Huanglongbing and Foliar Spray Programs Affect the Chemical Profile of "Valencia" Orange Peel Oil.

Florida orange trees have been affected by huanglongbing (HLB) for more than a decade. To alleviate disease-caused tree decline, maintain fruit productivity, and reduce disease transmission, enhanced foliar spray programs combining vector control and nutritional supplementation have been applied to healthy and diseased trees. The aim of this research was to discover if the various foliar sprays affect fruit peel oil chemical components. In this study, "Valencia" orange trees, with or without HLB (HLB±), were treated with the grower standard program (control, C) or one of four proprietary enhanced foliar spray programs (N1, N2, N3, and N4) over 16 months. Compared with HLB-, HLB+ samples had lower concentrations of typical peel oil components, including valencene, octanal, and decanal, and were abundant in oxidative/dehydrogenated terpenes, such as carvone and limonene oxide. However, limonene, the dominant component, was not affected by any treatment. Control and three out of four enhanced foliar spray programs, N2, N3, and N4, had very little influence on the chemical profiles of both HLB- and HLB+ samples, while N1 treatment greatly altered the chemical profile of HLB+ samples, resulting in peel oil similar to that of HLB- samples.

Report on the SCRA Nuts and Bolts Workshop II: case studies of citrus greening, Ultra-low Gossypol Cotton, and blight tolerant, low-acrylamide potato.

To be commercialized and grown in the US, genetically engineered (GE) crops typically go through an extensive food, feed, and environmental safety assessment process which, in certain instances, requires complex consultations with three different US regulatory agencies. Many small market, niche, and specialty crops have been genetically engineered using the modern tools of recombinant DNA but few have been commercialized due to real or perceived regulatory constraints. This workshop discussed the practical aspects of developing dossiers on GE specialty, niche, or small-market crops/products for submission to US regulatory agencies. This workshop focused on actual case studies, and provided an opportunity for public or private sector scientists and crop developers to spend time with regulatory officials to learn the specifics of compiling a dossier for regulatory approval. The objective of the workshop was to explain and demystify data requirements and regulatory dossier compilation by small companies, academics, and other developers.

Comparative analysis of the transcriptomes of the calyx abscission zone of sweet orange insights into the huanglongbing-associated fruit abscission.

Citrus greening disease or huanglongbing (HLB) is associated with excessive pre-harvest fruit drop. To understand the mechanisms of the HLB-associated fruit abscission, transcriptomes were analyzed by RNA sequencing of calyx abscission zones (AZ-C) of dropped "Hamlin" oranges from HLB-diseased trees upon shaking the trees (Dd), retained oranges on diseased trees (Rd), dropped oranges from healthy shaken trees (Dh), and retained oranges on healthy trees (Rh). Cluster analysis of transcripts indicated that Dd had the largest distances from all other groups. Comparisons of transcriptomes revealed 1047, 1599, 813, and 764 differentially expressed genes (DEGs) between Dd/Rd, Dd/Dh, Dh/Rh, and Rd/Rh. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated hormone signaling, defense response, and secondary metabolism were involved in HLB-associated fruit abscission. Ethylene (ET) and jasmonic acid (JA) synthesis/signaling-related genes were upregulated in Dd, while other phytohormone-related genes were generally downregulated. In addition, genes related to JA/ET-activated defense response were upregulated in Dd as well. Consistent with the phytohormone gene expression data, increased levels (p < 0.05) of ET and JA, and a decreased level (p < 0.05) of abscisic acid were found in Dd compared with Rd, Dh or Rh. Lasiodiploidia theobromae level in Dd AZ-C was higher than the other fruit types, confirmed by qPCR, indicating AZ-C secondary fungal infection of HLB fruit may exacerbate their abscission. This information will help formulate effective strategies to control HLB-related abscission.

Effect of Abscission Zone Formation on Orange ( Citrus sinensis) Fruit/Juice Quality for Trees Affected by Huanglongbing (HLB).

