Xylella fastidiosa: An in vivo system to study possible survival strategies within citrus xylem vessels based on global gene expression analysis

Xylella fastidiosa inhabits the plant xylem, a nutrient-poor environment, so that mechanisms to sense and respond to adverse environmental conditions are extremely important for bacterial survival in the plant host. Although the complete genome sequences of different Xylella strains have been determined, little is known about stress responses and gene regulation in these organisms. In this work, a DNA microarray was constructed containing 2,600 ORFs identified in the genome sequencing project of Xylella fastidiosa 9a5c strain, and used to check global gene expression differences in the bacteria when it is infecting a symptomatic and a tolerant citrus tree. Different patterns of expression were found in each variety, suggesting that bacteria are responding differentially according to each plant xylem environment. The global gene expression profile was determined and several genes related to bacterial survival in stressed conditions were found to be differentially expressed between varieties, suggesting the involvement of different strategies for adaptation to the environment. The expression pattern of some genes related to the heat shock response, toxin and detoxification processes, adaptation to atypical conditions, repair systems as well as some regulatory genes are discussed in this paper. DNA microarray proved to be a powerful technique for global transcriptome analyses. This is one of the first studies of Xylella fastidiosa gene expression in vivo which helped to increase insight into stress responses and possible bacterial survival mechanisms in the nutrient-poor environment of xylem vessels.

Morphological description of the mouthparts of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae)

Scanning (SEM) and transmission (TEM) electron microscopy were used to elucidate the morphology of the rostrum, as well as the mandibular and maxillary stylets of the psyllid Diaphorina cirri, vector of phloem-inhabiting bacteria associated with citrus huanglongbing (HLB) disease. D. cirri has a cone-shaped rostrum that extends behind the pair of prothoracic coxae. The stylet bundle comprises a pair of mandibular (Md) and maxillary (Mx) stylets with a mean length of 513.3 mu m; when retracted, their proximal portions form a loop and are stored in the crumena (Cr). Serial cross-sections of the rostrum revealed that the mandibles are always projected in front of the maxillary stylets. The two maxillary stylets form the food and salivary canals, with diameters of 0.9 mu m and 0.4 mu m respectively. These two canals merge at the end of the stylets forming a common duct with a length of 4.3 mu m and a mean diameter of 0.9 mu m. The acrostyle, a distinct anatomical structure present in the common duct of aphid maxillary stylets, was not observed by TEM in the ultrathin cross-sections of the common duct (CD) of D. citri. This study provides new information on D. citri mouthparts that may help to understand the behaviour of this important vector of HLB-associated bacteria. (C) 2011 Elsevier Ltd. All rights reserved.

Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.

Citrus aurantium L. essential oil exhibits anxiolytic-like activity mediated by 5-HT1A-receptors and reduces cholesterol after repeated oral treatment

Abstract Background The current treatments for anxiety disorders and depression have multiple adverse effects in addition to a delayed onset of action, which has prompted efforts to find new substances with potential activity in these disorders. Citrus aurantium was chosen based on ethnopharmacological data because traditional medicine refers to the Citrus genus as useful in diminishing the symptoms of anxiety or insomnia, and C. aurantium has more recently been proposed as an adjuvant for antidepressants. In the present work, we investigated the biological activity underlying the anxiolytic and antidepressant effects of C. aurantium essential oil (EO), the putative mechanism of the anxiolytic-like effect, and the neurochemical changes in specific brain structures of mice after acute treatment. We also monitored the mice for possible signs of toxicity after a 14-day treatment. Methods The anxiolytic-like activity of the EO was investigated in a light/dark box, and the antidepressant activity was investigated in a forced swim test. Flumazenil, a competitive antagonist of benzodiazepine binding, and the selective 5-HT1A receptor antagonist WAY100635 were used in the experimental procedures to determine the mechanism of action of the EO. To exclude false positive results due to motor impairment, the mice were submitted to the rotarod test. Results The data suggest that the anxiolytic-like activity observed in the light/dark box procedure after acute (5 mg/kg) or 14-day repeated (1 mg/kg/day) dosing was mediated by the serotonergic system (5-HT1A receptors). Acute treatment with the EO showed no activity in the forced swim test, which is sensitive to antidepressants. A neurochemical evaluation showed no alterations in neurotransmitter levels in the cortex, the striatum, the pons, and the hypothalamus. Furthermore, no locomotor impairment or signs of toxicity or biochemical changes, except a reduction in cholesterol levels, were observed after treatment with the EO. Conclusion This work contributes to a better understanding of the biological activity of C. aurantium EO by characterizing the mechanism of action underlying its anxiolytic-like activity.

