Citrange Research Articles

Transcription profiles of boron-deficiency-responsive genes in citrus rootstock root by suppression subtractive hybridization and cDNA microarray.

Boron (B) deficiency has seriously negative effect on citrus production. Carrizo citrange (CC) has been reported as a B-deficiency tolerant rootstock. However, the molecular mechanism of its B-deficiency tolerance remained not well-explored. To understand the molecular basis of citrus rootstock to B-deficiency, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes responsive to B-deficiency. Firstly four SSH libraries were constructed for the root tissue of two citrus rootstocks CC and Trifoliate orange (TO) to compare B-deficiency treated and non-treated plants. Then 7680 clones from these SSH libraries were used to construct a cDNA array and microarray analysis was carried out to verify the expression changes of these clones upon B-deficiency treatment at various time points compared to the corresponding controls. A total of 139 unigenes that were differentially expressed upon B-deficiency stress either in CC or TO were identified from microarray analysis, some of these genes have not previously been reported to be associated with B-deficiency stress. In this work, several genes involved in cell wall metabolism and transmembrane transport were identified to be highly regulated under B-deficiency stress, and a total of 23 metabolic pathways were affected by B-deficiency, especially the lignin biosynthesis pathway, nitrogen metabolism, and glycolytic pathway. All these results indicated that CC was more tolerant than TO to B-deficiency stress. The B-deficiency responsive genes identified in this study could provide further information for understanding the mechanisms of B-deficiency tolerance in citrus.

Early Evaluation of Compatibility between Commercial Citrus Varieties and Kaffir Lime (Citrus hystrix) and Carrizo Citrange (C. sinensis Osb. x P. trifoliata L. Raf.) Rootstocks at Mekong Delta, Vietnam

The results of investigation on early evaluation of compatibility between commercial citrus varieties, and Kaffir lime ( Citrus hystrix ) and Carrizo citrange ( C. sinensis Osb. x P. trifoliata L. Raf.) rootstocks, at Mekong Delta Vietnam, during four successive years from 2010 to 2013, reported that out of the tested combinations of the recent commercial citrus varieties, included Da xanh pumelo ( C. grandis Osbeck), seedless Mat orange ( C. sinensis L. Osbeck), Sanh orange ( C . nobilis Lour.) and Duong mandarin ( C. reticulata ), budded on Kaffir lime and Carrizo citrange, in the 3 rd and 4 th year after planting under the trial fields at Mekong Delta Vietnam, the trees of Sanh orange ( C. nobilis Lour.) budded on Kaffir lime ( Citrus hystrix ) were supperior with precocity, producing high number of fruits (16.67 fruits per tree ), and very good fruit quality of larger fruit size (334.6 g and 11.16 cm, fruit weight and diameter, respectively), higher total soluble solids (12.5 brix), better orange-yellow juice color development, and excellent flavor and aroma of juice, as compared to the typical characteristics; meanwhile these were followed by trees of seedless Mat orange budded on Kaffir lime, which produced the highest number of fruits (47.33 fruits per tree), with little inferior quality of seedy fruit (1-3 seeds per fruit), lower total soluble solids (7.33%), and no good flavor and aroma, as somewhat differed from the typical fruit traits.

Characterization of Citrus Rootstocks Under Sriganganagar Conditions of Zone 1B

Investigation was carried out to evaluate and characterize the available citrus rootstocks and to find out the genetic variability and correlation in between the qualitative/quantitative traits. Fully grown, eleven Citrus types i.e. C. maderaspatana, C. jambhiri var. Mitthi, C. Pectinifera, C. Reshni T, Citrumelo (P. trifoliata x C. sinensis), Carrizo Citrange (P. trifoliate x C. paradisi), C. limonia, C. Karna, C. aurantium, C. Jambhiri var. Jatti-katti and C. limon var. Bhadri Lemon were selected for the study. These exhibited variation for different physico-chemical fruit and seed attributes. Variation in seed characters were also recorded for seed shape, number and colour of cotyledons. Variation were noted for quantitative traits like fruit length (24–210 mm) fruit weight (19–1883 g), TSS (7–10); seed length (7–16 mm), hundred seed weight (16–31 g) and seed number per fruit (2–55). In character association analysis, positive and significant correlation were recorded for fruit weight with fruit size, seed weight with seed size and seed weight with seed germination. Thus, the study revealed that significant amount of genetic variability exists among the available rootstocks. Genetic variability and inter relationship of characters may lead to effective selection of rootstock for commercial multiplication and further improvement programme of citrus plants.

