Citrus Genotypes Research Articles

Identification of Gene Candidates Associated with Huanglongbing Tolerance, Using 'Candidatus Liberibacter asiaticus' Flagellin 22 as a Proxy to Challenge Citrus.

The 22-amino acid (flg22) pathogen-associated molecular pattern from the flagellin of Xanthomonas citri subsp. citri has been shown to induce defense responses correlated with citrus canker resistance. Here, flg22 of 'Candidatus Liberibacter asiaticus', the putative causal agent of Huanglongbing (HLB), elicited differential defense responses that were weaker than those from Xcc-flg22, between those of the HLB-tolerant mandarin cultivar Sun Chu Sha and susceptible grapefruit cultivar Duncan. Transcriptomics was used to compare the effect of CLas-flg22 and Xcc-flg22 between the citrus genotypes and identified 86 genes induced only by CLas-flg22 in the tolerant mandarin. Expression of 16 selected genes was validated, by reverse transcription-quantitative polymerase chain reaction, and was evaluated in citrus during 'Ca. L. asiaticus' infection. Differential expression of a number of genes occurred between tolerant and susceptible citrus infected with 'Ca. L. asiaticus', suggesting their involvement in HLB tolerance. In addition, several genes were similarly regulated by CLas-flg22 and 'Ca. L. asiaticus' treatments, while others were oppositely regulated in the tolerant mandarin, suggesting similarity and interplay between CLas-flg22 and 'Ca. L. asiaticus'-triggered defenses. Genes identified are valuable in furthering the study of HLB tolerance mechanisms and, potentially, for screening for HLB-tolerant citrus using CLas-flg22 as a pathogen proxy.

Citrus plants exude proline and phytohormones under abiotic stress conditions.

This article describes the root exudation of proline and phytohormones in citrus and their involvement in salt- and heat-stress responses. Plants are constantly releasing several compounds to the rhizosphere through their roots, including primary and secondary metabolites. Root exudation can be affected by growth conditions, including pH, nutrient availability, soil salinity, or temperature. In vitro-cultured plants of two citrus genotypes with contrasting tolerance to salt- and heat-stress conditions were used as plant material. Proline and phytohormone contents in root exudates from plants subjected to salt or high-temperature conditions were evaluated. In addition, tissue damage and lipid peroxidation together with endogenous levels of chloride, proline, and phytohormones were determined in roots and shoots. Proline was released in larger quantities to the rhizosphere when plants were subjected to salt or heat stress. In each stress condition, the concentration of this amino acid was higher in the exudates obtained from plants tolerant to this particular stress condition. On the other hand, root exudation of phytohormones salicylic acid, indole acetic acid, abscisic acid, and jasmonic acid generally increased under both adverse conditions. Results confirm a phytohormone exudation in citrus plants, which had not been described previously and can have an important role in the rhizosphere communication. Moreover, stress conditions and the different tolerance of each genotype to the particular stress significantly modify the exudation pattern both quantitatively and qualitatively.

Comparative transcriptome analysis during early fruit development between three seedy citrus genotypes and their seedless mutants

Identification of genes with differential transcript abundance (GDTA) in seedless mutants may enhance understanding of seedless citrus development. Transcriptome analysis was conducted at three time points during early fruit development (Phase 1) of three seedy citrus genotypes: Fallglo (Bower citrus hybrid (Citrus reticulata×C. reticulata×C. paradisi)×Temple (C. reticulata×C. sinensis)), grapefruit (C. paradisi), Pineapple sweet orange (C. sinensis), and their seedless mutants. Seed abortion in seedless mutants was observed at 26 days post anthesis (Time point 2). Affymetrix transcriptomic analysis revealed 359 to 1077 probe sets with differential transcript abundance in the comparison of seedless versus seedy fruits for each citrus genotypes and time points. The GDTA identified by 18 microarray probe sets were validated by qPCR. Hierarchical clustering analysis revealed a range of GDTA associated with development, hormone and protein metabolism, all of which may reflect genes associated with seedless fruit development. There were 14, 9 and 12 genes found exhibiting similar abundance ratios in all three seedless versus seedy genotype comparisons at time point 1, 2 and 3, respectively. Among those genes were genes coding for an aspartic protease and a cysteine protease, which may play important roles in seedless fruit development. New insights into seedless citrus fruit development may contribute to biotech approaches to create seedless cultivars.

