condensation and DNA fragmentation during the programmed cell death of secretory cavity cells. Caspase 3-like was found to be significantly located in both the cytoplasm and the nucleus of secretory cavity cells undergoing programmed cell death, and caspase 3-like is closely associated with chromatin condensation and DNA fragmentation. Interestingly, both caspase 3-like and DNA fragmentation were detected in the nucleoli. Our findings suggest that caspase 3-like may be involved in the programmed cell death of secretory cavity cells, especially in chromatin condensation, DNA fragmentation, nuclear degradation and the degradation of certain organelles.