'OMICS' AND CHEMICAL APPROACHES USED TO MONITOR IRON-DEFICIENCY IN CITRUS ROOTSTOCKS

Two different ‘omics’ approaches were performed to a better comprehension of biological mechanisms involved in citrus iron (Fe) deficiency. Tips roots from Swingle citrumelo and Carrizo citrange (sensitive and tolerant rootstocks, respectively), growing in pots with control and chlorotic soil, were used for transcriptomic and proteomic analysis. CombiMatrix array was performed to isolate differential genes, among which glutathione peroxidase (GPX), SAUR gene and glutamate dehydrogenase (GDH) showed to be the most involved ones. They were switched on Swingle grown on calcareous conditions compared to Carrizo (in the same soil) and to the same stock in the control soil. The over-expression of GPX could be the effort of plants to neutralize the oxidative environment produced by stress. The involvement of auxin (and as consequence of SAUR gene) in the regulation of Fe deficiency responses is also well known. Both genes were considered in association to peroxidase, ferric chelate reductase activities, iron and chlorophyll content, to monitor the degree of suffering of rootstocks. Among differentially expressed proteins, identified by means of 2D-PAGE and RP-HPLC/nESI-MSMS, a strong down-regulation of cytosolic pyrophosphate-dependent phosphofructokinase I²-subunit and NADPH-isocitrate dehydrogenase could produce plant inability to sustain the energetic request of cell roots.

Improved Salinity Tolerance in Carrizo Citrange Rootstock through Overexpression of Glyoxalase System Genes.

Citrus plants are widely cultivated around the world and, however, are one of the most salt stress sensitive crops. To improve salinity tolerance, transgenic Carrizo citrange rootstocks that overexpress glyoxalase I and glyoxalase II genes were obtained and their salt stress tolerance was evaluated. Molecular analysis showed high expression for both glyoxalase genes (BjGlyI and PgGlyII) in 5H03 and 5H04 lines. Under control conditions, transgenic and wild type plants presented normal morphology. In salinity treatments, the transgenic plants showed less yellowing, marginal burn in lower leaves and showed less than 40% of leaf damage compared with wild type plants. The transgenic plants showed a significant increase in the dry weight of shoot but there are no differences in the root and complete plant dry weight. In addition, a higher accumulation of chlorine is observed in the roots in transgenic line 5H03 but in shoot it was lower. Also, the wild type plant accumulated around 20% more chlorine in the shoot compared to roots. These results suggest that heterologous expression of glyoxalase system genes could enhance salt stress tolerance in Carrizo citrange rootstock and could be a good biotechnological approach to improve the abiotic stress tolerance in woody plant species.

Citrus Viroids: Symptom Expression and Performance of Washington Navel Sweet Orange Trees Grafted on Carrizo Citrange.

Citrus are natural hosts of several viroid species. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are the causal agents of two well-known diseases of citrus, exocortis and cachexia. Other viroids have been found to induce specific symptoms and different degrees of stunting in trees grafted on trifoliate orange and trifoliate orange hybrids. A field assay was initiated in 1989 to establish the effect of CEVd, HSVd, Citrus bent leaf viroid (CBLVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd) on Washington navel sweet orange trees grafted on Carrizo citrange rootstock. Here we report the effect of viroid infection on symptom expression, tree size, fruit production and quality evaluated from 2004 to 2007. Vegetative growth was affected by viroid infection with height and canopy volume being reduced. No bark scaling symptoms were observed in CEVd-infected trees albeit they presented lesions and blisters in the roots. Bark cracking symptoms were consistently observed in CBCVd-infected trees that were smaller with enhanced productivity and fruit size. No major effects were found as a result of infection with CBLVd, HSVd, or CDVd. The quality of the fruits was not affected by viroid infection, except for the low diameter of the fruits harvested from HSVd-infected trees. An interesting effect was identified in terms of tree productivity increase (yield/canopy volume) as a result of infection with CEVd, CDVd, and especially CBCVd.

