Diversity of "Candidatus Liberibacter asiaticus" strains in Texas revealed by prophage sequence analyses.

Prophages/phages are important components of the genome of 'Candidatus Liberibacter asiaticus' (CLas), an unculturable alphaproteobacterium associated with citrus Huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida, but different to those reported in California and China. Further studies were performed on within prophage type diversity. Analyses on Type 1 specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originated from Florida and Type 1-B, clustered with prophage P-SGCA5-1 detected in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2 specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. Results from this study provides an initial baseline information on genomic and population diversity of CLas in Texas.

Sensory quality of Citrus scion hybrids with Poncirus trifoliata in their pedigrees.

Hybrids of Poncirus trifoliata L. Raf. with Citrus have shown degrees of tolerance to the deadly citrus greening disease, hence prompting interest as potential commercial varieties. Although P. trifoliata is known to produce fruit that is inedible, fruit from many advanced hybrid trees have not been evaluated for their quality potential. The sensory quality of selected Citrus hybrids with varying degrees of P. trifoliata in their pedigrees is reported herein. Four Citrus × P. trifoliata hybrids developed through the USDA Citrus scion breeding program-1-76-100, 1-77-105, 5-18-24, and 5-18-31-had acceptable eating quality and sweet and sour taste, with mandarin, orange, fruity-noncitrus, and floral flavors. On the other hand, hybrids with higher proportion of P. trifoliata in their pedigrees, US 119 and 6-23-20, produced a juice characterized by green, cooked, bitter, and Poncirus-like flavor and aftertaste. Partial least square regressions revealed that the Poncirus-like off-flavor is likely due to a combination of higher than typical amounts of sesquiterpene hydrocarbons (woody/green odor), monoterpenes (citrus/pine), and terpene esters (floral) and a lack of aldehydes with typical citrus odor (octanal, nonanal, and decanal). Sweetness and sourness were mostly explained by high sugars and acids, respectively. Further, carvones and linalool contributed to sweetness in the samples from early and late seasons, respectively. In addition to highlighting chemical contributors to sensory descriptors in Citrus × P. trifoliata hybrids, this study provides useful information on sensory quality for future citrus breeding efforts. PRACTICAL APPLICATION: The relationships between the sensory quality and secondary metabolites of Citrus × P. trifoliata hybrids described in this study help identify disease-resistant Citrus scion hybrids with acceptable flavor and help mobilize this resistance in future breeding efforts. It also shows potential of such hybrids to be commercialized.

Genetic modulation of Valencia sweet orange field performance by 50 rootstocks under huanglongbing-endemic conditions.

Although the citrus scion cultivar primarily determines the characteristics of the fruit, the rootstock cultivar of the graft combination has a major role in determining the horticultural performance of the tree. The disease huanglongbing (HLB) is particularly devastating to citrus, and the rootstock has been demonstrated to modulate tree tolerance. However, no existing rootstock is entirely suitable in the HLB-endemic environment, and citrus rootstocks are particularly challenging to breed because of a long life cycle and several biological characteristics that interfere with breeding and commercial use. This study with Valencia sweet orange scion documents the multi-season performance of 50 new hybrid rootstocks and commercial standards in one trial that forms the first wave of a new breeding strategy, with the aim of identifying superior rootstocks for commercial use now, and mapping important traits to be used in selection for the next generation of outstanding rootstocks. A large assortment of traits were quantified for all trees in the study, including traits associated with tree size, health, cropping, and fruit quality. Among the quantitative traits compared between rootstock clones, all except one were observed to have significant rootstock influence. Multiple progeny from eight different parental combinations were included in the trial study, and significant differences between parental combinations of the rootstocks were observed for 27 of the 32 traits compared. Pedigree information was integrated with quantitative trait measurements to dissect the genetic components of rootstock-mediated tree performance. Results suggest there is a significant genetic component underlying rootstock-mediated tolerance to HLB and other critical traits, and that integration of pedigree-based genetic information with quantitative phenotypic data from trials should enable marker-based breeding approaches for the rapid selection of next-generation rootstocks with superior combinations of traits that are needed for commercial success. The current generation of new rootstocks included in this trial is a step toward this goal. Based on results from this trial, the new hybrids US-1649, US-1688, US-1709, and US-2338 were considered the four most promising new rootstocks. Release of these rootstocks for commercial use is being considered, pending the evaluation of continuing performance in this trial and the results from other trials.