Comparative analysis of juice volatiles in selected mandarins, mandarin relatives and other citrus genotypes

Citrus fruit flavor is an important attribute prioritized in variety improvement. The present study compared juice volatiles compositions from 13 selected citrus genotypes, including six mandarins (Citrus reticulata), three sour oranges (Citrus aurantium), one blood orange (Citrus sinensis), one lime (Citrus limonia), one Clementine (Citrus clementina) and one satsuma (Citrus unshiu). Large differences were observed with respect to volatile compositions among the citrus genotypes. 'Goutou' sour orange contained the greatest number of volatile compounds and the largest volatile production level. 'Ponkan' mandarin had the smallest number of volatiles and 'Owari' satsuma yielded the lowest volatile production level. 'Goutou' sour orange and 'Moro' blood orange were clearly distinguished from other citrus genotypes based on the analysis of volatile compositions, even though they were assigned into one single group with two other sour oranges by the molecular marker profiles. The clustering analysis based on the aroma volatile compositions was able to differentiate mandarin varieties and natural sub-groups, and was also supported by the molecular marker study. The gas chromatography-mass spectrometry analysis of citrus juice aroma volatiles can be used as a tool to distinguish citrus genotypes and assist in the assessment of future citrus breeding programs. The aroma volatile profiles of the different citrus genotypes and inter-relationships detected among volatile compounds and among citrus genotypes will provide fundamental information on the development of marker-assisted selection in citrus breeding. © 2017 Society of Chemical Industry.

Root morphology and the effect of rootstocks on leaf nutrient acquisition of Kinnow mandarin (Citrus nobilis Loureiro × Citrus reticulata Blanco)

ABSTRACTThe study aimed to understand the root morphological character of 1 year old seedling rootstock of citrus genotypes viz., rough lemon, Karna Khatta, Carrizo citrange, Rangpur lime, Troyer citrange, Jatti Khatti and sour orange as well as to investigate the effect of these rootstocks on the leaf mineral composition of Kinnow. Root morphological traits showed significant genotypic variation. Rough lemon seedlings had a higher root length, projected area, surface area, volume and the number of forks. Sour orange was moderate rootstock of the 7 genotypes for most of the root morphological characters, while citrange rootstocks expressed poor root morphology. The rootstocks also imparted significant influence on the leaf mineral composition. Leaf K, Ca and Mg content was recorded maximum in plants grown on rough lemon seedling, while N, P, Fe, Zn, Mn and Cu concentration was higher on sour orange rootstock. Kinnow trees on Rangpur lime rootstock accumulated more Na in their leaf tissue, where as Troyer citrange restricted the accumulation of Na. Considering the root morphological characters and nutrient accumulation pattern, rough lemon and sour orange for areas free from tristeza virus could be suggested as alternative rootstocks for the studied Kinnow mandarin under similar ecological conditions.

Modulation of Antioxidant Defense System Is Associated with Combined Drought and Heat Stress Tolerance in Citrus.

Drought and high temperatures are two major abiotic stress factors that often occur simultaneously in nature, affecting negatively crop performance and yield. Moreover, these environmental challenges induce oxidative stress in plants through the production of reactive oxygen species (ROS). Carrizo citrange and Cleopatra mandarin are two citrus genotypes with contrasting ability to cope with the combination of drought and heat stress. In this work, a direct relationship between an increased antioxidant activity and stress tolerance is reported. According to our results, the ability of Carrizo plants to efficiently coordinate superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) activities involved in ROS detoxification along with the maintenance of a favorable GSH/GSSG ratio could be related to their relative tolerance to this stress combination. On the other hand, the increment of SOD activity and the inefficient GR activation along with the lack of CAT and APX activities in Cleopatra plants in response to the combination of drought and heat stress, could contribute to an increased oxidative stress and the higher sensibility of this citrus genotype to this stress combination.