Assessing Five Citrus Rootstocks for NaCl Salinity Tolerance Using Mineral Concentrations, Proline and Relative Water Contents as Indicators

A B S T R A C T Changes in concentrations of Na, Cl and K, proline and Relative Water Contet (RWC) of five citrus species (Cleopatra mandarin, Carrizo citrange, Tiwanica, Bacraii and Shaker) grown in four NaCl salinity levels (0, 25, 50 and 75 mM NaCl) for the purpose of assessing their magnitude of susceptibility to salinity and estimating the salt tolerance degree were studied. The Na and Cl concentrations increased whilst K concentration in leaves decreased with increasing NaCl concentration in the rootzone. Shaker and Cleopatra mandarin accumulated optimal concentration of the ions in their tissues. Increasing NaCl concentration reduced RWC and increased proline content in plant tissues. Shaker and Cleopatra mandarin rootstocks maintained relatively higher leaf RWC and proline in comparison to other rootstocks. Cleopatra mandarin and Shaker showed to be markedly less affected by salinity up to 50 mM NaCl and could bear the highest salinity levels (75 mM) imposed in this study.

Genetic diversity revealed by physiological behavior of citrus genotypes subjected to salt stress

Different physiological behavior of a wide range of varieties and species belonging to the Citrus genus was analyzed when subjected to salt stress with the aim to seek new sources of tolerance that might be specie-specific. Our goal was to use physiological results obtained along a salt stress in order to clarify if it would be possible to associate them with the known citrus genetic diversity. For that purpose, we have selected 20 different genotypes representing the major species on the basis of the genetic diversity of Citrus genus complemented with one intergeneric hybrid Carrizo citrange (C. sinensis × P. trifoliata). A moderate salt stress of 75 mM of NaCl was applied for 12 weeks. For control plants, the main parameters contributing for more than 25 % to the diversity on the two axes of principal component analysis (PCA) were chlorophyll content, photosynthesis and Fv/Fm under light. However, the dispersal of species and varieties on the PCA did not show any particular structure. Under salt stress condition, four parameters (leaf chloride content, leaf chlorophyll content, photosynthesis and stomatal conductance) contributed more specifically to the dispersion on PCA representation with more than 15 % of contribution for each parameter. Large differences were observed within citrus genus: mandarin and pummelo presented good tolerance to salt stress while citron was very sensitive. Furthermore, all secondary genotypes that presented good tolerance to salt tolerance shared mandarin or pummelo as female parent.

Evidence that ‘flying dragon’ trifoliate orange delays HLB symptom expression for four sweet orange cultivars, Tahiti lime and Okitsu mandarin

Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus and vectored by Diaphorina citri, was first reported in 2004 in Brazil and is currently widespread in Sao Paulo State. Brazil is the world’s largest sweet orange producer and has 49,000 ha cultivated with ‘Tahiti’ lime acid lime. Mandarin cultivation represents 5.5% of total citrus production in the country. In 2001, three experiments were planted in the Citrus Experimental Station (EECB), Bebedouro, Northern Sao Paulo State, where the first HLB symptomatic tree was detected in 2006. The initial objective was to evaluate the performance of ‘Folha Murcha’ sweet orange, ‘Tahiti’ acid lime and ‘Okitsu’ mandarin grafted on twelve rootstocks including Rangpur lime, Swingle citrumelo, Rubidoux and Flying Dragon (FD) trifoliate oranges. Cumulative HLB incidence (CI) was calculated in 2009. Folha Murcha and Tahiti lime trees on FD had lower CI values (6.7 and 10%) than trees on Rangpur lime (33.3 and 80%), Swingle citrumelo (46.7 and 66.7%) and Rubidoux (46.7 and 60%). CI was similar for Okitsu on FD, Carrizo citrange and HRS 827 (11.1%). In another field trial at EECB, Valencia, Hamlin and Natal were planted on FD in November 1994. Drip irrigation was installed in 2001. The first symptomatic plants in the area were detected in November 2008. In 2010, the 16 yr-old trees on FD have a lower CI (5.6%) than 20 yr-old sweet orange trees on Swingle citrumelo in an adjacent trial (CI = 35.4%). Our results indicated that FD rootstock could prolong the longevity of citrus orchards but more well-controlled studies of scion-rootstocks combinations with and without vector management are required.

Genetic transformation of sweet orange to overexpress a CsPR-8 gene aiming for Candidatus Liberibacter asiaticus resistance

A strategy to produce HLB-resistant citrus using genetic engineering is the overexpression of genes identified in the citrus genome. Plants respond to pathogen attacks by producing several pathogenesis-related (PR) proteins. Therefore, individual PR overexpression in transgenic plants can lead to an increased resistance. In this study, we have chosen to use one PR-8 isoform cloned from Citrus sinensis (CsPR-8). The PR-8 is an endochitinase that also has lysozyme activity, to be potentially used against bacterial attacks. We constructed an expression transformation vector (pCAMBIA2201) containing the CsPR-8 gene and the selection gene nptII that confers kanamycin resistance in plants, both driven by the CaMV35S constitutive promoter. Epicotyl segments collected from in vitro seedlings of ‘Hamlin’ sweet orange (Citrus sinensis L. Osbeck) were used for transformation via Agrobacterium tumefaciens strain EHA105. The developed shoots were excised from the explants and in vitro grafted onto Carrizo citrange [C. sinensis x Poncirus trifoliata (L.) Raf] seedlings. The grafted plants were analyzed by PCR, using specific primers for detection of the nptII gene. Acclimation of transgenic plants is under way in order to be transferred to the greenhouse. These plants will be analyzed by Southern blot to confirm the integration of the transgene and by RT-qPCR to evaluate the transgene expression, prior to their evaluation for Candidatus Liberibacter asiaticus resistance.

Fruit yield of common orange varieties in south-east Australia: Initial evaluation versus long term performance

Fruit yield of 12 common orange varieties on either ‘sweet orange’ rootstock or ‘Carrizo citrange’ rootstock growing on light textured soil in south-east Australia were measured from 2000 to 2012. The varieties covered the range from early maturing (April) through to late hanging (March), and the trial was conducted to identify suitable varieties to ensure continuity of common orange supply to service the Australian fresh orange juice market. Based on the initial four seasons’ yield data, ‘Hamlin’ would have been selected for the June–August period, and ‘Salustiana’, which cropped relatively poorly in the initial four years, would have been rejected. Subsequent yields of fruit by ‘Salustiana’ trees meant that its overall cropping levels were similar to ‘Hamlin’, calling into question the adequacy of recommending a variety based on only an initial four years of observation. Although all varieties demonstrated some degree of irregular cropping, only three varieties could confidently be called biennial bearers. The tendency to irregular bearing (biennial or otherwise) was unrelated to a selection's seasonality, suggesting an inadequacy in our current understanding of the importance of the presence of fruit in determining the size of the return crop.

