GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the beta-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

LIFE TABLE ANALYSIS OF DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) INFESTING SWEET ORANGE (CITRUS SINENSIS) IN SAO PAULO

An ecological life table for eggs and nymphs of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) was constructed with data obtained from orange orchards (Citrus sinensis Osbeck) in 2 regions of the State of Sao Paulo, over 4 generations in the period from XI-2006 to V-2007, comprising spring, summer, and fall seasons. Young growing shoots with D. citri eggs present were identified, and live individuals were counted until adult emergence. No predatory arthropods were observed in association with D. citri eggs and nymphs during the study. The mean parasitism of fourth- and fifth-instar nymphs by Tamarixia radiata Waterston (Hymenoptera: Eulophidae) was 2.3%. The durations of the egg-adult period were similar among the 4 generations, ranging from 18.0 to 24.7 d (at mean temperatures ranging from 21.6 to 26.0 degrees C) and followed the temperature requirement models obtained in the laboratory for D. citri. However, survival from the egg to the adult stage for the same period varied considerably from 1.7 to 21.4%; the highest mortalities were observed in the egg and small nymphal (first- to thirdinstar) stages, which were considered to be key phases for population growth of the pest.

Relationship between sweet orange yield and intensity of Citrus Variegated Chlorosis

Citrus Variegated Chlorosis (CVC) is currently present in approximately 40% of citrus plants in Brazil and causes an annual loss of around 120 million US dollars to the Brazilian citrus industry. Despite the fact that CVC has been present in Brazil for over 20 years, a relationship between disease intensity and yield loss has not been established. In order to achieve this, an experiment was carried out in a randomized block design in a 3 x 2 factorial scheme with 10-year-old Natal sweet orange. The following treatments were applied: irrigation with 0, 50 or 100% of the evapotranspiration of the crop, combined with natural infection or artificial inoculation with Xylella fastidiosa, the causal agent of CVC. The experiment was evaluated during three seasons. A negative exponential model was fitted to the relationships between yield versus CVC severity and yield versus Area Under Disease Progress Curve (AUDPC). In addition, the relationship between yield versus CVC severity and canopy volume was fitted by a multivariate exponential model. The use of the AUDPC variable showed practical limitations when compared with the variable CVC severity. The parameter values in the relationship of yieldCVC severity were similar for all treatments unlike in the multivariate model. Consequently, the yieldCVC intensity relationship (with 432 data points) could be described by one single model: y = 114.07 exp(-0.017 x), where y is yield (symptomless fruit weight in kg) and x is disease severity (R2 = 0.45; P < 0.01).

Weedy Hosts and Prevalence of Potential Leafhopper Vectors (Hemiptera: Cicadellidae) of a Phytoplasma (16SrIX group) Associated With Huanglongbing Symptoms in Citrus Groves

Huanglongbing (HLB) is a severe citrus (Citrus spp.) disease associated with the bacteria genus Candidatus Liberibacter, detected in Brazil in 2004. Another bacterium was found in association with HLB symptoms and characterized as a phytoplasma belonging to the 16SrIX group. The objectives of this study were to identify potential leafhopper vectors of the HLB-associated phytoplasma and their host plants. Leafhoppers were sampled every other week for 12 mo with sticky yellow cards placed at two heights (0.3 and 1.5 m) in the citrus tree canopy and by using a sweep net in the ground vegetation of two sweet orange, Citrus sinensis (L.) Osbeck, groves infected by the HLB-phytoplasma in Sao Paulo state. Faunistic analyses indicated one Agalliinae (Agallia albidula Uhler) and three Deltocephalinae [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (Stal), and Scaphytopius (Convelinus) marginelineatus (Stal)] species, as the most abundant and frequent leafhoppers (Hemiptera: Cicadellidae). Visual observations indicated an association of leafhopper species with some weeds and the influence of weed species composition on leafhopper abundance in low-lying vegetation. S. marginelineatus and P. flavicosta were more frequent on Sida rhombifolia L. and Althernantera tenella Colla, respectively, whereas A. albidula was observed more often on Conyza bonariensis (L.) Cronq. and B. hebe only occurred on grasses. DNA samples of field-collected S. marginelineatus were positive by polymerase chain reaction and sequencing tests for the presence of the HLB-phytoplasma group, indicating it as a potential vector. The association of leafhoppers with their hosts may be used in deciding which management strategies to adopt against weeds and diseases in citrus orchards.