Gas exchange of citrus rootstocks in response to intensity and duration of saline stress

Soil and water salinity cause physiological disorders in sensitive plants, such as altered gas exchange in citrus genotypes. However, it is possible to analyse these effects and to identify genotypes tolerant to salt stress. An experiment was carried out in order to evaluate the tolerance of citrus genotypes considering the irrigation with saline water during rootstock formation. The study took place under greenhouse conditions in Pombal county, Paraiba, Brazil. A randomised block design was used, with treatments arranged in a factorial scheme (5 x 8). The two factors were: [i] five salinity levels of irrigation water (0.8, 1.6, 2.4, 3.2 and 4.0 dS m-1) and [ii] eight genotypes of citrus rootstocks: 1. ‘Rangpur Santa Cruz’ lime (LCRSTC); 2. common ‘Sunki’ mandarin (TSKC) x ‘Swingle’ citrumelo (CTSW) - 028; 3. TSKC x CTSW - 033; 4. TSKC x CTSW - 041; 5. ‘Volkamer’ lemon (LVK) x ‘Rangpur’ lime (LCR) - 038; 6. ‘Sunki of Florida’ mandarin (TSKFL); 7. TSKC and 8. ‘Florida’ rough lemon (LRF). Gas exchange was evaluated at 15, 30 and 60 days after the beginning of the saline water application. High water salinity reduces gas exchange in citrus genotypes, which was more evident at 15 days from the beginning of stress. The genotypes ‘Santa Cruz Rangpur’ lime, TSKC x CTSW - 041, LVK x LCR - 038 and ‘Florida’ rough lemon exhibit a satisfactory physiological behaviour during the first 30 days of exposure to the stress, showing moderate tolerance to salt stress. The genotypes TSKC x CTSW - 033 and common ‘Sunki’ mandarin are the most sensitive to salinity.

Biochemical components and dry matter of lemon and mandarin hybrids under salt stress

ABSTRACT The objective was to study the biochemical changes and dry matter content in lemon and mandarin hybrids under salt stress during rootstock formation. For this, a study was conducted in randomized complete block, using a 2 x 5 factorial scheme, with two salinity levels (0.3 and 4.0 dS m-1) applied in five citrus rootstock genotypes (1. TSKC x CTARG - 019; 2. LRF; 3. TSKC x (LCR x TR) - 040; 4. LCRSTC and 5. LVK), with three replicates and four plants per plot. At 90 days after sowing, saline treatments started to be applied and continued until 120 days after sowing, the moment in which the plants were collected for evaluation of biochemical characteristics and phytomass accumulation. The increase in water salinity negatively affected the biochemical components and dry matter accumulation of citrus genotypes. The genotypes TSKC x (LCR x TR) - 040, LCRSTC and LVK were the least affected by salt stress, standing out as the materials most tolerant to salinity.

Evaluation of parameters affecting Agrobacterium-mediated transient expression in citrus

Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple, easy to perform, and achieve gene expression rapidly. This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope. These factors included the composition of the infiltration buffer, the Agrobacterium cell density, the leaf development stage, the incubation temperature, and plant genotype. The highest transient expression level of yellow fluorescent protein (YFP) was detected in Mexican lime (Citrus aurantifolia) on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1 (15 mmol L−1 2-(N-morpholino) ethanesulfonic acid, 10 mmol L−1 MgCI2, and 200 pmol L−1 acetosyringone), which had an optical density of 0.8 and was incubated at 22°C. Additionally, this transient expression assay was applied to other citrus genotypes. Of note, trifoliate orange (Poncirus trifoliata) and kumquat (Fortunella obovate) had higher expression efficiency than other six genotypes of the Citrus genus. Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.