Effects of supplementary potassium nitrate on growth and gas-exchange characteristics of salt-stressed citrus seedlings

We investigated the effects of supplementary KNO3 and NaCl on one-year-old, potted Valencia orange (Citrus sinensis) scions grafted on Iranian mandarin Bakraii [Citrus reticulate × Citrus limetta] (Valencia/Bakraii) and Carrizo citrange [C. sinensis × Poncirus trifoliata] (Valencia/Carrizo) rootstocks. After watering plants for 60 days with 50 mM NaCl, the lowest reduction in dry mass, stomatal conductance, and chlorophyll (Chl) content was found in Valencia/Bakraii. Bakraii accumulated more Cl− and Na+ in roots and transferred less to Valencia leaves compared with Carrizo rootstock. Moreover, higher net photosynthetic rate was found in Valencia/Bakraii than those on Carrizo rootstock. NaCl caused a decrease in the maximal efficiency of PSII photochemistry (Fv/Fm) and effective quantum yield (ΦPSII) but elevated coefficient of nonphotochemical quenching. Salinity reduced Ca2+, Mg2+, and total N contents, and increased Na+/K+ ratio in leaves and roots of both grafting combinations. Salinity increased K+ and proline content in leaves and decreased K+ concentrations in roots of both grafting combinations. In salinized plants, nitrate supplementation (10 mM KNO3) reduced leaf abscission, Cl−, Na+, Na+/K+, and Ca2+ concentrations in leaves and roots of both combinations. K+ and N concentrations and proline increased in leaves of the nitrate-supplemented salinized plants. Supplementary nitrate increased leaf number and area, stem elongation, Chl content, Fv/Fm, and ΦPSII and stimulated photosynthetic activity. Thus, nitrate ameliorated the deleterious effects of NaCl stress and stimulated the plant metabolism and growth. It can be used as a vital treatment under such condition.

First Report of Armillaria Root Rot Caused by Armillaria mellea Infecting Carrizo Citrange and Sour Orange Rootstocks in Turkey.

Citrus rootstocks, Carrizo citrange (Citrus sinensis [L.] Osb. × Poncirus trifoliata [L.] Raf.) and sour orange (C. aurantium L.) grown in containers filled with 5 liters of potting mix of 40% peat and 60% volcanic tuff declined in a 0.2-ha commercial nursery in Adana, Turkey, between 2004 and 2007. Seedlings with symptoms of root rot were found with an average disease incidence of 20% among 1,000 Carrizo citrange seedlings and 10% among 15,000 sour orange seedlings. The potting mixture preparation unit was located next to an oak tree (Quercus sp.) showing symptoms of Armillaria root rot. Six- to 12-month-old seedlings of both rootstocks were stunted and the crowns were necrotic with the presence of white mycelium. Mycelial fans were observed beneath the bark of infected roots and they expanded into the crown. The root systems and nearby potting mix contained rhizomorphs. Thus, Armillaria spp. was suspected as a possible causal agent. Three diseased crowns and three rhizomorphs were surface-sterilized with 1% NaClO for 1 min and cultured on benomyl-dichloran-streptomycin containing selective medium (3) at 25°C in the dark for 1 week. Six isolates transferred to 1.5% malt extract agar at 33°C in the dark for 7 weeks consistently yielded abundant aerial hyphae and mean diameter growth range was 4 to 21 mm and the mycelium margin was regular (1). To confirm pathogen identity, total DNA was extracted using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., CA) directly from 7-day-old cultures grown in potato dextrose broth (PDB). The ribosomal DNA internal transcribed spacer (ITS) region was amplified by PCR using the primer pair ITS1 and ITS4 (5) and sequenced. The sequences were 99% identical to that of Armillaria mellea isolates from Japan (AB510880) and China (KF032535). This confirmed the identity of the causal agent as A. mellea (Vahl.) P. Kumm. Ten 3-month-old seedlings of Carrizo citrange and sour orange were transplanted into steam-sterilized potting mix and inoculated with wood pieces of oak (Quercus sp.) colonized by the fungus (two pieces for each container) (2). The oak wood pieces were sterilized prior to the colonization by the pathogen. Plants were maintained in a greenhouse (23 to 25°C) until symptoms appeared. Ten non-inoculated seedlings from each rootstock served as controls and were maintained in the same environment. After 4 months, the crowns of the seedlings developed necrotic areas and root systems contained rhizomorphs on all inoculated seedlings and fungus was re-isolated from crowns and rhizomorphs. All control plants remained disease-free and no fungus was re-isolated. A. mellea was reported to infect citrus rootstocks in Spain in 1999 (4). To our knowledge, this is the first report of Armillaria root rot caused by A. mellea infecting Carrizo citrange and sour orange rootstocks in Turkey. This indicates that citrus rootstocks could be at risk for infection and sterilization of the potting mix and good sanitation practices in nurseries are very important.