SuperSour: A New Strategy for Breeding Superior Citrus Rootstocks.

Citrus crops have a long history of cultivation as grafted trees on selected rootstock cultivars, but all current rootstocks have significant limitations and traditional methods of rootstock breeding take at least 2-3 decades to develop and field test new rootstocks. Citrus production in the United States, and other parts of the world, is impaired by a wide range of biotic and abiotic problems, with especially severe damage caused by the disease huanglongbing (HLB) associated with Candidatus Liberibacter asiaticus. All major commercial citrus scion cultivars are damaged by HLB, but tree tolerance is significantly improved by some rootstocks. To overcome these challenges, the USDA citrus breeding program has implemented a multi-pronged strategy for rootstock breeding that expands the diversity of germplasm utilized in rootstock breeding, significantly increases the number of new hybrids evaluated concurrently, and greatly reduces the time from cross to potential cultivar release. We describe the key components and methodologies of this new strategy, termed "SuperSour," along with reference to the historical favorite rootstock sour orange (Citrus aurantium), and previous methods employed in citrus rootstock breeding. Rootstock propagation by cuttings and tissue culture is one key to the new strategy, and by avoiding the need for nucellar seeds, eliminates the 6- to 15-year delay in testing while waiting for new hybrids to fruit. In addition, avoiding selection of parents and progeny based on nucellar polyembryony vastly expands the potential genepool for use in rootstock improvement. Fifteen new field trials with more than 350 new hybrid rootstocks have been established under the SuperSour strategy in the last 8 years. Detailed multi-year performance data from the trials will be used to identify superior rootstocks for commercial release, and to map important traits and develop molecular markers for the next generation of rootstock development. Results from two of these multi-year replicated field trials with sweet orange scion are presented to illustrate performance of 97 new hybrid rootstocks relative to four commercial rootstocks. Through the first 7 years in the field with endemic HLB, many of the new SuperSour hybrid rootstocks exhibit greatly superior fruit yield, yield efficiency, canopy health, and fruit quality, as compared with the standard rootstocks included in the trials.

Linking metabolic phenotypes to pathogenic traits among "Candidatus Liberibacter asiaticus" and its hosts.

Candidatus Liberibacter asiaticus (CLas) has been associated with Huanglongbing, a lethal vector-borne disease affecting citrus crops worldwide. While comparative genomics has provided preliminary insights into the metabolic capabilities of this uncultured microorganism, a comprehensive functional characterization is currently lacking. Here, we reconstructed and manually curated genome-scale metabolic models for the six CLas strains A4, FL17, gxpsy, Ishi-1, psy62, and YCPsy, in addition to a model of the closest related culturable microorganism, L. crescens BT-1. Predictions about nutrient requirements and changes in growth phenotypes of CLas were confirmed using in vitro hairy root-based assays, while the L. crescens BT-1 model was validated using cultivation assays. Host-dependent metabolic phenotypes were revealed using expression data obtained from CLas-infected citrus trees and from the CLas-harboring psyllid Diaphorina citri Kuwayama. These results identified conserved and unique metabolic traits, as well as strain-specific interactions between CLas and its hosts, laying the foundation for the development of model-driven Huanglongbing management strategies.

An In Vitro Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-"Candidatus Liberibacter asiaticus" Properties.

Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus plants, making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems, and leaves using traditional microbial culturing integrated with culture-independent methods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an in vitro agar diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against Liberibacter crescens, a culturable surrogate for the nonculturable "Candidatus Liberibacter asiaticus" bacterium associated with HLB. We identified microbes with robust inhibitory activity against L. crescens that include the fungi Cladosporium cladosporioides and Epicoccum nigrum and bacterial species of Pantoea, Bacillus, and Curtobacterium Purified bioactive natural products with anti-"Ca. Liberibacter asiaticus" activity were identified from the fungus C. cladosporioides Bioassay-guided fractionation of an organic extract of C. cladosporioides yielded the natural products cladosporols A, C, and D as the active agents against L. crescens This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand the functional roles of members of the microbiome, with the long-term goal of developing anti-"Ca Liberibacter asiaticus" bioinoculants that thrive in the citrus holosystem.IMPORTANCE Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens, a culturable surrogate for the unculturable HLB-associated bacterium "Candidatus Liberibacter asiaticus," we tested the hypothesis that members of the citrus microbiome produce potential anti-"Ca Liberibacter asiaticus" natural products with potential anti-"Ca Liberibacter asiaticus" activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens.