Inhibitory activity of liposomal flavonoids during oxidative metabolism of human neutrophils upon stimulation with immune complexes and phorbol ester

Context and objective: The massive production of reactive oxygen species by neutrophils during inflammation may cause damage to tissues. Flavonoids act as antioxidants and have anti-inflammatory effects. In this study, liposomes loaded with these compounds were evaluated as potential antioxidant carriers, in attempt to overcome their poor solubility and stability. Materials and methods: Liposomes containing quercetin, myricetin, kaempferol or galangin were prepared by the ethanol injection method and analyzed as inhibitors of immune complex (IC) and phorbol ester-stimulated neutrophil oxidative metabolism by luminol (CLlum) and lucigenin-enhanced (CLluc) chemiluminescence (CL) assays. The mechanisms involved this activity of liposomal flavonoids, such as cytotoxicity and superoxide anion scavenging capacity, and their effect on phagocytosis of ICs were also investigated. Results and discussion: The results showed that the inhibitory effect of liposomal flavonoids on CLlum and CLluc is inversely related to the number of hydroxyl groups in the flavonoid B ring. Moreover, phagocytosis of liposomes by neutrophils does not seem to necessarily promote such activity, as the liposomal flavonoids are also able to reduce CL when the cells are pretreated with cytochalasin B. Under assessed conditions, the antioxidant liposomes are not toxic to the human neutrophils and do not interfere with IC-induced phagocytosis. Conclusion: The studied liposomes can be suitable carriers of flavonoids and be an alternative for the treatment of diseases in which a massive oxidative metabolism of neutrophils is involved.

EVALUATION OF PHOTODEGRADABLE TAPE ON GRAFTING IN CITRUS NURSERY TREES

The use of a photodegradable tape was evaluated on 'Valencia' sweet orange nursery trees budded both on Rangpur lime and Swingle citrumelo in a greenhouse in Bebedouro-SP, Brazil, from September to November 2009. On both rootstocks three wrapping procedures were evaluated: i) conventional polyethylene tape wrapped around the bud eye; ii) photodegradable tape wrapped around the bud eye, and iii) photodegradable tape wrapped around the graft junction without covering the bud eye. The following variables were measured: time spent for wrapping, percentage of bud sprouting, length and stem diameter of the scion shoot, and percentage of commercially valuable nursery trees. The trial was conducted following a randomized complete block design, with six treatments, four replications and 12 trees per plot. The use of photodegradable tape, with or without covering the bud eye, anticipated bud sprouting; despite of the longer time spent with wrapping when the photodegradable tape was used. Plants grafted onto the less vigorous Swingle citrumelo rootstock showed lower bud sprout percentages when the bud eye was covered with the photodegradable tape.

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.

Schistosomicidal evaluation of flavonoids from two species of Styrax against Schistosoma mansoni adult worms

Context: Schistosomiasis is a major health problem worldwide. Thus, the search for new schistosomicidal agents from natural sources can provide prototypes for drug discovery. Objective: The present study investigated the chemical composition of the EtOAc fractions of Styrax pohlii Pohl (Styracaceae) (EF-SP) aerial parts and S. camporum A. DC. leaves (EF-SC), as well as schistosomicidal activities against Schistosoma mansoni adult worms, which have not yet been studied. Materials and methods: The crude ethanol extracts of S. camporum leaves and S. pohlii aerial parts (EE-SC and EE-SP) were partitioned with n-hexane, EtOAc, and n-BuOH. The EtOAc fractions were purified by preparative HPLC. The crude extracts, EtOAc fractions and pure compounds were tested against S. mansoni adult worms in vitro. Results: The purification procedure resulted in the isolation of kaempferol-3-O-(2 '',4 ''-di-O-(E)-p-coumaroyl)-beta-D-glucopyranoside (1), kaempferol-3-O-(2 '',6 ''-di-O-(E)-p-coumaroyl)-beta-D-glucopyranoside (2), quercetin (3), and kaempferol (4). The bioassay results indicated that EE-SC, EF-SC, EF-SP, and compounds 2 and 4 are able to separate coupled S. mansoni adult worms. Additionally, EE-SC, EF-SP, and compound 4 killed the adult schistosomes in vitro at 100 mu g/mL and 100 mu M. Discussion and conclusion: This is the first time that the presence of compounds 1-2 in S. pohlii and 3-4 in S. camporum has been reported. Additionally, biological results indicated that S. pohlii and S. camporum have great potential as a source of active compounds.

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model "diploid embryogenic callus protoplast + diploid mesophyll-derived protoplast". Protoplasts were isolated from embryogenic calli of 'Pera' and 'Westin' sweet orange cultivars (Citrus sinensis) and from young leaves of 'Fremont', Nules', and 'Thomas' mandarins (C. reticulata), and 'Nova' tangelo [C. reticulata x (C. paradisi x C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when 'Pera' sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of 'Pera' sweet orange + 'Fremont' mandarin, 3 'Pera' sweet orange + 'Nules' mandarin, and 2 'Pera' sweet orange + 'Nova' tangelo, and all the diploid regenerated plants showed the 'Pera' sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96 h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.