Ectopic accumulation of linalool confers resistance to Xanthomonas citri subsp. citri in transgenic sweet orange plants.

In order to clarify whether high linalool content in citrus leaves alone induces strong field resistance to citrus canker caused by Xanthomonas citri subsp. citri (Xcc), and to assess whether this trait can be transferred to a citrus type highly sensitive to the bacterium, transgenic 'Hamlin' sweet orange (Citrus sinensis L. Osbeck) plants over-expressing a linalool synthase gene (CuSTS3-1) were generated. Transgenic lines (LIL) with the highest linalool content showed strong resistance to citrus canker when spray inoculated with the bacterium. In LIL plants inoculated by wounding (multiple-needle inoculation), the linalool level was correlated with the repression of the bacterial titer and up-regulation of defense-related genes. The exogenous application of salicylic acid, methyl jasmonate or linalool triggered responses similar to those constitutively induced in LIL plants. The linalool content in Ponkan mandarin leaves was significantly higher than that of leaves from six other representative citrus genotypes with different susceptibilities to Xcc. We propose that linalool-mediated resistance might be unique to citrus tissues accumulating large amounts of volatile organic compounds in oil cells. Linalool might act not only as a direct antibacterial agent, but also as a signal molecule involved in triggering a non-host resistance response against Xcc.

English

Mathematical models were developed, using 22 different genotypes of citrus, to estimate leaf area. The information of the relationship between leaf length and width (L/W)2 for simple leaf blade form (eliptic, ovate, obovate, lanceolate); and length of the three folioles (L2+L3)/L1 for a compound leaf (trifoliate leaves), was used with the purpose to separate group of similarities of leaf blade form and promote high accuracy of estimate. The best models presented an excellent precision with errors varying from 1.2 to 6.2 (%) and r2 higher than 0.95 for the majority of the models tested. Considering a single leaf blade, the linear model (Y = β . L . W) presented the lower mean deviation and lower square deviation. For the compound leaves, the potential models are simple to use, since use only the information of length of central foliole L1 (Y= β L1µ), although the use of linear models gave the best precision, as observed by using the model Y = β . L1 . W1. Furthermore the model might be used as a single model independent of the relation (L2+L3)/L1∶ {Y=β⋅(L1⋅W1 + L2⋅W2 + L3⋅W3), r² = 0.98}. Key words: Citrus, compound leaf, hybrids, leaf area modeling.

Activation of Secondary Metabolism in Citrus Plants Is Associated to Sensitivity to Combined Drought and High Temperatures.

Drought and heat stresses are two of the most frequent environmental factors that take place simultaneously in the field constraining global crop productivity. Metabolism reconfiguration is often behind the adaptation of plants to adverse environmental conditions. Carrizo citrange and Cleopatra mandarin, two citrus genotypes with contrasting ability to tolerate combined heat and drought conditions, showed different metabolite patterns. Increased levels of phenylpropanoid metabolites were observed in Cleopatra in response to stress, including scopolin, a metabolite involved in defense mechanisms. Tolerant Carrizo accumulated sinapic acid and sinapoyl aldehyde, direct precursors of lignins. Finally, Cleopatra showed an accumulation of flavonols and glycosylated and polymethoxylated flavones such as tangeritin. The activation of flavonoid biosynthesis in Cleopatra could be aimed to mitigate the higher oxidative damage observed in this genotype. In general, limonoids were more severely altered in Cleopatra than in Carrizo in response to stress imposition. To conclude, all metabolite changes observed in Cleopatra suggest the activation of energy metabolism along with metabolic pathways leading to the accumulation of photoprotective and antioxidant secondary metabolites, oriented to mitigate the damaging effects of stress. Conversely, the higher ability of Carrizo to retain a high photosynthetic activity and to cope with oxidative stress allowed the maintenance of the metabolic activity and prevented the accumulation of antioxidant metabolites.

