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.

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.

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.

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.

Genetic transformation of three sweet orange cultivars from explants of adult plants

The difficulty in adult tissue genetic transformation in woody species is still an obstacle to be overcome, including in most sweet orange cultivars of the Brazilian citrus industry. This work reports that, after in vitro culture adjustments, transgenic adventitious buds of 'Hamlin', 'Pra', and 'Valencia' sweet oranges (Citrus sinensis L. Osbeck) were recovered using adult material as explant source, in genetic transformation experiments via Agrobacterium tumefaciens. The transgenic buds were identified by the GUS histochemical analysis and confirmed by PCR analysis, which indicated the presence of an amplified fragment of 817 bp corresponding to the uidA gene sequence. The efficiencies of genetic transformation for 'Hamlin', 'Pra', and 'Valencia' sweet orange cultivars were 2.5, 1.4, and 3.7%, respectively. Media supplemented with auxins and cytokinins during co-culture, and media with high concentrations of cytokinins (3 mg L-1) during transgenic selection led to the transformation and, consequently, the regeneration of adequate number of adventitious buds for the three cultivars. The use of sonication during the explant disinfection was not effective to reduce endophytic contamination and reduced transformation efficiency.

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.

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.

Somatic embryogenesis from ovaries of sweet orange cv. Tobias

Callogenesis, somatic embryogenesis, and regeneration were obtained from tissues of unfertilized ovaries of sweet orange (Citrus sinensis Osbeck.) cv. Tobias. The influence of two modified basal media, woody plant medium (WPM) and N6 medium, to induce callus formation from pistils was determined. Overall, high frequencies of callogenesis were observed when either medium was used. However, initial culture of explants in WPM medium followed by transfer of callus to N6 medium resulted in higher frequency of callus induction (of 2.30 callus per explant that were larger than 0.5 cm in size), and of subsequent development of embryogenic callus (10%). A total of 125 somatic embryos were obtained. After 6 months of culture, 72% of somatic embryos germinated into plantlets. These plantlets were subsequently micrografted in vitro, and then acclimatized. Ploidy of these plants were determined using flow cytometry and TRAPS molecular markers were used to confirm their maternal origin.

Potencial do flavedo (epicarpo) de Citrus aurantifolia cv. Tahiti no controle do bolor verde e da antracnose em citros

O Brasil é considerado o maior produtor de citros e o maior exportador de suco de laranja. Doenças de pós-colheita representam uma grande perda para a citricultura, sendo que para a exportação de frutos são rígidas as exigências com relação a isenção de resíduos químicos nos mesmos. Patógenos de importância em pós-colheita de citros incluem o Penicillium digitatum, agente causal do bolor-verde e o Colletotrichum gloeosporioides, agente causal da antracnose. Dada a importância econômica que representam estas doenças dos frutos cítricos, tanto em termos de comprometimento da qualidade e dificuldade de controle, a busca de alternativas adicionais que possam viabilizar a capacidade produtiva e garantir a obtenção de frutos com excelentes padrões de qualidade torna-se imprescindível. Portanto, estudou-se os efeitos dos extratos aquosos do flavedo de Citrus aurantifolia var. Tahiti, Lentinula edodes, Agaricus subrufescens (syn. Agaricus brasiliensis), albedo de Citrus sinensis var. Valência e do ácido jasmônico no controle póscolheita do bolor verde e da antracnose e na indução de resistência em frutos de laranjeira Valência (Citrus sinensis). Foi possível observar que o extrato aquoso do flavedo (C. aurantifolia) apresentou efeito inibitório sobre os patógenos, quando tratados em pós-colheita, em função da redução dos sintomas e esporulação. Porém, os extratos de albedo (C. sinensis), L. edodes, A. subrufescens e o ácido jasmônico não apresentaram efeitos sobre P. digitatum e C. gloeosporioides.

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.

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.

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.

