Genetic structure and biology of Xylella fastidiosa strains causing disease in citrus and coffee in Brazil

Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most X. fastidiosa populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of X. fastidiosa that have diverged but are genetically homogenized by frequent recombination.

Performance of a multi-level TDR system exposed to tropical field conditions - A time-space comparison with tensiometry

Time-domain reflectometry (TDR) is an important technique to obtain series of soil water content measurements in the field. Diode-segmented probes represent an improvement in TDR applicability, allowing measurements of the soil water content profile with a single probe. In this paper we explore an extensive soil water content dataset obtained by tensiometry and TDR from internal drainage experiments in two consecutive years in a tropical soil in Brazil. Comparisons between the variation patterns of the water content estimated by both methods exhibited evidences of deterioration of the TDR system during this two year period at field conditions. The results showed consistency in the variation pattern for the tensiometry data, whereas TDR estimates were inconsistent, with sensitivity decreasing over time. This suggests that difficulties may arise for the long-term use of this TDR system under tropical field conditions. (c) 2008 Elsevier B.V. All rights reserved.

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil To elucidate some biochemical processes during infection in the pathosystem Puccinia psidii x eucalyptus, the defense metabolism in different-stage leaves was compared between rust-resistant and susceptible clones, respectively. In addition, chitinase and peroxidase activities were assayed. Each treatment consisted of 4 replicates, in a completely randomized design: 2 clones, inoculated and not inoculated with P. psidii; sprayed with acibenzolar-S-methyl (ASM) and distilled water; and represented by the 1(st) leaf pair (size equivalent to 1/5 total leaf development), 2(nd) pair (2/5 total development), and 4(th) pair (4/5 total leaf length). Leaves were harvested in 4 periods: 0, 24, 72 and 96 hours after inoculation. Results indicated that ASM treatment or P. psidii action led to higher chitinase and peroxidase activity level but did not alter the expression of these activities in developed leaves (4(th) pair) during the experiment. Alterations in enzyme levels after inoculation were only observed in developing leaves (1(st) and 2(nd) pairs), which suggests that the response to infection was concomitant to chitinase and peroxidase synthesis. The highest increases in enzymatic activities were observed in resistant clones at 72 hours after inoculation and in susceptible ones previously treated with ASM and later inoculated with the pathogen.

Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker

Transgenic Citrus sinensis (L.) Osb. cv. Hamlin plants expressing the hrpN gene were obtained by Agrobacterium tumefaciens (Smith and Towns) Conn-mediated transformation. hrpN encodes a harpin protein, which elicits the hypersensitive response and systemic acquired resistance in plants. The gene construct consisted of gst1, a pathogen-inducible promoter, a signal peptide for protein secretion to the apoplast, the selection genes nptI1 or aacC1 and the Nos terminator. The function of gst1 in citrus was evaluated in transgenic C. sinensis cv. Valencia harboring the reporter gene uidA (gus) driven by this promoter. Histochemical analysis for gus revealed that gst1 is activated in citrus leaves by both wounding and inoculation with Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al. Genetic transformation was confirmed by Southern blot hybridization in eight cv. Hamlin acclimatized plants. RT-PCR confirmed hrpN gene expression in seven cv. Hamlin transgenic lines before pathogen inoculation. Some hrpN transgenic lines showed severe leaf curling and abnormal growth. Six hrpN transgenic lines were propagated and evaluated for susceptibility to X axonopodis pv. citri. RT-PCR confirmed gene expression in all six hrpN transgenic lines after pathogen inoculation. Several of the hrpN transgenic lines showed reduction in susceptibility to citrus canker as compared with non-transgenic plants. One hrpN transgenic line exhibited normal vegetative development and displayed very high resistance to the pathogen, estimated as up to 79% reduction in disease severity. This is the first report of genetic transformation of citrus using a pathogen-inducible promoter and the hrpN gene. Further evaluations of the transgenic plants under field conditions are planned. Nevertheless, the evidence to date suggests that the hrpN gene reduces the susceptibility of citrus plants to the canker disease. (C) 2009 Elsevier B.V. All rights reserved.

Bacterial soil community in a Brazilian sugarcane field

The assessment of bacterial communities in soil gives insight into microbial behavior under prevailing environmental conditions. In this context, we assessed the composition of soil bacterial communities in a Brazilian sugarcane experimental field. The experimental design encompassed plots containing common sugarcane (variety SP80-1842) and its transgenic form (IMI-1 - imazapyr herbicide resistant). Plants were grown in such field plots in a completely randomized design with three treatments, which addressed the factors transgene and imazapyr herbicide application. Soil samples were taken at three developmental stages during plant growth and analyzed using 16S ribosomal RNA (rRNA)-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and clone libraries. PCR-DGGE fingerprints obtained for the total bacterial community and specific bacterial groups - Actinobacteria, Alphaproteobacteria and Betaproteobacteria - revealed that the structure of these assemblages did not differ over time and among treatments. Nevertheless, slight differences among 16S rRNA gene clone libraries constructed from each treatment could be observed at particular cut-off levels. Altogether, the libraries encompassed a total of eleven bacterial phyla and the candidate divisions TM7 and OP10. Clone sequences affiliated with the Proteobacteria, Actinobacteria, Firmicutes and Acidobacteria were, in this order, most abundant. Accurate phylogenetic analyses were performed for the phyla Acidobacteria and Verrucomicrobia, revealing the structures of these groups, which are still poorly understood as to their importance for soil functioning and sustainability under agricultural practices.