Apparent Tolerance to Huanglongbing in Citrus and Citrus-related Germplasm

In a Fort Pierce, FL, field planting, plant growth, and Huanglongbing (HLB) severity were assessed as indicators of HLB tolerance on progenies of 83 seed-source accessions of Citrus and Citrus relatives mainly from the Riverside, CA, genebank. The HLB-associated pathogen [Candidatus Liberibacter asiaticus (CLas)] and vector [asian citrus psyllid (ACP), Diaphorina citri] were abundant, and trees were naturally challenged for 6 years before metrics (leaf mottle, percent canopy mottle, overall health, canopy density, canopy width, canopy height, and trunk diameter) were collected in Oct. and Nov. 2015. The healthiest trees with low or no HLB symptoms were distant citrus relatives: Balsamocitrus dawei, Bergera koenigii, Casimiroa edulis, Clausena excavata, Murraya paniculata, and one accession of Severinia buxifolia. Within Citrus, most of the healthiest trees with densest canopies, little leaf loss, and greater growth were those with pedigrees that included Citrus medica (citron). These included progenies of Citrus hybrid (‘Limon Real’), Citrus limetta, Citrus limettioides, Citrus limonia, C. medica, Citrus volkameriana, and some Citrus limon accessions. Trees in this category exhibited distinct leaf-mottle characteristic of HLB and substantial pathogen titers, but maintained dense canopies and exhibited good growth. Trees from seed-source accessions in the genus Citrus without citron in their background were generally among the least healthy overall with less dense canopies. The exceptions were progenies of two Citrus aurantium accessions, which were markedly healthier than progenies of other Citrus seed-source accessions not derived from citron. Linear regression analysis, between metrics collected and pedigree of seed parent, indicated that percentage of citron in the pedigree significantly correlated with measures of tolerance. Although no commercial Citrus genotypes yielded progenies with strong HLB resistance, in this field experiment several progenies maintained dense canopies and good growth, and may be useful for breeding HLB tolerant cultivars.

Morphological and genetic diversity in citrus genotypes to substantiate rootstock breeding for root rot resistance

Investigations were conducted on six citrus rootstocks, viz., rough lemon (RL), trifoliate orange (TO), Swingle citrumelo (SC), X 639 (X), Rangpur lime (RA) and Gou Tou (GT), to assess the morphological and genetic variability; and reaction against Phytophthora. Smooth trunk surface was recorded in all the genotypes. Three genotypes had erect growth habit, while it was spreading in Gou Tou (GT), Rangpur lime (RA) and rough lemon (RL). The leaves of GT, RA and RL were of unifoliate, while trifoliate orange (TO) had trifoliate leaf and Swingle citrumelo (SC) and X 639 (X) had multifoliate leaf division. No variation was recorded with respect to flowering season in all citrus rootstocks, i.e. mid February to last week of March. Fifty-five SSR markers were used for evaluation of genetic diversity amongst the six rootstocks. Twenty markers exhibited high polymorphism and showed wide allelic diversity. Capacity of each SSR to show polymorphic loci, varied from 0.29 (F98) to 0.83 (CCSME46 and CCSMEc4) with an average PIC value of 0.61. The resolving power (Rp) was highest for the primer CCSME43 (8.33) and was lowest for F90 (1.33). Significant differences were observed in the value of MI and were found to be the highest for primer F40 (9.25), while minimum MI was recorded for F98 (0.38). The PIC values of a marker vary with the crop and the set of the genotypes used. The reduction in the number of sporangia and lesion size in trifoliate orange and Swingle citrumelo indicated their tolerance against Phytophthora nicotianae var. parasitica. The number of sporangia counted after 48 h of incubation showed that all leaf baits of each rootstock were attacked by large number of sporangia. The number of sporangia on each leaf disc of rootstocks decreased after 48 h as the sporangia germinated into mycelium on the edges of leaf discs.