Exogenous application of the plant signalers methyl jasmonate and salicylic acid induces changes in volatile emissions from citrus foliage and influences the aggregation behavior of Asian citrus psyllid (Diaphorina citri), vector of Huanglongbing.

Huanglongbing, also known as citrus greening, is a destructive disease that threatens citrus production worldwide. It is putatively caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (Las). Currently, the disease is untreatable and efforts focus on intensive insecticide use to control the vector, Asian citrus psyllid (Diaphorina citri). Emerging psyllid resistance to multiple insecticides has generated investigations into the use of exogenously applied signaling compounds to enhance citrus resistance to D. citri and Las. In the present study, we examined whether foliar applications of methyl jasmonate (MJ), a volatile signaling compound associated with the induced systemic resistance pathway, and salicylic acid, a constituent of the systemic acquired resistance pathway, would elicit the emission of defense-related volatiles in citrus foliage, and what effect this might have on the host-plant searching behavior of D. citri. Comparisons were made of volatiles emitted from growing shoots of uninfected and Las-infected 'Valencia' sweet orange (Citrus sinensis) trees over two consecutive sampling days. A settling behavioral assay was used to compare psyllid attraction to MJ-treated vs. Tween-treated citrus sprigs. All three main effects, Las infection status, plant signaler application, and sampling day, influenced the proportions of individual volatile compounds emitted in different treatment groups. MJ- and SA-treated trees had higher emission rates than Tween-treated trees. Methyl salicylate (MeSA) and ß-caryophyllene were present in higher proportions in the volatiles collected from Las-infected + trees. On the other hand, Las-infected + MJ-treated trees emitted lower proportions of MeSA than did Las-infected + Tween-treated trees. Because MeSA is a key D. citri attractant, this result suggests that MJ application could suppress MeSA emission from Las-infected trees, an approach that could be used to discourage psyllid colonization during shoot growth. MJ application enhanced emission of E-ß-ocimene, indole, volatiles attractive to many of the psyllid's natural enemies, indicating that MJ application could be used in an 'attract and reward' conservation biological control strategy. Volatile emissions in SA-treated trees were dominated by MeSA. MJ application elicited aggregation behavior in D. citri. Similar numbers of psyllids settled on MJ-treated versus Tween-treated sprigs, but a significantly greater percentage of the MJ-treated sprigs had aggregations of nine or more psyllids on them. Taken together, the results of this study indicate that exogenous applications of MJ or SA could be used to influence Asian citrus psyllid settling behavior and attract its natural enemies.

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.

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.

Grafting and Paladin Pic-21 for Nematode and Weed Management in Vegetable Production.

Two years of field trials conducted in a Meloidogyne incognita-infested field evaluated grafting and Paladin Pic-21 (dimethyl disulfide:chloropicrin [DMDS:Pic] 79:21) for root-knot nematode and weed control in tomato and melon. Tomato rootstocks evaluated were; 'TX301', 'Multifort', and 'Aloha'. 'Florida 47' was the scion and the nongrafted control. A double crop of melon was planted into existing beds following tomato harvest. Melon rootstocks, C. metulifer and 'Tetsukabuto', were evaluated with nongrafted 'Athena' in year 1. In year 2, watermelon followed tomato with scion variety 'Tri-X Palomar' as the control and also grafted onto 'Emphasis' and 'Strongtosa' rootstocks. Four soil treatments were applied in fall both years under Canslit metalized film; Paladin Pic-21, methyl bromide:chloropicrin (MeBr:C33, 67:33), Midas (iodomethane:chloropicrin 50:50), and a herbicide-treated control. M. incognita J2 in soil were highest in herbicide control plots and nongrafted tomato. All soil treatments produced similar tomato growth, which was greater than the herbicide control. All treatments reduced M. incognita J2 in roots compared to the herbicide control. 'Multifort' rootstock produced the largest and healthiest roots; however, the number of M. incognita isolated from roots did not differ among the tomato rootstocks tested. Galling on tomato was highest in herbicide control plots and nongrafted plants. In melon, M. incognita J2 in soil did not differ among melon rootstocks, but numbers isolated from melon rootstocks increased in 'Tetsukabuto' compared with C. metuliferus. 'Tetsukabuto' were larger root systems than nongrafted 'Athena'. All fumigants provided protection for all melon rootstocks against galling by M. incognita compared to the herbicide control. Galling on C. metuliferus rootstock was less in all fumigant treatments compared with nongrafted 'Athena' and 'Tetsukabuto'. In watermelon, M. incognita in soil and roots did not differ among soil treatments or watermelon rootstocks, and yield was lower in both grafted rootstocks compared with the nongrafted control. All soil treatments increased average fruit weight of watermelon compared with the herbicide control, and provided effective weed control, keeping the most predominant weed, purple nutsedge (Cyperus rotundus L.), density at or below 1/m row. Grafting commercial scions onto M. incognita-resistant rootstocks has potential for nematode management combined with soil treatments or as a stand-alone component in crop production systems.