Pollen analysis, particle size analysis, end-member modelling and x-ray fluorescence measurements from sediment cores off Cape Ghir, Morocco

Understanding past human-climate-environment interactions is essential for assessing the vulnerability of landscapes and ecosystems to future climate change. This is particularly important in southern Morocco where the current vegetation is impacted by pastoralism, and the region is highly sensitive to climate variability. Here, we present a 2000-year record of vegetation, sedimentation rate, XRF chemical element intensities, and particle size from two decadal-resolved, marine sediment cores, raised from offshore Cape Ghir, southern Morocco. The results show that between 650 and 850 AD the sedimentation rate increased dramatically from 100 cm/1000 years to 300 cm/1000 years, and the Fe/Ca and pollen flux doubled, together indicating higher inputs of terrestrial sediment. Particle size measurements and end-member modelling suggest increased fluvial transport of the sediment. Beginning at 650 AD pollen levels from Cichorioideae species show a sharp rise from 10% to 20%. Pollen from Atemisia and Plantago, also increase from this time. Deciduous oak pollen percentages show a decline, whereas those of evergreen oak barely change. The abrupt increase in terrestrial/fluvial input from 650 to 850 AD occurs, within the age uncertainty, of the arrival of Islam (Islamisation) in Morocco at around 700 AD. Historical evidence suggests Islamisation led to population increase and development of southern Morocco, including expanded pastoralism, deforestation and agriculture. Livestock pressure may have changed the vegetation structure, accounting for the increase in pollen from Cichorioideae, Plantago, and Artemisia, which include many weedy species. Goats in particular may have played a dominant role as agents of erosion, and intense browsing may have led to the decline in deciduous oak; evergreen oak is more likely to survive as it re-sprouts more vigorously after browsing. From 850 AD to present sedimentation rates, Fe/Ca ratios and fluvial discharge remain stable, whereas pollen results suggest continued degradation. Pollen results from the past 150 years suggest expanded cultivation of olives and the native argan tree, and the introduction of Australian eucalyptus trees. The rapidly increasing population in southern Morocco is causing continued pressure to expand pastoralism and agriculture. The history of land degradation presented here suggests that the vegetation in southern Morocco may have been degraded for a longer period than previously thought and may be particularly sensitive to further land use changes. These results should be included in land management strategies for southern Morocco.

Sum Insured Determination for Cereal, Citrus and Vineyards in the Spanish Agricultural Insurance System.

In general, insurance is a form of risk management used to hedge against a contingent loss. The conventional definition is the equitable transfer of a risk of loss from one entity to another in exchange for a premium or a guaranteed and quantifiable small loss to prevent a large and possibly devastating loss being agricultural insurance a special line of property insurance. Agriculture insurance, as actually are designed in the Spanish scenario, were established in 1978. At the macroeconomic insurance studies scale, it is necessary to know a basic element for the insurance actuarial components: sum insured. When a new risk assessment has to be evaluated in the insurance framework, it is essential to determinate venture capital in the total Spanish agriculture. In this study, three different crops (cereal, citrus and vineyards) cases are showed to determinate sum insured as they are representative of the cases found in the Spanish agriculture. Crop sum insured is calculated by the product of crop surface, unit surface production and crop price insured. In the cereal case, winter as spring cereal sowing, represents the highest Spanish crop surface, above to 6 millions of hectares (ha). Meanwhile, the four citrus species (oranges, mandarins, lemons and grapefruits) occupied an extension just over 275.000 ha. On the other hand, vineyard target to wine process shows almost one million of ha in Spain.

Study of packing lines for stone fruits and citrus using two instrumented spheres in some cooperatives in the region of Murcia (Spain).

Two instrumented spheres IS 100 were used to evaluate the quality of post-harvest operations. Results obtained from measurements made with both IS (8.8 cm 0 and 6.2 cm 0) show significant differences. Both IS measure the same values of the same variables for soft materials, but not for hard surfaces. Four packing lines belonging to different cooperatives of the region of Murcia (two for stone fruits and two for citrus) were tested. IS values obtained in transfers belonging to the tested lines lay well above 50 g's in most of them Much higher impact intensities are registered in citrus lines than in stone fruit packing lines. To study the incidence of a certain packing line on different products an interaction fruit-packing line test was perf01med. In all cases, more than 50% of fruits belonging to the post-handling sample showed some kind of damage. Bruises evolve after 48 hours storage at room temperature.