Effect of bacterial inoculation, plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (Solanum tuberosum)

Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage.

Citrus Sudden Death Is Transmitted by Graft-Inoculation and Natural Transmission Is Prevented by Individual Insect-Proof Cages

Citrus sudden death (CSD) transmission was studied by graft-inoculation and under natural conditions. Young sweet orange trees on Rangpur rootstock were used as indicator plants. They were examined regularly for one or two characteristic markers of CSD: (i) presence of a yellow-stained layer of thickened bark on the Rangpur rootstock, and (ii) infection with the CSD-associated marafivirus. Based on these two markers, transmission of CSD was obtained, not only when budwood for graft-inoculation was taken from symptomatic, sweet orange trees on Rangpur, but also when the budwood sources were asymptomatic sweet orange trees on Cleopatra mandarin, indicating that the latter trees are symptomless carriers of the CSD agent. For natural transmission, 80 young indicator plants were planted within a citrus plot severely affected by CSD. Individual insect-proof cages were built around 40 indicator plants, and the other 40 indicator plants remained uncaged. Only two of the 40 caged indicator plants were affected by CSD, whereas 17 uncaged indicator plants showed CSD symptoms and were infected with the marafivirus. An additional 12 uncaged indicator plants became severely affected with citrus variegated chlorosis and were removed. These results strongly suggest that under natural conditions, CSD is transmitted by an aerial vector, such as an insect, and that the cages protected the trees against infection by the vector.

Hand position on the bunch and source-sink ratio influence the banana fruit susceptibility to crown rot disease

The postharvest development of crown rot of bananas depends notably on the fruit susceptibility to this disease at harvest. It has been shown that fruit susceptibility to crown rot is variable and it was suggested that this depends on environmental preharvest factors. However, little is known about the preharvest factors influencing this susceptibility. The aim of this work was to evaluate the extent to which fruit filling characteristics during growth and the fruit development stage influence the banana susceptibility to crown rot. This involved evaluating the influence of (a) the fruit position at different levels of the banana bunch (hands) and (b) changing the source-sink ratio (So-Si ratio), on the fruit susceptibility to crown rot. The fruit susceptibility was determined by measuring the internal necrotic surface (INS) after artificial inoculation of Colletotrichum musae. A linear correlation (r = -0.95) was found between the hand position on the bunch and the INS. The So-Si ratio was found to influence the pomological characteristics of the fruits and their susceptibility to crown rot. Fruits of bunches from which six hands were removed (two hands remaining on the bunch) proved to be significantly less susceptible to crown rot (INS = 138.3 mm 2) than those from bunches with eight hands (INS = 237.9 mm 2). The banana susceptibility to crown rot is thus likely to be influenced by the fruit development stage and filling characteristics. The present results highlight the importance of standardising hand sampling on a bunch when testing fruit susceptibility to crown rot. They also show that hand removal in the field has advantages in the context of integrated pest management, making it possible to reduce fruit susceptibility to crown rot while increasing fruit size.

Molecular dynamics, density functional, ADMET predictions, virtual screening, and molecular interaction field studies for identification and evaluation of novel potential CDK2 inhibitors in cancer therapy

In this work, we have used molecular dynamics, density functional theory, virtual screening, ADMET predictions, and molecular interaction field studies to design and propose eight novel potential inhibitors of CDK2. The eight molecules proposed showed interesting structural characteristics that are required for inhibiting the CDK2 activity and show potential as drug candidates for the treatment of cancer. The parameters related to the Rule of Five were calculated, and only one of the molecules violated more than one parameter. One of the proposals and one of the drug-like compounds selected by virtual screening indicated to be promising candidates for CDK2-based cancer therapy.

Evaluation of the Experimental Inoculation of Cryptococcus albidus and Cryptococcus laurentii in Normal Mice: Virulence Factors and Molecular Profile Before and After Animal Passage

The genus Cryptococcus includes free-developing species, a few of which are of medical importance. Some, such as C. neoformans and C. gattii, cause infections in man frequently and C. albidus and C. laurentii cause less so. The aims of this study were to evaluate organ colonization after inoculation of C. albidus and C. laurentii isolates in normal BALB/c mice, the virulence factors (growth at 37A degrees C, capsule, melanin, proteinase, and phospholipase production) and the molecular profile (PCR-fingerprinting) of the yeasts before and after infection. The importance of different profiles (virulence and molecular) was considered in relation to the distribution in different organs and to the time intervals of isolation from organs. C. albidus was isolated from animal organs 2 to 10 days after inoculation and C. laurentii from 2 to 120 days. Most isolates of the two species kept the virulence factors showed before inoculation. The high homogeneity of the molecular profile of C. albidus and the high heterogeneity of C. laurentii were kept through the passages in animals. It is concluded that most isolates of both species were recovered from the animal organs after 5 or more days, and phenotypes were not altered by inoculation. No molecular alteration was detected and the virulence factors were not related to the time intervals before isolation from organs.