Genetic transformation of sweet oranges to over-express SABP2 gene

Background The history of the world citrus industry is marked by a series of diseases caused by different etiologic agents. Several of them are characterized as biotrophic pathogens like Xanthomonas citri subsp. citri ,t hreeCandidatus Liberibacter spp and Citrus leprosis virus C (CiLV-C). In plant-pathogen interactions, the role of salicylic acid (SA) in activating defense related genes is well recognized [1]. The SABP2 (Salicylic acid-binding protein 2) is required to convert methyl salicylate to SA as part of the signal transduction pathway that activates systemic acquired resistance, induces PR expression, and enhances disease resistance. Due to the reduced or absence of genetic resistance to these pathogens in commercial sweet orange cultivars, the genetic transformation to over-express a gene involved in the defense response of plants is a possible alternative to produce tolerant or resistant plants to biotrophic pathogens. Methods The aim of this study was to produce ‘Hamlin’ sweet orange (Citrus sinensis L. Osb.) transgenic plants, via Agrobacterium tumefaciens, over-expressing the SABP2 gene from sweet orange driven by the constitutive promoter ubiquitin (Ubq10). The SABP2 gene was cloned into pCambia 2301 and inserted into A. tumefaciens EHA 105. The genetic transformation was performed using epicotyl segments from seedlings [2]. Results and conclusions A total of 620 explants in three independent experiments were introduced and approximately 336 shoots were regenerated. The GUS histochemical test was performed and confirmed the transformation of 30 positive shoots. These shoots are being grafted onto Carrizo citrange [Citrus sinensis x Poncirus trifoliata (L.) Raf.] seedlings grown in test tubes containing MS culture medium. The presence of the transgene will be evaluated by PCR using specifics primers that amplify part of the ubiquitin promoter and part of the gene. SABP2 expression levels in transgenic plants will be assessed through qPCR. After bud multiplication, transgenic plants will be evaluated for their response to citrus canker, HLB and leprosis.

Production of Common Sour Orange × Carrizo Citrange Hybrids Using Embryo Rescue

Citrus rootstock breeding is important in improving resistance to diseases, insect pests, and adverse environmental conditions. The majority of citrus species and cultivars have nuceller embryony, which causes some problems for breeding programs. This disadvantage can be eliminated by using embryo rescue. In this research, Common sour orange × Carrizo citrange were crossed in order to produce new rootstocks and immature embryos. The embryos were gathered from the crossed plants 80, 100, and 120 days after pollination. The immature embryos were transferred to 0, 0.5, and 1 mg/L GA3 containing medium to germinate them. In general, the later after pollination that the embryos were harvested, the higher the germination rate. Those harvested at 80 days averaged 32% over all treatments, while those harvested at 100 days averaged 77%, and those harvested at 120 days averaged 98% germination. Embryos on control media averaged 67% germination over all treatments, while those with 0.5 mg/L GA3 averaged 62%, and those with 1 mg/L GA3 in the media averaged 78% germination. The percentage of embryos developing trifoliate leaves for those harvested at 80 days averaged 51% over all treatments, while those harvested at 100 days averaged 24%, and those harvested at 120 days averaged 41%. Embryos on control media averaged 36% trifoliate leaf development over all treatments, while those with 0.5 mg/L GA3 averaged 35%, and those with 1 mg/L GA3 in the media averaged 44% germination.