Fine Mapping for Identification of Citrus Alternaria Brown Spot Candidate Resistance Genes and Development of New SNP Markers for Marker-Assisted Selection.

Alternaria brown spot (ABS) is a serious disease affecting susceptible citrus genotypes, which is a strong concern regarding citrus breeding programs. Resistance is conferred by a recessive locus (ABSr) previously located by our group within a 3.3 Mb genome region near the centromere in chromosome III. This work addresses fine-linkage mapping of this region for identifying candidate resistance genes and develops new molecular markers for ABS-resistance effective marker-assisted selection (MAS). Markers closely linked to ABSr locus were used for fine mapping using a 268-segregating diploid progeny derived from a heterozygous susceptible × resistant cross. Fine mapping limited the genomic region containing the ABSr resistance gene to 366 kb, flanked by markers at 0.4 and 0.7 cM. This region contains nine genes related to pathogen resistance. Among them, eight are resistance (R) gene homologs, with two of them harboring a serine/threonine protein kinase domain. These two genes along with a gene encoding a S-adenosyl-L-methionine-dependent-methyltransferase protein, should be considered as strong candidates for ABS-resistance. Moreover, the closest SNP was genotyped in 40 citrus varieties, revealing very high association with the resistant/susceptible phenotype. This new marker is currently used in our citrus breeding program for ABS-resistant parent and cultivar selection, at diploid, triploid and tetraploid level.

Influence of Photoperiod Duration and Phloem Disruption through Scoring on Growth, Disease Symptoms, and Bacterial Titer in Citrus Graft Inoculated with Candidatus Liberibacter asiaticus

Plants inoculated with the huanglongbing (HLB)-associated bacterium, Candidatus Liberibacter asiaticus (CLas) typically must be monitored for 8–10 months to identify differences in susceptibility between genotypes. Continuous light is reported to accelerate development of HLB symptoms and field observations suggest that trees girdled by tags or tree ties showed greater symptoms. Therefore, an experiment was conducted assessing HLB susceptibility as influenced by light/dark periods of 12 hours: 12 hours and 24 hours: 0 hours, in combination with scoring tree trunks to disrupt phloem. Sixty trees of each of three citrus genotypes (‘Kuharske’, previously shown to be HLB resistant; rough lemon, previously shown to be HLB tolerant; and ‘Valencia’, highly HLB susceptible) were bud grafted using two CLas-infected buds (rough lemon and citron) per tree on 26 Mar. 2012, and were placed in controlled growth rooms (one 12 hour light: 12 hour dark and one constant light) on 4 June 2012. Ten trees of each genotype in each growth room were scored 10 cm above the soil (cutting through the bark but not the wood) with a knife on 18 July 2012 and the scoring was repeated at the same scoring wounds on 30 Aug. 2012. Trees were removed from growth rooms on 12 Dec. 2012 and subsequently maintained in a greenhouse. At two to three month intervals between June 2012 and May 2013, HLB symptoms and stem diameter at 5 cm above the soil were assessed, and three leaves per tree were collected for quantitative polymerase chain reaction (qPCR) determination of CLas titer. Six months after inoculation and 3 months following imposition of treatments, the ‘Valencia’ scored in the 12 hour light: 12 hour dark regime, the ‘Valencia’ non scored trees in 24 hours of light and the ‘Kuharske’ scored trees in 24 hours of light displayed higher CLas titers than most other trees. After an additional two months, both scored and non-scored trees of all three genotypes in 24 hours of light had significantly elevated CLas titers compared with trees in 12 hour light: 12 hour dark regime, but within most treatments all three genotypes had titers which were not statistically different from each other. Growth of ‘Kuharske’ and rough lemon was enhanced; whereas ‘Valencia’ growth was reduced when graft-inoculated plants were maintained in continuous light. Scoring enhanced early CLas development in ‘Kuharske’ when combined with continuous light, had no effect in rough lemon, and showed inconsistent effects in ‘Valencia’. Although continuous lighting enhanced disease progression, it did not reveal differences in HLB susceptibility.