Development of delayed bitterness and effect of harvest date in stored juice from two complex citrus hybrids.

BACKGROUND: Mandarins and mandarin hybrids have excellent flavor and color attributes, making them good candidates for consumption as fresh fruit. When processed into juice, however, they are less palatable, as they develop delayed bitterness when stored for a period of time. In this study the kinetics of delayed bitterness in two citrus mandarin hybrid siblings, 'Ambersweet' and USDA 1-105-106, was explored by sensory and instrumental analyses. In addition to the bitter limonoids, other quality factors (i.e. sugars, acids, pH, soluble solids content (SSC), titratable acidity (TA) and the ratio SSC/TA) were also measured. RESULTS: The two citrus hybrid siblings had different chemical profiles, which were perceived by taste panels. USDA 1-105-106 developed delayed bitterness when the juice was stored for more than 4 h, similar to juice from 'Navel' oranges, but 'Ambersweet' did not. Bitterness in 'Ambersweet' was more affected by harvest maturity, as juice from earlier harvest had lower SSC but higher TA and bitter limonoids. CONCLUSION: Since juice of USDA 1-105-106 shows delayed bitterness when stored for more than 4 h, this cultivar is not suitable for juice processing. Our finding that siblings can differ in chemical and sensory properties emphasize the importance of post-processing storage studies before releasing cultivars for juice.

Changes in Volatile and Non-Volatile Flavor Chemicals of "Valencia" Orange Juice over the Harvest Seasons.

Florida "Valencia" oranges have a wide harvest window, covering four months after first reaching the commercial maturity. However, the influence of harvest time on juice flavor chemicals is not well documented, with the exception of sugars and acids. Therefore, we investigated the major flavor chemicals, volatile (aroma), non-volatile (taste) and mouth feel attributes, in the two harvest seasons (March to June in 2007 and February to May in 2012). Bitter limonoid compounds, limonin and nomilin, decreased gradually. Out of a total of 94 volatiles, 32 increased, 47 peaked mid to late season, and 15 decreased. Juice insoluble solids and pectin content increased over the season; however, pectin methylesterase activity remained unchanged. Fruit harvested in the earlier months had lower flavor quality. Juice from later harvests had a higher sugar/acid ratio with less bitterness, while, many important aroma compounds occurred at the highest concentrations in the middle to late season, but occurred at lower concentrations at the end of the season. The results provide information to the orange juice processing industry for selection of optimal harvest time and for setting of precise blending strategy.

Susceptibility of Sixteen Citrus Genotypes to 'Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB) disease is the most serious threat to citrus production worldwide and, in the last decade, has devastated the Florida citrus industry. In the United States, HLB is associated with the phloem-limited α-proteobacterium 'Candidatus Liberibacter asiaticus' and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri). Significant effort is being put forth to develop novel citrus germplasm that has a lower propensity to succumb to HLB than do currently available varieties. Effective methods of screening citrus germplasm for susceptibility to HLB are essential. In this study, we exposed small, grafted trees of 16 citrus types to free-ranging ACP vectors and 'Ca. L. asiaticus' inoculum in the greenhouse. During 45 weeks of exposure to ACP, the cumulative incidence of 'Ca. L. asiaticus' infection was 70%. Trees of Citrus macrophylla and C. medica were most susceptible to 'Ca. L. asiaticus', with 100% infection by the end of the test period in three trials, while the complex genetic hybrids 'US 1-4-59' and 'Fallglo' consistently were least susceptible, with approximately 30% infection. Results obtained in this greenhouse experiment showed good agreement with trends observed in the orchard, supporting the validity of our approach for screening citrus germplasm for susceptibility to HLB.