Identification of Water Stress in Citrus Leaves Using Sensing Technologies

Water stress is a serious concern in the citrus industry due to its effect on citrus quality and yield. A sensor system for early detection will allow rapid implementation of control measures and management decisions to reduce any adverse effects. Laser-induced breakdown spectroscopy (LIBS) presents a potentially suitable technique for early stress detection through elemental profile analysis of the citrus leaves. It is anticipated that the physiological change in plants due to stress will induce changes in the element profile. The major goal of this study was to evaluate the performance of laser-induced breakdown spectroscopy as a method of water stress detection for potential use in the citrus industry. In this work, two levels of water stress were applied to Cleopatra (Cleo) mandarin, Carrizo citrange, and Shekwasha seedlings under the controlled conditions of a greenhouse. Leaves collected from the healthy and stressed plants were analyzed using LIBS, as well as with a spectroradiometer (visible-near infrared spectroscopy) and a thermal camera (thermal infrared). Statistical classification of healthy and stressed samples revealed that the LIBS data could be classified with an overall accuracy of 80% using a Naïve-Bayes and bagged decision tree-based classifiers. These accuracies were lower than the classification accuracies acquired from visible-near infrared spectra. An accuracy of 93% and higher was achieved using a bagged decision tree with visible-near infrared spectral reflectance data.

The physiological and nutritional responses of seven different citrus rootstock seedlings to boron deficiency

Carrizo citrange was the most tolerant citrus rootstock to B-deficiency and some physiological performance could be attributed to the decreased mineral nutrient concentrations caused by B-deficiency. Boron (B) is an essential microelement for normal growth and development in vascular plants, and adequate B nutrition is crucial for agricultural production. Although citrus plants are not classified as the most sensitive species to B-deficiency, the occurrence of B-deficiency has been reported in the major citrus producing countries of the world, including the east and south of China. In this study, in order to evaluate the effects of B-deficiency on plant growth, root-morphological traits, B and other nutritional responses of citrus rootstock and to investigate the relationship between this physiological performance and mineral nutrients seven common rootstock seedlings, including Trifoliate orange (TO), Carrizo citrange (CC), Chongyi tangerine (CT), Red tangerine (RT), Cleopatra mandarin (CM), Fragrant citrus (FC), and Sour orange (SO), were treated by B-deficiency (0 mg L−1) or moderate B (0.25 mg L−1). All the seedlings were grown in hydroponics situation with modified 1/2-strength Hoagland’s solution under greenhouse conditions for 10 weeks. The results showed that B-deficiency inhibited the growth and development of all tested citrus rootstocks, but substantial differences were observed among these rootstocks. Different visible symptoms were observed both in the leaf and root. Corking of the leaf veins and leaf yellowing symptoms were observed on all rootstock genotypes except on CC, which exhibited a little discoloration at the end of the experiment. In addition, root growth of the citrus seedlings were also decreased by B-deficiency, but the decreases were more obvious in TO and FC. It was worth noting that B-deficiency inhibited lateral root growth and development more significantly than tap root, but not in lateral root initiation. The different performance of these rootstock genotypes indicated that CC was the most tolerant while TO was the most sensitive to B-deficiency. In addition, under B-deficiency conditions, not only the B concentration, but also the other mineral nutrient concentrations were influenced, especially in Mg, Fe and Mn. This change in nutrient concentrations might partly contribute to the seedlings’ physiological performances under B-deficiency.

A Transcriptomic Analysis of Sensitive and Tolerant Citrus Rootstocks under Natural Iron Deficiency Conditions