Effects of precooling and degreening treatments on the susceptibility of peach and citrus to handling damage.

Mechanical damage to fruits results from a combination of fruit properties and of damage inflicting effects by the handling equipment. Treatments were applied to fruits which affect mechanical damage susceptibility: precooling for stone fruits and degreening to citrus fruits. Laboratory tests (compression, impact, tumbling, abrasion) and field tests (damage in the handling lines) were applied to (3) peach, (2) apricot, (2) orange and (1) lemon varieties. Hydroor-air-cooling influence positively peach and apricot firmness and cause a significant reduction in: number of bruised fruits, and size of visible bruise, when combined with a low level of loading during handling. Degreening also affects the resistance of citrus fruits to handling, when compared to green fruits.

Models for the prediction of handling damage in citrus and peaches grading lines, related to their physical properties.

Fruit damage during harvesting and handling is a standing problem, particularly for susceptible fruits like peaches and apricots. The resulting mechanical damage is a combination of fruit properties and damage inflicting effects due to procedures and to the equipment. Nine packing lines in the region of Murcia (SE Spain) have been tested with the aid of two different-size electronic fruits IS-100. Probabilities of impacts above three preset thresholds (50 g's, 100 g's and 150 g's) were calculated for each transfer point. Interaction fruit-packing line tests have been also performed in order to study the real incidence of packing lines on natural produce: apricots (1 variety), peaches (3 v.), lemons (1 v.) and oranges (3 v.). Bruises of handled and not handled samples of fruits were compared.

`Candidatus Liberibacter asiaticus´ titers in citrus and acquisition rates by Diaphorina citri are decreased by higher temperature

`Candidatus Liberibacter asiaticus´ is the most prevalent Liberibacter sp. associated with huanglongbing (HLB) in Brazil. Within São Paulo state (SP), HLB has spread more rapidly to and reached higher incidence in regions with relatively mild (cooler) summer temperatures. This suggests that climate can influence disease spread and severity. ?Ca. L. asiaticus? titers on soft, immature leaves from infected ?Valencia? sweet orange plants exposed to different temperature regimes and adult Diaphorina citri fed for 48 h on these plants for ?Ca. L. asiaticus? acquisition were determined by quantitative polymerase chain reaction in two experiments. The first experiment included plants with three levels of infection, three incubation periods (IPs), and air temperatures favorable (14.6 to 28°C) and unfavorable (24 to 38°C) to ?Ca. L. asiaticus?. The second included plants with severe late-stage infections, 10 IPs (based on 3-day intervals over 27 days), and three air temperature regimes (12 to 24, 18 to 30, and 24 to 38°C). Overall, ?Ca. L. asiaticus? titers and the percentages of ?Ca. L. asiaticus?-positive psyllids were lower in plants maintained at the warmer temperature regime (24 to 38°C) than in plants maintained in the cooler regimes. The results suggest that the lower incidence and slower spread of ?Ca. L. asiaticus? to warmer regions of SP are related to the influence of ambient temperatures on titers of ?Ca. L. asiaticus? in leaves.

Optimisation of analytical methods for the characterisation of oranges, clementines and citrus hybrids cultivated in Spain on the basis of their composition in ascorbic acid, citric acid and major sugars

Three HPLC methods were optimised for the determination of citric acid, succinic acid and ascorbic acid using a photodiode array detector and fructose, glucose and sucrose using a refractive index in twenty eight citrus juices. The analysis was completed in <16 min. Two different harvests were taken into account for this study. For the season 2011, ascorbic acid content was comprised between 19.4 and 59 mg vitamin C/100 mL; meanwhile for the season 2012, the content was slightly higher for most of the samples ranging from 33.5 to 85.3 mg vitamin C/100 mL. Moreover, the citric acid content in orange juices ranged between 9.7 and 15.1 g L−1, while for clementines the content was clearly lower (i.e. from 3.5 to 8.4 g L−1). However, clementines showed the highest sucrose content with values near to 6 g/100 mL. Finally, a cluster analysis was applied to establish a classification of the citrus species.