High incidence of preharvest colonization of huanglongbing-symptomatic citrus sinensis fruit by Lasiodiplodia theobromae (Diplodia natalensis) and exacerbation of postharvest fruit decay by that fungus.

Huanglongbing (HLB), presumably caused by the bacterium "Candidatus Liberibacter asiaticus," is a devastating citrus disease associated with excessive preharvest fruit drop. Lasiodiplodia theobromae (diplodia) is the causal organism of citrus stem end rot (SER). The pathogen infects citrus fruit under the calyx abscission zone (AZ-C) and is associated with cell wall hydrolytic enzymes similar to plant enzymes involved in abscission. By means of DNA sequencing, diplodia was found in "Ca. Liberibacter asiaticus"-positive juice from HLB-symptomatic fruit (S) but not in "Ca. Liberibacter asiaticus"-negative juice. Therefore, the incidence of diplodia in fruit tissues, the impact on HLB-related postharvest decay, and the implications for HLB-related preharvest fruit drop were investigated in Hamlin and Valencia oranges. Quantitative PCR results (qPCR) revealed a significantly (P < 0.001) greater incidence of diplodia in the AZ-C of HLB-symptomatic (S; "Ca. Liberibacter asiaticus" threshold cycle [CT] of <30) than in the AZ-C of in asymptomatic (AS; "Ca. Liberibacter asiaticus" CT of ≥30) fruit. In agreement with the qPCR results, 2 weeks after exposure to ethylene, the incidences of SER in S fruit were 66.7% (Hamlin) and 58.7% (Valencia), whereas for AS fruit the decay rates were 6.7% (Hamlin) and 5.3% (Valencia). Diplodia colonization of S fruit AZ-C was observed by scanning electron microscopy and confirmed by PCR test and morphology of conidia in isolates from the AZ-C after surface sterilization. Diplodia CT values were negatively correlated with ethylene production (R = -0.838 for Hamlin; R = -0.858 for Valencia) in S fruit, and positively correlated with fruit detachment force (R = 0.855 for Hamlin; R = 0.850 for Valencia), suggesting that diplodia colonization in AZ-C may exacerbate HLB-associated preharvest fruit drop.

Cyclic LIPopeptides from Bacillus subtilis ABS-S14 elicit defense-related gene expression in citrus fruit.

Effects of cyclic lipopeptides (CLPs) obtained from Bacillus subtilis ABS-S14 on eliciting defense-related gene transcription and activity of defense-related enzymes; glucanase (GLU), chitinase (CHI), peroxidase (POX) and lipoxygenase (LOX) in Citrus sinensis cv. Valencia fruit were determined. The maximum level of GLU transcripts induced in fruit treated with fengycin was significantly greatest among treatments at 48 h. Surfactin enhanced the LOX and POX transcripts. In parallel, corresponding enzyme activities were correlated with changes in gene expression observed in fruit inoculated with Penicillium digitatum following treatment with individual CLPs. Synergistic effects of fengycin and iturin A, fengycin and surfactin were shown in gene transcript of GLU and CHI, respectively, and surfactin induced POX and LOX gene expression of citrus flavedo without pathogen infection. These results suggest that fengycin and surfactin act as elicitors of defense-related gene expression in "Valencia" fruit following infection.

A dark incubation period is important for Agrobacterium-mediated transformation of mature internode explants of sweet orange, grapefruit, citron, and a citrange rootstock.

BACKGROUND: Citrus has an extended juvenile phase and trees can take 2-20 years to transition to the adult reproductive phase and produce fruit. For citrus variety development this substantially prolongs the time before adult traits, such as fruit yield and quality, can be evaluated. Methods to transform tissue from mature citrus trees would shorten the evaluation period via the direct production of adult phase transgenic citrus trees. METHODOLOGY/PRINCIPAL FINDINGS: Factors important for promoting shoot regeneration from internode explants from adult phase citrus trees were identified and included a dark incubation period and the use of the cytokinin zeatin riboside. Transgenic trees were produced from four citrus types including sweet orange, citron, grapefruit, and a trifoliate hybrid using the identified factors and factor settings. SIGNIFICANCE: The critical importance of a dark incubation period for shoot regeneration was established. These results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid.

Metabolomic analysis of citrus infection by 'Candidatus Liberibacter' reveals insight into pathogenicity.

