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Comparative analysis on the distribution of heterochromatin in Citrus, Poncirus and Fortunella chromosomes.

Double fluorochrome staining with chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI) was used to characterize and compare the distribution of constitutive heterochromatin along chromosomes of Citrus, Poncirus and Fortunella species. Only CMA-positive bands were distinguishable in metaphase chromosomes. Preferential distribution of heterochromatin in terminal regions, mainly of the long arm, and centromeric regions of a few long chromosomes was a common feature of these genera. Heteromorphism between possible homologous chromosomes was present in the majority of species. Citrus and Poncirus revealed some remarkably uniform chromosomes without any intensively fluorescing region, whereas Fortunella cultivars were differentiated by the presence of CMA bands in all chromosomes. Through measurements assisted by a computer, amounts of CMA-positive regions were shown to be highest in Fortunella. Similarities between Citrus and Poncirus suggest little heterochromatin diversification among karyotypes of these genera, whereas Fortunella, with higher amounts and more homogenous distribution of heterochromatin, is more divergent.

Chromomycin A3↗

Effect of insecticides and fungicides on the interaction between members of the mite families Phytoseiidae and Stigmaeidae on citrus.

The study was carried out to evaluate the effect of some insecticides and fungicides on the interaction between predacious mites of the families Phytoseiidae and Stigmaeidae on citrus. Euseius citrifolius Denmark and Muma, Euseius concordis (Chant) (Phytoseiidae), and Agistemus aff. bakeri (Stigmaeidae) were the most abundant species of predacious mites. No significant reduction of the population of stigmaeids was detected following the pesticide treatment. On the contrary, the population of those mites increased after the application of methidathion, petroleum oil, deltamethrin, cupric oxychloride and cuprous oxide, as compared to the population of those mites in the control plots. The chemicals promoting increased stigmaeid populations are some of those that promoted reduction of phytoseiid populations. A significant negative correlation was found between the numbers of phytoseiids and stigmaeids on citrus leaves immediately before treatments, and 33, 50, 83 and 105 days after treatment. A clear negative linear correlation between the number of predacious mites from both families was detected for benomyl, cupric oxychloride, cuprous oxide, thiophanate methyl, and control. Phytoseiid-stigmaeid interaction on citrus orchard is discussed.

Animals↗

Resistance to hexythiazox in Brevipalpus phoenicis (Acari: Tenuipalpidae) from Brazilian citrus.

The objective of this study was to collect baseline information for implementing an acaricide resistance management program of Brevipalpus phoenicis (Geijskes) to hexythiazox in Brazilian citrus groves. The egg susceptibility of B. phoenicis to hexythiazox was measured by a direct contact bioassay. The estimated LC50 for the S strain was 0.89 mg hexythiazox L(-1) of water (95% FL 0.75-1.03). After hexythiazox selection of a field-collected population associated with intense hexythiazox use, a resistance ratio greater than 10,000-fold was detected. Results from a survey revealed a great variability in the frequency of resistance in populations of B. phoenicis collected from citrus groves located in the State of São Paulo. No relationship was observed between the intensity of hexythiazox use and the frequency of resistance. Studies on dynamics of resistance showed that the resistance of B. phoenicis to hexythiazox is stable under laboratory conditions. Therefore, there is an urgent need to implement resistance management of B. phoenicis to hexythiazox in order to prolong its effective use in Brazilian citrus groves.

Animals↗

Sensitivity to Alternaria alternata toxin in citrus because of altered mitochondrial RNA processing.

Specificity in the interaction between rough lemon (Citrus jambhiri Lush.) and the fungal pathogen Alternaria alternata rough lemon pathotype is determined by a host-selective toxin, ACR-toxin. Mitochondria from rough lemon are sensitive to ACR-toxin whereas mitochondria from resistant plants, including other citrus species, are resistant. We have identified a C. jambhiri mitochondrial DNA sequence, designated ACRS (ACR-toxin sensitivity gene), that confers toxin sensitivity to Escherichia coli. ACRS is located in the group II intron of the mitochondrial tRNA-Ala and is translated into a SDS-resistant oligomeric protein in C. jambhiri mitochondria but is not translated in the toxin-insensitive mitochondria. ACRS is present in the mitochondrial genome of both toxin-sensitive and -insensitive citrus. However, in mitochondria of toxin-insensitive plants, the transcripts from ACRS are shorter than those in mitochondria of sensitive plants. These results demonstrate that sensitivity to ACR-toxin and hence specificity of the interaction between A. alternata rough lemon pathotype and C. jambhiri is due to differential posttranscriptional processing of a mitochondrial gene.