Proteomic analysis highlights the role of detoxification pathways in increased tolerance to Huanglongbing disease.

BackgroundHuanglongbing (HLB) disease is still the greatest threat to citriculture worldwide. Although there is not any resistance source in the Citrus germplasm, a certain level of moderated tolerance is present. A large-scale analysis of proteomic responses of Citrus may help: 1) clarifying physiological and molecular effects of disease progression, 2) validating previous data at transcriptomic level, and 3) identifying biomarkers for development of early diagnostics, short-term therapeutics and long-term genetic resistance.ResultsIn this work we have conducted a proteomic analysis of mature leaves of two Citrus genotypes with well-known differing tolerances to HLB: Navel orange (highly susceptible) and Volkameriana (moderately tolerant). Pathway enrichment analysis showed that amino acid degradation processes occurred to a larger degree in the Navel orange. No clear differences between the two genotypes were observed for primary metabolic pathways. The most important finding was that four glutathione-S-transferases were upregulated in Volkameriana and not in Navel orange. These proteins are involved in radical ion detoxification.ConclusionsUpregulation of proteins involved in radical ion detoxification should be considered as an important mechanism of increased tolerance to HLB.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0858-5) contains supplementary material, which is available to authorized users.

The evolution of citrate metabolism in acidic and acidless citrus genotypes during fruit development and ripening

In the present work, citric acid accumulation and degradation was monitored during fruit development and ripening in two acidless varieties, Tarocco Ferreri (TF) and Vaniglia biondo (VB), in comparison with three acidic ones, namely Lanelate (L), Tarocco Meli (TM) and Mandalate [Fortune mandarin (2x) × Avana mandarin (4x)] (M), a tryploid hydrid. We evaluated titratable acidity (TA), pH and expression of genes involved in the critical steps of Krebs cycle, GABA shunt, acetyl-CoA metabolism and 'acid trap' from about 100 days post anthesis (DPA) until ripening. Based on the correlation between TA and pH, TF could not be considered a true ''acidless' as VB: even if the acidity levels of TF and VB at maturity reached lower values compared to acidic varieties (0.1 versus 1.6), the relation between TA and pH was negatively correlated in acidic varieties and TF, but weakly positively correlated in VB. The maximum of acidity in VB was identified at 160 DPA (0.28), while surprisingly TF anticipated the peak of acidity at 100 DPA, showing a higher value (2.35). Gene expression data showed marked differences between acidic and acidless varieties, as well as within acidic and within acidless ones. The main differences between acidic and acidless varieties were related to genes involved in Krebs cycle, as citrate synthase (CS), mitochondrial isoforms of aconitase (ACO) and NADP-isocitrate dehydrogenase (NADP-IDH), higher expressed in acidic varieties. We also observed that differences in gene expression within the acidic varieties are likely associated with differences in their citrate content. Specifically, CS, NADP-IDH and glutamate decarboxylase (GAD) are higher expressed in M, while ACO and ATP citrate lyase (ATP-CL) in TM, all compared to L. Differences between TF and VB were found in the expression of NADP-IDH, GAD, ATP-CL and H+ transporting ATPase isoform I, supporting the hypothesis of a different genetic basis of the acidless character in the two genotypes.