Período de incubação de Guignardia citricarpa em diferentes estádios fenológicos de frutos de laranjeira 'Valência'

A mancha preta dos citros (MPC), causada pelo fungo Guignardia citricarpa, produz lesões em frutos, os quais ficam depreciados para o mercado interno e os restringem para a exportação. O grande período de suscetibilidade dos frutos cítricos, em adição ao fato de G. citricarpa causar infecções latentes, dificulta o entendimento sobre o período de incubação da doença. O objetivo do trabalho foi determinar o período de incubação da MPC inoculando frutos de laranjeira 'Valência' em diferentes estádios fenológicos. Para a inoculação foram empregadas suspensões de conídios de G. citricarpa (10³, 10(4), 10(5) e 10(6) conídios mL-1) em diferentes diâmetros dos frutos (1,5; 2,0; 2,5; 3,0; 5,0 e 7,0 cm). O período de incubação da MPC para os diferentes diâmetros dos frutos inoculados apresentou uma relação polinomial negativa. Em frutos com até 3 cm de diâmetro o período de incubação médio foi superior a 200 dias, enquanto que em frutos com diâmetros superiores a 5 cm o período de incubação médio foi inferior a 84 dias. A MPC apresenta período de incubação variável dependente do estádio fenológico em que os frutos são infectados. A concentração de conídios de G. citricarpa, na infecção, não interfere no período de incubação da doença.

Histopathology of black spot symptoms in sweet oranges

In Brazil, citrus black spot (CBS) caused by Guignardia citricarpa is a major disease that has different symptoms on fruit. In this study, fruit of Citrus sinensis infected by G. citricarpa and showing the symptoms false melanosis, freckle spot and hard spot were cross-sectioned and analysed anatomically and histochemically by light microscopy. Immuno-histological assays were performed. All symptoms were accompanied by a thickening of the cuticle. False melanosis lesions did not contain pycnidia and remained restricted to the epicarp or to the first layers of the mesocarp. The stomata in this type of lesion showed phenolic compounds in the guard cells and in the sub-stomatal chamber. In some samples, the guard cells and their surrounding cells lysed, and a wound meristem began to form underneath them. Freckle spot and hard spot lesions had very similar histological alterations to the epicarp and mesocarp, but in our samples only hard spot lesions contained pycnidia. Both of these symptoms were accompanied by protein inclusions. Epidermal and sub-epidermal cells located in the oil-gland region were obliterated, causing alterations in these structures. All symptoms had regions that stained strongly for lipids and phenols.

Seasonal effects on the relationship between photosynthesis and leaf carbohydrates in orange trees

To understand the effect of summer and winter on the relationships between leaf carbohydrate and photosynthesis in citrus trees growing in subtropical conditions, 'Valencia' orange trees were subjected to external manipulation of their carbohydrate concentration by exposing them to darkness and evaluating the maximal photosynthetic capacity. In addition, the relationships between carbohydrate and photosynthesis in the citrus leaves were studied under natural conditions. Exposing the leaves to dark conditions decreased the carbohydrate concentration and increased photosynthesis in both seasons, which is in accordance with the current model of carbohydrate regulation. Significant negative correlations were found between total non-structural carbohydrates and photosynthesis in both seasons. However, non-reducing sugars were the most important carbohydrate that apparently regulated photosynthesis on a typical summer day, whereas starch was important on a typical winter day. As a novelty, photosynthesis stimulation by carbohydrate consumption was approximately three times higher during the summer, i.e. the growing season. Under subtropical conditions, citrus leaves exhibited relatively high photosynthesis and high carbohydrate levels on the summer day, as well as a high nocturnal consumption of starch and soluble sugars. A positive association was determined between photosynthesis and photoassimilate consumption/exportation, even in leaves showing a high carbohydrate concentration. This paper provides evidence that photosynthesis in citrus leaves is regulated by an increase in sink demand rather than by the absolute carbohydrate concentration in leaves.