Base Sequence↗

Effects of 15N application frequency on nitrogen uptake efficiency in citrus trees.

Two irrigation systems were used to compare nitrogen uptake efficiency in citrus trees and to evaluate the NO3- runoff in "Navelina" orange trees [Citrus sinensis (L.) Osbeck] on Carrizo citrange rootstock (Citrus sinensis x Poncirus trifoliata Raf.). These were fertilized with 125 g N as labelled K15NO3 and grown outdoors in containers filled with a sand-loamy soil. Two groups of 3 trees received this N dose either in five equally split applications by a flooding irrigation system or in 66 applications by drip. Trees were harvested at the end of the vegetative cycle (December) and the isotopic ratios of 15N/14N were measured in the soil-plant system. The N uptake efficiency of the whole tree was higher with drip irrigation (75%) than with flooding system (64%). In the 0-90 cm soil profile, the N immobilized in the organic fraction was similar for both irrigation methods (around 13 %), whereas the N retained as NO3- was 1% of the N applied under drip and 10% under flooding. In the last case, most of NO3- remained under root system and it could be lost to leaching either by heavy rainfalls or excessive water applications. These results showed that a drip irrigation system was more efficient for improving water use and N uptake from fertilizer, in addition to potentially reduced leaching losses.

Citrus↗

Natural enemies of three mealybug species (Hemiptera: Pseudococcidae) found on citrus and effects of some insecticides on the mealybug parasitoid Coccidoxenoides peregrinus (Hymenoptera: Encyrtidae) in South Africa.

The population density of mealybug species in some South African citrus orchards has increased to pest status in recent years. The characterization of the natural enemy complex and quantification of their contribution to the control of Planococcus citri (Risso), Pseudococcus longispinus (Targioni-Tozzetti) and Pseudococcus calceolariae (Maskell) on Citrus limon (L.) and Citrus reticulata (Blanco) was investigated through intensive sampling. Eight primary and four secondary parasitoids, and two predator species were identified from P. citri and P. calceolariae. Anagyrus pseudococci (Girault) and Coccidoxenoides peregrinus (Timberlake) were the most common species, accounting for 44% and 21% of the total. Of the five primary parasitoids reared from P. longispinus, A. pseudococci and Anagyrus sp. were predominant, comprising 41% and 30%. Nymphal and adult parasitism (range = 0-26% vs. 0-66%) and predation (range = 0-5.6% vs. 0-4.1%) varied significantly between host trees and mealybug species (P < 0.001). The numbers of nymphal instars and adult stages of P. calceolariae and P. longispinus and the nymphal stage of P. citri that were parasitized and killed by predators correlated significantly with the total number of hosts on which they acted (P < 0.01), suggesting a density-dependent association. Laboratory bioassay of nine contact insecticides (methidathion, methomyl, methyl-parathion, parathion, profenofos and prothiofos) against C. peregrinus indicated that all were highly toxic, causing 98-100% mortality in < 6 h of treatment. The IGRs fenoxycarb and triflumuron did not cause significant parasitoid mortality (P > 0.05). However, a mixture of pyriproxyfen and mineral oil caused a marginally significant mortality (P < 0.05).

Animals↗

Cover crops and natural vegetation mulch effect achieved by mechanical management with lateral rotary mower in weed population dynamics in citrus.