Genetic transformation of Citrus sinensis with Citrus tristeza virus (CTV) derived sequences and reaction of transgenic lines to CTV infection

Transgenic Citrus sinensis (L.) Osb. plants, cvs. Valencia and Hamlin, expressing Citrus tristeza virus (CTV) derived sequences were obtained by genetic transformation. The gene constructs were pCTV-CP containing the 25 kDa major capsid protein gene (CTV-CP), pCTV-dsCP containing the same CTV-CP gene in an intron-spliced hairpin construct, and pCTV-CS containing a 559 nt conserved region of the CTV genome. The transgenic lines were identified by PCR and the transgene integration was confirmed by Southern blot. Transgene mRNA could be detected in most transgenic lines containing pCTV-CP or pCTV-CS transgene. The mRNA of pCTV-dsCP transgene was almost undetectable, with very light bands in most analyzed plants. The transgene transcription appears to be closely linked to the type of gene construct. The virus challenge assays reveals that all transgenic lines were infected. However, it was possible to identify propagated clones of transgenic plants of both cultivars studied with a low virus titer, with values similar to the non-inoculated plants (negative control). These results suggested that the transgenic plants present some level of resistance to virus replication. The higher number of clones with low virus titer and where mRNA could not be detected or was presented in a very light band was found for pCTV-dsCP-derived transgenic lines.

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.

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

Glyphosate has low toxicity to citrus plants growing in the field

Gravena, R., Filho, R. V., Alves, P. L. C. A., Mazzafera, P. and Gravena, A. R. 2012. Glyphosate has low toxicity to citrus plants growing in the field. Can. J. Plant Sci. 92: 119-127. There has been controversy over whether glyphosate used for weed management in citrus fields causes significant toxicity to citrus plants. Glyphosate may be toxic to non-target plants exposed to accidental application or drift. This work evaluated glyphosate toxicity in plants of Valencia citrus (Citrus sinensis. L. Osbeck) grafted onto 'Rangpur lime' (Citrus limonia L. Osbeck) and citrumelo 'Swingle' (Poncirus trifoliata (L.) Raf x Citrus paradisi Mad) by trunk- or foliar-directed herbicide applications under field conditions. In the first experiment, glyphosate was sprayed at rates of 0, 90, 180, 260, 540, 1080 and 2160 g a.e. ha(-1) directly on the trunk to a height of 5 cm above the grafting region. In the second experiment, glyphosate was sprayed on the plant canopies at rates of 0, 0.036, 0.36, 3.6, 36, 360 and 720 g a.e. ha(-1). There was no visual damage caused by glyphosate applied directly to the trunk, but the plants were affected by glyphosate sprayed directly on the canopies at rates over 360 g a.e. ha(-1). The main symptom was observed in the new shoots formed after the application, indicating an effect on meristems. Little or no effect was observed in mature leaves. Eight days after application the levels of shikimate, total free amino acids and total phenolic compounds were unaffected. All plants affected by glyphosate recovered between 6 and 12 mo after the treatments. Therefore, despite some transient symptoms Valencia citrus grafted onto 'Rangpur lime' and citrumelo 'Swingle' were tolerant to glyphosate.

Non-substituted B-ring flavonoids and an indole alkaloid from Piper aleyreanum (Piperaceae)

Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)

Antioxidant capacity and mineral content of pulp and peel from commercial cultivars of citrus from Brazil

Four Citrus species (C. sinensis, cvs. Pera and Lima; C. latifolia Tanaka cv. Tahiti; C limettioides Tanaka cv. Sweet lime and C. reticulate, cv. Ponkan) grown in Brazil were characterised in relation to contents of minerals, ascorbic acid, total polyphenols and antioxidant capacity of pulps and peels. In general, the peels demonstrated significantly higher contents of all compounds than the pulps (p < 0.05), with the exception of the Pera orange pulp that presented the highest acid ascorbic content (68 mg/100 ml), while the Tahiti lime peel presented the lowest (8 mg/100 g). Citrus showed high levels of potassium, calcium and magnesium, and the peels were considered sources of these minerals. The Ponkan mandarin peel presented the highest antioxidant capacity. The antioxidant capacity of citrus was correlated both to vitamin C and phenolics. Aside from citrus pulps, the peels are also good sources of bioactive compounds and minerals, and can be explored for their health promoting values in food products. (C) 2012 Elsevier Ltd. All rights reserved.