Influence of Citrus Source and Test Genotypes on Inoculations with Candidatus Liberibacter asiaticus

Assessments of the resistance of citrus germplasm to huanglongbing (HLB) can be expedited by inoculating plants under laboratory or greenhouse settings with the HLB bacterium, Candidatus Liberibacter asiaticus (CLas). Consistent rapid screening is critical to efficiently assess disease resistance among plant materials; however, a number of factors may govern the efficacy of such inoculations. Despite the rapidity at which HLB can spread in a grove, it often takes 8 to 10 months for high levels of CLas and HLB symptoms to develop even in highly susceptible sweet orange. Therefore, two experiments were conducted to assess factors that might influence efficiency in screening for HLB resistance. In one experiment, three test citrus genotypes (‘Kuharske’, previously shown to be HLB resistant; rough lemon, previously shown to be HLB tolerant; and ‘Valencia’, HLB susceptible) were bud grafted using CLas-infected buds from four different source genotypes. All bud source genotypes had similar levels of CLas titer, but citron, rough lemon, and Volkamer lemon were hypothesized to be better bud inoculum sources as they are more tolerant of HLB than ‘Valencia’. Among the three test genotypes over all sources of infected buds, inoculations of ‘Kuharske’ resulted in lower CLas titers and fewer HLB symptoms than inoculations of rough lemon or ‘Valencia’. Inoculations of rough lemon resulted in higher CLas titers and more pronounced HLB symptoms when it was inoculated using infected buds from rough lemon or ‘Valencia’. Grafting ‘Valencia’ with infected buds from Volkamer lemon resulted in less disease than when ‘Valencia’ was grafted with infected citron, rough lemon, or ‘Valencia’ buds. Overall, these results suggest that the source of CLas-infected buds used to graft-inoculate some genotypes will influence disease development. Trunk cross-sectional area increase for the year following infection was 3× higher in ‘Kuharske’ and rough lemon, compared with ‘Valencia’. ‘Kuharske’ had very low levels of CLas (30 CLas/µg DNA), whereas ‘Valencia’ (43,000 CLas/µg DNA) and rough lemon (6700 CLas/ µg DNA) had relatively high levels. As an alternative to graft-inoculating plants with CLas-infected buds, plants can be subjected to infestations of CLas-infected Asian citrus psyllid (ACP) as occurs naturally. Of interest is if transmission rates of CLas and the development of HLB in a genotype are greater when the ACP have been feeding on the same host genotype. An experiment was therefore conducted to assess transmission of CLas by ACP reared on CLas-infected rough lemon to five different genotypes (‘Carrizo’, ‘Flame’ grapefruit, rough lemon, ‘Temple’, and ‘Valencia’). These assessments were made using a detached leaf assay recognized as a faster method of gauging transmission rates of CLas than using whole plants. Higher percentages of ACP died when they were transferred from infected rough lemon to healthy ‘Carrizo’, and lower percentages died when they were transferred to rough lemon or ‘Flame’. However, CLas transmission by infected ACP occurred to at least some leaves of each genotype in each of the five different assays, with 70% or more leaves of each genotype becoming infected in at least one assay. Over all assays, there was relatively little variation among genotypes in the percentage of leaves becoming CLas infected and in the titer of CLas developing in infected leaves. However, there were relatively large differences in transmission rates among individual assays unrelated to differences among test genotypes. Because of the rapidity of the detached leaf assay, efforts are merited to improve consistency of this inoculation method.

Essential Oil Variation from Twenty Two Genotypes of Citrus in Brazil-Chemometric Approach and Repellency Against Diaphorina citri Kuwayama.

The chemical composition of volatile oils from 22 genotypes of Citrus and related genera was poorly differentiated, but chemometric techniques have clarified the relationships between the 22 genotypes, and allowed us to understand their resistance to D. citri. The most convincing similarities include the synthesis of (Z)-β-ocimene and (E)-caryophyllene for all 11 genotypes of group A. Genotypes of group B are not uniformly characterized by essential oil compounds. When stimulated with odor sources of 22 genotypes in a Y-tube olfactometer D. citri preferentially entered the arm containing the volatile oils of Murraya paniculata, confirming orange jasmine as its best host. C. reticulata × C. sinensis was the least preferred genotype, and is characterized by the presence of phytol, (Z)-β-ocimene, and β-elemene, which were not found in the most preferred genotype. We speculate that these three compounds may act as a repellent, making these oils less attractive to D. citri.