The aim of this study was to obtain information on practical weed management in order to reduce the use of herbicides thereby contributing to the sustainable development of citrus crop. The experiment was carried out under field conditions at the experimental area of the Department of Vegetal Production at the College of Agriculture "Luiz de Queiroz," Piracicaba, SP, Brazil, during the season 2002-2003. Influence of mulches produced by four types of vegetations on the dynamic population of weeds in the line of citrus crop (Citrus sinensis (L.) Osbeck) was evaluated. The experimental design was of factorial randomized blocks (4 x 2), where the treatments were: (i) four types of vegetation: Dolichos lablab L., Cajanus cajan (L.) Millsp, Penisetum glaucum (L.) Leeke, and the natural infestation composed basically by Panicum maximum Jacq.; and (ii) two types of fertilization: directed under canopy and broadcast. Mechanical management of the different vegetations was accomplished using a lateral rotary mower, KAMAQ, I model NINJA MAC 260, projected to release the green cut material under crop canopy, forming a mulch layer. The studied parameters were: (i) counting of weeds per m2 in the crop line after 30, 60, 90, 180, and 210 days following the cutting of existing vegetation; (ii) percentage of covered area by weeds; and (iii) some chemical properties of the soil. It was observed that the natural infestation showed a better weed control when compared with the other treatments, and that the broadcast fertilization, regardless of coverage used, presented a lower number of weeds.

Agriculture↗

Early events in Agrobacterium-mediated genetic transformation of citrus explants.

BACKGROUND AND AIMS: Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so-called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co-cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co-cultivation medium as possible enhancers of gene transfer was also studied. METHODS: A highly responsive citrus genotype and well-established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co-cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry. KEY RESULTS: It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants. Furthermore, co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase, the stage in which cells are more prone to integrate foreign DNA. CONCLUSIONS: Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.

Agrobacterium tumefaciens↗

Effects of aluminum on light energy utilization and photoprotective systems in citrus leaves.

BACKGROUND AND AIMS: Under high photon flux, excitation energy may be in excess in aluminum (Al)-treated leaves, which use a smaller fraction of the absorbed light in electron transport due to decreased CO2 assimilation compared with normal leaves. The objectives of this study were to test the hypothesis that the antioxidant systems are up-regulated in Al-treated citrus leaves and correlate with protection from photoxidative damage, and to test whether xanthophyll cycle-dependent thermal energy dissipation is involved in dissipating excess excitation energy. * METHODS: 'Cleopatra' tangerine seedlings were fertilized and irrigated daily for 8 weeks with quarter-strength Hoagland's nutrient solution containing Al at a concentration of 0 or 2 mM from Al2(SO4)3.18H2O. Thereafter, leaf absorptance, chlorophyll (Chl) fluorescence, Al, pigments, antioxidant enzymes and metabolites were measured on fully expanded leaves. * KEY RESULTS: Compared with control leaves, energy was in excess in Al-treated leaves, which had smaller thermal energy dissipation, indicated by non-photochemical quenching (NPQ). In contrast, conversion of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z) at midday increased in both treatments, but especially in Al-treated leaves, although A + Z accounted for less 40 % of the total xanthophyll cycle pool in them. Activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and catalase (CAT), and concentrations of ascorbate (AsA), dehydroascorbate (DASA), reduced glutathione (GSH) and oxidized glutathione (GSSG) were higher in Al-treated than in control leaves. * CONCLUSIONS: These results corroborate the hypothesis that, compared with control leaves, antioxidant systems are up-regulated in Al-treated citrus leaves and protect from photoxidative damage, whereas thermal energy dissipation was decreased. Thus, antioxidant systems are more important than thermal energy dissipation in dissipating excess excitation energy in Al-treated citrus leaves.

Aluminum↗

Monitoring butocarboxim resistance of the woolly whitefly (Homoptera: Aleyrodidae) in citrus from Valencia, Spain.

Glass vials coated internally with an insecticide were used as a resistance monitoring technique for testing field populations of the woolly whitefly, Aleurothrixus floccosus (Maskell), collected from citrus. The distribution of resistance to the insecticide butocarboxim in citrus orchards from Valencia (Spain) during 1993 and 1994 was determined by means of this technique. Adults resting on citrus shoots were captured with a portable vacuum cleaner and introduced into the vials. The technique provides control mortality of <25% when assessed 7 h after the insects are captured. In 21 populations tested, LC50s ranged from 1.8 to 42.3 mg/ml. This represents resistance ratios >20-fold among populations. Lower slopes of the concentration-mortality line were found in whitefly populations that exhibited a low level of the LC50. Resistance was widespread in the Valencia areabut spatially irregular, with nearby orchards occasionally showing wide differences in resistance levels. The levels of resistance to butocarboxim obtained with this technique closely matched the field efficacy of the insecticide. This residue bioassay provides a convenient and rapid method to monitor insecticide resistance in A. floccosus populations.