Huanglongbing (HLB), considered the most serious citrus disease in the world, is associated with the nonculturable bacterium 'Candidatus Liberibacter asiaticus' (Las). Infection of citrus by this pathogen leads to reduced plant vigor and productivity, ultimately resulting in death of the infected tree. It can take up to two years following initial infection before outward symptoms become apparent, making detection difficult. The existing knowledge gap in our understanding of Las and its pathogenesis leading to HLB has stymied development of treatments and methods to mitigate the pathogen's influence. To evaluate the influence of Las on fruit quality in both symptomatic and asymptomatic fruit, and gain further insight into the pathogenesis of the disease, a 1H NMR metabolomics investigation, complemented with physicochemical and analyte-specific analyses, was undertaken. Comparison of the juice obtained from oranges gathered from Las+ (symptomatic and asymptomatic) and Las- (healthy) trees revealed significant differences in the concentrations of sugars, amino and organic acids, limonin glucoside, and limonin. This study demonstrates differing metabolic profiles in the juice of oranges from Las+ and Las- and proposes how Las may be able to evade citrus defense responses.

Effect of Liberibacter infection (huanglongbing or "greening" disease) of citrus on orange juice flavor quality by sensory evaluation.

UNLABELLED: Some anecdotal reports suggest that infection of citrus trees with Candidatus Liberibacter asiaticus (Las), the suspected causal agent of huanglongbing (HLB) disease, imparts off flavor to orange juice. It is of interest to the industry to know how Las infection affects juice quality with respect to cultivar, maturity, or processing method. Hamlin, Midsweet, and Valencia oranges were harvested over 2 y from trees that tested negative (Las-) or positive (Las+) for Las from different groves and included normal looking (nonsymptomatic) and symptomatic fruit (small, green, and lopsided) from Las+ trees. In the 1st year, fruit were manually juiced, while in the 2nd year, a commercial process was used. Juice from Las+ trees was compared to juice from Las- trees in difference-from-control tests, and by descriptive analysis. Results showed large variability due to tree, harvest date, and cultivar. Juice from Hamlin Las+ trees tended to be more bitter and sour than its Las- counterpart. In contrast, hand processed Valencia juice from Las+ trees was perceived to have some off-flavor and bitterness compared to control, but the following year, commercially processed Valencia juice from Las+ trees was perceived to be only slightly more sour than juice from Las- trees for the April harvest, and to be sweeter for the June harvest. When juice from individual replicates was pooled to be more representative of a commercial situation, there was no difference between Las+ and Las- juice in Valencia. Trained panel differences were noted for juice from Hamlin Las+ fruit, especially for symptomatic fruit. PRACTICAL APPLICATION: Assumptions that juice made from oranges harvested from Huanglongbing (from infection with Liberibacter sp.) affected trees is off-flavored appeared to be generally more true for Hamlin juice than for Midsweet or Valencia, especially for Hamlin juice made from symptomatic fruit. For Midsweet and Valencia, flavor differences between juice made from fruit harvested from diseased or healthy trees varied greatly between trees, season, and even processing method. Under a commercial processing situation, where juice is blended from several varieties, seasons, and multiple locations, it is expected that off-flavor will not be a major problem.

Effect of liberibacter infection (huanglongbing disease) of citrus on orange fruit physiology and fruit/fruit juice quality: chemical and physical analyses.

More than 90% of oranges in Florida are processed, and since Huanglongbing (HLB) disease has been rumored to affect fruit flavor, chemical and physical analyses were conducted on fruit and juice from healthy (Las -) and diseased (Las +) trees on three juice processing varieties over two seasons, and in some cases several harvests. Fruit, both asymptomatic and symptomatic for the disease, were used, and fresh squeezed and processed/pasteurized juices were evaluated. Fruit and juice characteristics measured included color, size, solids, acids, sugars, aroma volatiles, ascorbic acid, secondary metabolites, pectin, pectin-demethylating enzymes, and juice cloud. Results showed that asymptomatic fruit from symptomatic trees were similar to healthy fruit for many of the quality factors measured, but that juice from asymptomatic and especially symptomatic fruits were often higher in the bitter compounds limonin and nomilin. However, values were generally below reported taste threshold levels, and only symptomatic fruit seemed likely to cause flavor problems. There was variation due to harvest date, which was often greater than that due to disease. It is likely that the detrimental flavor attributes of symptomatic fruit (which often drop off the tree) will be largely diluted in commercial juice blends that include juice from fruit of several varieties, locations, and seasons.