Iron chlorosis is one of the most serious abiotic stresses affecting citrus (Citrus sp.) culture in the Mediterranean Basin. A trial was performed with potted tolerant and sensitive rootstocks that were grown in volcanic and calcareous soils. Microarray analysis allowed for the identification of differentially expressed genes putatively involved in iron (Fe) deficiency. Most of the differentially expressed genes isolated from the root tips were of unknown function; the remaining genes were related to the oxidative stress response (e.g., glutathione peroxidase), hormone metabolism and signaling (e.g., small auxin up RNA family protein genes), biological regulation, protein turnover, and the tricarboxylic acid cycle (e.g., aconitase). Additionally, the majority of the Fe stress-related genes expressed in the sensitive Swingle citrumelo (Citrus paradisi × Poncirus trifoliata) and tolerant Carrizo citrange (Citrus sinensis × P. trifoliata) rootstocks identified using real-time reverse transcription–polymerase chain reaction (RT-PCR) were related to regulation, the oxidative stress response, and hormone metabolism and signaling, thereby confirming the array data. Furthermore, validation of the differentially expressed genes in seven tolerant and sensitive rootstocks grown in a field trial under chlorotic conditions was performed. In general, the gene expression profiles reflect the different responses of rootstocks, possibly as a result of the various genetic mechanisms involved in the response to Fe deficiency. Moreover, the expression of aconitase was analyzed in the roots and juice to evaluate the implication of the different aconitase isoforms (Aco), which are derived from specific cellular compartments, in the different tissues. The involvement of the mitochondrial isoform (Aco2) was directly correlated with the acidity of the juice, whereas the cytosolic one (Aco3), which corresponds to the aconitase isolated during the microarray analysis, was found specifically in the roots.

The Prevalence of the Citrus tristeza virus Trifoliate Resistance Breaking Genotype Among Puerto Rican Isolates.

Citrus tristeza virus (CTV) isolates have been grouped into six genotypes: T3, T30, T36, VT, B165, and resistance breaking (RB) based on symptoms, host range, and genomic sequence data. The RB genotype has recently been identified with the novel property of replicating in trifoliate orange trees, a resistant host for the other five genotypes. Puerto Rican CTV isolate B301 caused mild vein clearing symptoms in Mexican lime but did not induce seedling yellows or stem pitting reactions in appropriate indicator Citrus spp., which are typical host reactions of the isolate T30. The isolate B301 was not detected by the genotype specific primer (GSP), which identifies the CTV-T3, -T30, -T36, -VT, and B165 genotypes. A primer pair for reverse transcription polymerase chain reaction (RT-PCR) amplification of the CTV-RB genotype was designed from the heat shock protein (p65) region based on the complete genomic sequences of trifoliate RB isolates from New Zealand available in the GenBank databases. The amplicon sequence from isolate B301 was 98% identical to that of the other trifoliate RB isolates. In addition, B301 was successfully inoculated into 'Carrizo citrange' (a trifoliate hybrid) but did not induce any symptoms. Furthermore, the complete genome sequence of B301 followed by the phylogenetic analysis revealed that the isolate is part of the RB clade with other CTV-RB isolates from New Zealand and Hawaii. Additional CTV isolates obtained from Puerto Rico were tested with the RB-GSP and confirmed the presence of trifoliate RB isolates in mixed infection with known CTV genotypes. Although this is the first report of a CTV trifoliate RB genotype from Puerto Rico, this genotype was present there prior to 1992.

Root Protein Profiles of Two Citrus Rootstocks Grown under Iron Sufficiency/Deficiency Conditions

Two citrus rootstocks, one sensitive to iron deficiency (Swingle Citrumelo (SCO)) and the other tolerant (Carrizo Citrange, (CC)), were studied to characterize variation in their root protein profile induced by iron-deficient conditions. Plants of both rootstocks were grown in two different soils, one volcanic (v) and the other calcareous (c), containing 0% and 10% active Lime, respectively. To evaluate the effects of the calcareous soil on the protein accumulation of both rootstocks, the root protein profiles (SCc vs. SCv and CCc vs. CCv) were characterized by two-dimensional gel electrophoresis, thus obtaining, for the first time, a reference map of this previously uncharacterized proteome. A total of 219 spots, significantly changed in abundance, were analyzed by high-performance Liquid chromatography nano electrospray ionization tandem mass spectrometry. The identified proteins were classified according to their putative function and known biosynthetic pathways. Principal component analysis, comparing the four sets of data, indicated that each group clustered together with low variance and that CCv and CCc data sets were well differentiated, whereas SCv and SCc were similar.