Chemical composition, acetylcholinesterase inhibitory and antifungal activities of Pera glabrata (Schott) Baill. (Euphorbiaceae)

Pera glabrata (Schott) Baill. was selected for this study after showing a preliminary positive result in a screening of Atlantic Forest plant species in the search for acetylcholinesterase inhibitors and antifungal compounds. The bioassays were conducted with crude ethanol extract of the leaves using direct bioautography method for acetylcholinesterase and antifungal activities. This extract was partitioned with hexane, chloroform and ethyl acetate solvents. The active chloroform fraction was submitted to silica gel chromatography column affording 12 groups. Caffeine, an alkaloid, which showed detection limits of 0.1 and 1.0 µg for anticholinesterasic and antifungal activities, respectively, was isolated from group nine. After microplate analyses, only groups four, nine, 10, 11 and 12 showed acetylcholinesterase inhibitory activity of 40% or higher. The group 12 was purified by preparative layer chromatography affording four sub-fractions. Two sub-fractions from this group were analyzed by gas chromatography-mass spectrometry and gas chromatography-flame ionization detector. The first sub-fraction showed anticholinesterasic activity and contained two major compounds: 9-hydroxy-4-megastigmen-3-one (84%) and caffeine (6%). The second sub-fraction presented five major compounds identified as 9-hydroxy-4-megastigmen-3-one, isololiolide, (-) loliolide, palmitic acid and lupeol and did not show activity.

Elemental composition changes in citrus affected by the CVC disease

The citrus variegated chlorosis (CVC) disease results in serious economical losses for the Brazilian citriculture. The influence of CVC disease on the elemental composition of citrus plants was investigated. Leaves of sweet orange varieties Hamlin, Pera Rio and Valencia were collected from healthy and CVC-affected trees for chemical characterization by instrumental neutron activation analysis (INAA). Significant differences between healthy and CVC-affected leaves were identified for Ca, Ce, Co, Eu, Fe, K, La, Na, Nd, Rb, Sc and Sm. Rare earth elements presented consistently higher mass fractions in the healthy leaves.

Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings

Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem-limited bacteria (Candidatus Liberibacter spp.) associated with citrus `huanglongbing` by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pera (Rutaceae) by using the electrical penetration graph (EPG-DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem-limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G-like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8-h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non-probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.

The Effects of Host, Geographic Origin, and Gender on the Thermal Requirements of Diaphorina citri (Hemiptera: Psyllidae)

Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the vector of the bacteria that causes citrus greening and is considered one of the world`s most important citrus diseases. We examined how host, geographic region, and gender affect the thermal requirements of D. citri. The insects were reared in climatic chambers at constant temperatures of 18, 20, 22, 25, 28, 30, and 32 1 degrees C, 70 +/- 10% RH, and a 14 h photophase. Host plants for D. citri included orange (Citrus sinensis [Rutaceae]) varieties Pera and Natal, the rootstock, Rungpur lime (C. limonia [Rutaceae]) and the natural host, Orange jessamine (Murraya paniculata [Rutaceae]). To study the influence of geographic origin on thermal requirements, we studied D. citri populations from Piracicaba, SP (warmer region) and Itapetininga, SP (cooler region). The duration and survival of the development stages and the duration of the total development (egg-adult) did not differ significantly on the different hosts, but it did vary with temperature. Nymphs of D. citri created on the different hosts have the same thermal requirements. The thermal requirements for this species collected from the two climate regions were identical; males and females also had the same thermal requirements.

Yield loss caused by huanglongbing in different sweet orange cultivars in So Paulo, Brazil

Citrus huanglongbing (HLB) reduces an affected orchard`s economic life. This work aimed to characterize yield loss due to HLB for different sweet orange cultivars and determine the relationship between disease severity and yield. Disease severity and yield were assessed on 949 individual trees distributed in 11 different blocks from sweet orange cultivars Hamlin, Westin, Pera and Valencia. In each block, plants showing a range of HLB severity levels and asymptomatic plants were selected. Total yield (weight of harvested fruit), mean weight of asymptomatic and symptomatic fruit, relative yield (symptomatic tree yield/mean yield of asymptomatic trees from the same block) and relative number of fruits (fruit number from symptomatic tree/mean number of fruits from asymptomatic trees from the same block) were determined. The weight of symptomatic fruit was lower than the weight of asymptomatic fruit, but the weights of asymptomatic and symptomatic fruit were not correlated with disease severity, indicating that the effects of HLB were restricted to symptomatic branches. The relationship of the relative yield with HLB severity can be satisfactorily described by a negative exponential model. The rates of yield decrease as a function of disease severity were similar for all assessed cultivars. A relative yield (up to 19%) was observed even for trees where disease severity was 100%. The strong linear relationship between relative number of fruits per tree and the relative yield per tree suggested that the yield reduction was due primarily to early fruit drop or lack of fruit set on affected branches.

Incidence of black spot in citrus fruits from different processing stages in packinghouses and from Sao Paulo`s wholesale market

The purposes of this work were a) to evaluate citrus black spot (CBS) incidence in `Valencia` oranges and `Murcott` tangors aimed at the export market, and in Pera`, `Lima` and `Natal` oranges, and `Murcott` tangors, aimed at the domestic market after different processing stages in packinghouses in 2004/05 and 2005/06; b) to evaluate CBS incidence in Pera` and `Lima` oranges and `Murcott` tangors sold at Ceagesp-SP, the biggest wholesale market in the State of Sao Paulo, in 2006. Citrus fruits were collected at the packinghouse, on their arrival, after pre-washing and de-greening, from the packing table, from the pallet and at Ceagesp. They were stored for 14 to 21 days at 25 degrees C and 85-90% RH. The incidence of CBS was visually evaluated after one day and at the end of the storage period. CBS incidence in fruits aimed at the export market decreased, with values under 2.0% on arrival and no CBS symptoms observed on fruits from the pallet. The average incidence of CBS in `Pera`, `Lima` and `Natal` oranges, and `Murcott` tangors in the packinghouse aimed at the domestic market were 64.1, 39.0, 32.1 and 19.3%, respectively, after one day of storage, then remaining constant in all processing stages. The incidence of CBS in Ceagesp fruits was low in winter months and increased in the spring. The increase in disease incidence during the storage period (21 days) was not significant in collected fruits.