Molecular dynamics, flexible docking, virtual screening, ADMET predictions, and molecular interaction field studies to design novel potential MAO-B inhibitors

Monoamine oxidase is a flavoenzyme bound to the mitochondrial outer membranes of the cells, which is responsible for the oxidative deamination of neurotransmitter and dietary amines. It has two distinct isozymic forms, designated MAO-A and MAO-B, each displaying different substrate and inhibitor specificities. They are the well-known targets for antidepressant, Parkinson`s disease, and neuroprotective drugs. Elucidation of the x-ray crystallographic structure of MAO-B has opened the way for the molecular modeling studies. In this work we have used molecular modeling, density functional theory with correlation, virtual screening, flexible docking, molecular dynamics, ADMET predictions, and molecular interaction field studies in order to design new molecules with potential higher selectivity and enzymatic inhibitory activity over MAO-B.

The use of transcriptional profiles to predict adult mosquito age under field conditions

Age is a critical determinant of an adult female mosquito's ability to transmit a range of human pathogens. Despite its central importance, relatively few methods exist with which to accurately determine chronological age of field-caught mosquitoes. This fact is a major constraint on our ability to fully understand the relative importance of vector longevity to disease transmission in different ecological contexts. It also limits our ability to evaluate novel disease control strategies that specifically target mosquito longevity. We report the development of a transcriptional profiling approach to determine age of adult female Aedes aegypti under field conditions. We demonstrate that this approach surpasses current cuticular hydrocarbon methods for both accuracy of predicted age as well as the upper limits at which age can be reliably predicted. The method is based on genes that display age-dependent expression in a range of dipteran insects and, as such, is likely to be broadly applicable to other disease vectors.

Reliability and validity of physical fitness field tests for adults aged 55 to 70 years

The aim of this study was to examine the reliability and validity of field tests for assessing physical function in mid-aged and young-old people (55–70 y). Tests were selected that required minimal space and equipment and could be implemented in multiple field settings such as a general practitioner's office. Nineteen participants completed 2 field and 1 laboratory testing sessions. Intra-class correlations showed good reliability for the tests of upper body strength (lift and reach, R= .66), lower body strength (sit to stand, R= .80) and functional capacity (Canadian Step Test, R= .92), but not for leg power (single timed chair rise, R= .28). There was also good reliability for the balance test during 3 stances: parallel (94.7% agreement), semi-tandem (73.7%), and tandem (52.6%). Comparison of field test results with objective laboratory measures found good validity for the sit to stand (cf 1RM leg press, Pearson r= .68, p< .05), and for the step test (cf PWC140, r= −.60, p< .001), but not for the lift and reach (cf 1RM bench press, r= .43, p> .05), balance (r= −.13, −.18, .23) and rate of force development tests (r= −.28). It was concluded that the lower body strength and cardiovascular function tests were appropriate for use in field settings with mid-aged and young-old adults.

Host age effect and expression of cytoplasmic incompatibility in field populations of Wolbachia-superinfected Aedes albopictus

The Asian tiger mosquito, Aedes albopictus (Skuse), is a known vector of dengue in South America and Southeast Asia. It is naturally superinfected with two strains of Wolbachia endosymbiont that are able to induce cytoplasmic incompatibility (CI). In this paper, we report the strength of CI expression in crosses involving field-caught males. CI expression was found to be very strong in all crosses between field males and laboratory-reared uninfected or wAlbA infected young females. In addition, crossing experiments with laboratory colonies showed that aged super- infected males could express strong CI when mated with young uninfected or wAlbA infected females. These results provide additional evidence that the CI properties of Wolbachia infecting Aedes albopictus are well suited for applied strategies that seek to utilise Wolbachia for host population modification.

Field prevalence of Wolbachia in the mosquito vector Aedes albopictus

The endosymbiotic bacteria in the genus Wolbachia have been proposed as a potential candidate to deliver pathogen-blocking genes into natural populations of medically important insects. The successful application of Wolbachia in insect vector control depends on the ability of the agent to successfully invade and maintain itself at high frequency under field conditions. Here, we evaluated the prevalence of Wolbachia infections in a field population of the Wolbachia-superinfected mosquito Aedes albopictus. A field prevalence of 100% (n = 1,016) was found in a single population in eastern Thailand via polymerase chain reaction (PCR) testing of Wolbachia both from individual parent females and their corresponding F1 offspring. This is the first report of accurate Wolbachia prevalence in a field population of an insect disease vector. The prevalence of superinfection was estimated to be 99.41%. All single-infected individual mosquitoes (n = 6) were found to harbor group A Wolbachia. For this particular population, none was found to be single-infected with group B Wolbachia. Our results also show that PCR testing of field materials alone without checking F1 offspring overestimated the natural prevalence of single infection. Thus, the confirmation of infection status by means of F1 offspring was critical to the accurate estimates of Wolbachia prevalence under field conditions.