Aldicarb↗

The chromosomes of Citrus and Poncirus species and hybrids: identification of characteristic chromosomes and physical mapping of rDNA loci using in situ hybridization and fluorochrome banding.

In situ hybridization of 18S-5.8S-25S rDNA probes labeled with biotin or rhodamine and 5S rDNA probes labeled with digoxigenin was used to locate rDNA sites on root-tip metaphase chromosomes of Citrus sinensis L. (2n = 2x = 18), Poncirus trifoliata L. Raf. (2n = 2x = 18), and Citrus x Poncirus hybrids (2n = 2x = 18). Counterstaining with the fluorochromes chromomycin A3 and DAPI uniquely identified many but not all chromosomes. C. sinensis had five 18S-25S rDNA sites, P. trifoliata had seven, and three different Citrus x Poncirus hybrids had five or six sites. Four 5S rDNA sites were detected, mostly linked to 18S-25S rDNA sites. Overall we observed high levels of chromosomal heterozygosity in all accessions examined.

Chromosome Banding↗

Citrus flavonoids stimulate secretion by human colonic T84 cells.

Flavonoids, compounds containing a 2-phenylbenzo(gamma)pyrane nucleus, are universally distributed among vascular plants. Even though flavonoids are ingested in a normal diet in average quantities of 1 g daily, their effects on the digestive system have only been recently investigated. This study used an in vitro model of colonic secretion, monolayers of T84 colonic adenocarcinoma cells mounted in Ussing chambers, to demonstrate that 100 mumol/L of either tangeritin or nobiletin, polymethoxylated flavonoids contained in citrus fruits, stimulated sustained electrogenic chloride secretion with a maximal short-circuit current of 3.3 microA/cm2. In contrast, naringin and hesperidin, glycosylated citrus flavonoids, stimulated minimal secretion, suggesting that carbohydrate substitutions inhibited their secretory potential. The secretion stimulated by tangeritin and nobiletin was synergistic with carbachol but not with vasoactive intestinal peptide and was inhibited by barium chloride, bumetanide, H-89, and Cl- depletion. These properties suggest that tangeritin and nobiletin stimulated Cl- secretion via the cAMP pathway; however, these flavonoids did not stimulate cAMP production to the extent seen with vasoactive intestinal peptide. These flavonoids did not autooxidize, suggesting that reactive oxygen species did not mediate this secretion. These observations suggest that dietary citrus flavonoids may modulate colonic secretion, possibly through direct interaction with intracellular secretory pathways.

Adenocarcinoma↗

Involvement of ethylene biosynthesis and perception in the susceptibility of citrus fruits to Penicillium digitatum infection and the accumulation of defence-related mRNAs.

Citrus fruits infected with the fungus Penicillium digitatum substantially increase the production of the plant hormone ethylene. In this study, the regulation of ethylene biosynthesis in Citrus sinensis-infected fruits and its putative involvement in an active defence response against P. digitatum infection is examined. Ethylene production is demonstrated as being the result of the co-ordinated and differential up-regulation of at least three ethylene biosynthetic genes: ACS1, ACS2, and ACO. Blocking ethylene perception by 1-MCP resulted in an increased ethylene production and ACS2 expression during infection and mechanical wounding, suggesting that this gene is negatively regulated by ethylene. ACO expression was induced by ethylene in the absence of wounding or infection, although further results indicate that its induction during the course of infection may not be primarily mediated by ethylene. Treatment with 1-MCP also increased susceptibility to Penicillium decay, showing an involvement of ethylene perception in promoting defence responses in citrus fruits. The changes in the expression of two defence-related genes up-regulated during infection were also studied: the ones coding for phenylalanine ammonia-lyase (PAL) and an acidic class II chitinase (ACR311). The onset of PAL expression after mechanical wounding or inoculation was not changed in 1-MCP-pretreated fruits, while its later increase during the course of infection was abolished. Chitinase gene induction was more related to mechanical damage and was partially repressed by ethylene. These studies indicate distinct possible regulatory mechanisms of plant fruit defence genes in the context of fungal infection and ethylene perception.