Postharvest diseases in citrus and characterization of the fungal population in Sao Paulo`s wholesale market

The purposes of this workwere to characterize postharvest injuries and to evaluate the physicochemical characteristics of`Nra` and `Lima`oranges and `Murcott` tangor at Ceagesp market, as well as to characterize the environmental mycoflora in retail points at Ceagesp in 2006. Fruits collected at retail points were stored for 14 days at 25 degrees C and 85-90% RH. The incidence of injuries was visually evaluated every three days. The physicochemical characteristics analyzed were titratable acidity and soluble solids amount. The environmental mycoflora was sampled according to the gravimetric method, using Petri dishes containing potato-dextrose-agar medium+pentabiotic opened for two minutes. The average rot incidences in `Pera` and `Lima` oranges and `Murcott` tangor were 12.8, 14.9 and 25.8%, respectively, at the end of the storage period, and green mold was the main postharvest disease. Associations between physicochemical parameters and rot incidence was, in general, not significant. The environmental fungal population varied significantly between the sampling months in retail points with an average of 25.3 cfu/plate. Penicillium and Cladosporium were the most recorded genera of fungi. Positive correlation (r=0.96) was observed between frequency of P digitatum found in the environment of retail points and the green mold in on-sale fruits of `Pera` orange. However, for `Lima` orange and `Murcott` tangor such a correlation was not verified.

Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp citri

Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and spray-inoculated with an Xcc suspension (10(6) cfu mL(-1)). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (non-transgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16-0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen.

Virulence features of atypical enteropathogenic Escherichia coli identified by the eae(+) EAF-negative stx(-) genetic profile

This study characterized 76 atypical enteropathogenic Escherichia coli (aEPEC) strains, previously classified by the eae(+) EAF-negative stx(-) genotype, isolated from children with diarrhea in Brazil. Presence of bfpA and bfpA/perA was detected in 2 and 6 strains, respectively. The expression of bundle-forming pilus (BFP), however, was observed by immunofluorescence in 1 bfpA and 3 bfpA/perA strains, classifying them as typical EPEC (tEPEC). The remaining 72 aEPEC strains were characterized by serotyping, intimin typing, adherence patterns to HEp-2 cells, capacity to induce actin aggregation (fluorescent actin staining test), and antimicrobial resistance. Our results show that aEPEC comprise a very heterogeneous group that does not present any prevalence or association regarding the studied characteristics. It also suggest that tEPEC and aEPEC must not be classified only by the reactivity with the EAF probe, and that the search of other markers present in pEAF, as well as the BFP expression, must be considered for this matter. (C) 2009 Elsevier Inc. All rights reserved.

The pst operon of enteropathogenic Escherichia coli enhances bacterial adherence to epithelial cells

Enteropathogenic Escherichia coli (EPEC) adheres in vivo and in vitro to epithelial cells. Two main adhesins, the bundle-forming pilus and intimin, encoded by the Up operon and eae, respectively, are responsible for the localized and the intimate adherence phenotypes. Deletion of the pst operon of EPEC abolishes the transport of inorganic phosphate through the phosphate-specific transport system and causes the constitutive expression of the PHO regulon genes. In the absence of pst there is a decrease in the expression of the main EPEC adhesins and a reduction in bacterial adherence to epithelial cells in vitro. This effect is not related to PHO constitutivity, because a Delta pst phoB double mutant that is defective in the transcription of the PHO genes also displayed low levels of adherence and expression of adhesins. Likewise, a PHO-constitutive phoR mutation did not affect bacterial adherence. The expression of the per operon, which encodes the Up and ler regulators PerA and PerC, is also negatively affected by the pst deletion. Overall, the data presented here demonstrate that the pst operon of EPEC plays a positive role in the bacterial adherence mechanism by increasing the expression of perA and perC and consequently the transcription of bfp and eae.

Microbiopsies of Surface Dental Enamel as a Tool to Measure Body Lead Burden

Lead (Pb) poisoning is preventable but continues to be a public health problem in several countries. Measuring Pb in the surface dental enamel (SDE) using microbiopsies is a rapid, safe, and painless procedure. There are different protocols to perform these microbiopsies, but the reliability of dental enamel lead levels (DELL) determination is dependent upon biopsy depth (BD). It is established that DELL decrease from the outermost superficial layer to the inner layer of dental enamel. The aim of this study was to determine DELL obtained by two different microbiopsy techniques on SDE termed protocol I and protocol II. Two consecutive enamel layers were removed from the same subject group (n = 138) for both protocols. Protocol I consisted of a biopsied site with a diameter of 4 mm after the application of 10 l HCl for 35 s. Protocol II involved a biopsied site of 1.6 mm diameter after application of 5 l HCl for 20 s. The results demonstrated that there were no significant differences for BD and DELL between homologous teeth using protocol I. However, there was a significant difference between DELL in the first and second layers using both protocols. Further, the BD in protocol II overestimated DELL values. In conclusion, SDE analyzed by microbiopsy is a reliable biomarker in protocol I, but the chemical method to calculate BD in protocol II appeared to be inadequate for measurement of DELL. Thus, DELL could not be compared among studies that used different methodologies for SDE microbiopsies.