Citrus sinensis↗

Fruit set dependence on carbohydrate availability in citrus trees.

We generated source-sink imbalances by defoliation and sucrose supplementation by stem injection, to investigate effects of carbohydrate availability on fruitlet growth and abscission in cv. Okitsu of Satsuma mandarins (Citrus unshiu (Mak.) Marc.). Partial defoliation promoted fruitlet abscission, whereas sucrose supplementation increased citrus fruit set by more than 10%. Moreover, when applied together, sucrose supplementation counteracted the effect of partial defoliation on fruit set. When sucrose was supplied continuously from flowering until harvest, it increased the concentrations of soluble and insoluble sugars in fruits. We conclude that fruit set in citrus is highly dependent on carbohydrate availability.

Carbohydrate Metabolism↗

Endopolygalacturonase is essential for citrus black rot caused by Alternaria citri but not brown spot caused by Alternaria alternata.

Alternaria citri, the cause of Alternaria black rot, and Alternaria alternata rough lemon pathotype, the cause of Alternaria brown spot, are morphologically indistinguishable pathogens of citrus: one causes rot by macerating tissues and the other causes necrotic spots by producing a host-selective toxin. To evaluate the role of endopolygalacturonase (endoPG) in pathogenicity of these two Alternaria spp. pathogens, their genes for endoPG were mutated by gene targeting. The endoPGs produced by these fungi have similar biochemical properties, and the genes are highly similar (99.6% nucleotide identity). The phenotypes of the mutants, however, are completely different. An endoPG mutant of A. citri was significantly reduced in its ability to cause black rot symptoms on citrus as well as in the maceration of potato tissue and could not colonize citrus peel segments. In contrast, an endoPG mutant of A. alternata was unchanged in pathogenicity. The results indicate that a cell wall-degrading enzyme can play different roles in the pathogenicity of fungal pathogens. The role of a cell wall-degrading enzyme depends upon the type of disease but not the taxonomy of the fungus.

Alternaria↗

Lack of kinetic interaction between valproic acid and citrus pectin.

To determine the influence of ingestion of dietary fiber (citrus pectin) in the kinetic disposition of valproic acid, two studies were conducted in adult volunteers. Twelve healthy subjects took single oral doses of 500 mg valproic acid in a form of sodium valproate as coated tablets, on two occasions: under fasting conditions (treatment A) or immediately after ingestion of 14 g citrus pectin (treatment B). In each study, a randomized, single-dose, two-way crossover design was used, and 8 days elapsed between the two trials. Assays were carried out for valproic acid in plasma samples, which were collected from zero to 48 hours after drug administration. The peak plasma concentration (60.52 vs. 55.74 micrograms x ml-1), the time to peak concentration (3.47 vs. 3.91 h), the absorption rate constant (0.12 vs. 0.12 h-1), and the area under the plasma concentration-time curve (1324.0 vs. 1268.5 micrograms x h x ml-1) did not differ significantly between treatments A and B (median, Wilcoxon test). These results suggest that the administration of citrus pectin does not alter the rate and extent of absorption of valproic acid administered as a single dose to healthy volunteers.

Adult↗

Host-directed processing of Citrus exocortis viroid.

Prolonged infection of tomato hybrid (Lycopersicon esculentum x Lycopersicon peruvianum) by Citrus exocortis viroid (CEVd) resulted in viroid-like enlarged structures, detected by gel electrophoresis. This population included two new enlarged variants or D-variants, D-87 and D-76, and three transient species or D-forms, D-38, D-40 and D-43. Sequence analyses exposed a locus near the terminal repeat region where major changes appeared consistently. In transmission tests to CEVd hosts, a variety of progeny populations were recovered, including progeny enlargements of and reversions to CEVd, as well as sequence fidelity to the inoculum. Transmission tests to citrus hosts of the genera Citrus, Poncirus or Fortunella were unsuccessful. The importance of host specificity to the recovery and processing of the various CEVd-related structures, as well as the temporal variability of progeny populations, was demonstrated.

Base Sequence↗

Stable transformation of the Xylella fastidiosa citrus variegated chlorosis strain with oriC plasmids.

Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan(r)). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kan(r) gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.

Base Sequence↗