Orange trees affected by huanglongbing (HLB) exhibit excessive fruit drop, and fruit loosely attached to the tree may have inferior flavor. Fruit were collected from healthy and HLB-infected ( Candidatus liberibacter asiaticus) 'Hamlin' and 'Valencia' trees. Prior to harvest, the trees were shaken, fruit that dropped collected, tree-retained fruit harvested, and all fruit juiced. For chemical analyses, sugars and acids were generally lowest in HLB dropped (HLB-D) fruit juice compared to nonshaken healthy (H), healthy retained (H-R), and healthy dropped fruit (H-D) in early season (December) but not for the late season (January) 'Hamlin' or 'Valencia' except for sugar/acid ratio. The bitter limonoids, many flavonoids, and terpenoid volatiles were generally higher in HLB juice, especially HLB-D juice, compared to the other samples. The lower sugars, higher bitter limonoids, flavonoids, and terpenoid volatiles in HLB-D fruit, loosely attached to the tree, contributed to off-flavor, as was confirmed by sensory analyses.

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.

Electronic Tongue Response to Chemicals in Orange Juice that Change Concentration in Relation to Harvest Maturity and Citrus Greening or Huanglongbing (HLB) Disease.

In an earlier study, an electronic tongue system (e-tongue) has been used to differentiate between orange juice made from healthy fruit and from fruit affected by the citrus greening or Huanglongbing (HLB) disease. This study investigated the reaction of an e-tongue system to the main chemicals in orange juice that impact flavor and health benefits and are also impacted by HLB. Orange juice was spiked with sucrose (0.2-5.0 g/100 mL), citric acid (0.1%-3.0% g/100 mL) and potassium chloride (0.1-3.0 g/100 mL) as well as the secondary metabolites nomilin (1-30 µg/mL), limonin (1-30 µg/mL), limonin glucoside (30-200 µg/mL), hesperidin (30-400 µg/mL) and hesperetin (30-400 µg/mL). Performance of Alpha MOS sensor sets #1 (pharmaceutical) and #5 (food) were compared for the same samples, with sensor set #1 generally giving better separation than sensor set #5 for sucrose, sensor set #5 giving better separation for nomilin and limonin, both sets being efficient at separating citric acid, potassium chloride, hesperitin and limonin glucoside, and neither set discriminating hesperidin efficiently. Orange juice made from fruit over the harvest season and from fruit harvested from healthy or HLB-affected trees were separated by harvest maturity, disease state and disease severity.

Prophage-mediated dynamics of 'Candidatus Liberibacter asiaticus' populations, the destructive bacterial pathogens of citrus huanglongbing.

Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of Candidatus Liberibacter asiaticus' (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease development.

Extraction of DNA from orange juice, and detection of bacterium Candidatus Liberibacter asiaticus by real-time PCR.

Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium. The current standard to confirm CLas for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and use a quantitative real-time polymerase chain reaction (qPCR) test to detect the 16S rDNA gene of CLas. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high sugar and pectin concentration, low pH, and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice and detection of CLas by qPCR. Homogenization using a sonicator increased DNA yield by 86% in comparison to mortar and pestle extraction. It is difficult to separate DNA from pectin; however, DNA was successfully extracted by treating the juice with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. This work provided a protocol to extract DNA from whole orange juice and detect CLas in HLB-affected fruit.

Candidate gene markers for Candidatus Liberibacter asiaticus for detecting citrus greening disease.

Citrus Huanglongbing (HLB) also known as citrus greening is one of the most devastating diseases of citrus worldwide. The disease is caused by Candidatus Liberibacter bacterium, vectored by the psyllid Diaphorina citri Kuwayama and Trioza erytreae Del Guercio. Citrus plants infected by the HLB bacterium may not show visible symptoms sometimes for years following infection. The aim of this study was to develop effective gene-specific primer pairs for polymerase chain reaction based method for quick screening of HLB disease. Thirty-two different gene-specific primer pairs, across the Ca. Liberibacter asiaticus genome, were successfully developed. The possibility of these primer pairs for cross-genome amplification across 'Ca. Liberibacter africanus' and 'Ca. Liberibacter americanus' were tested. The applicability of these primer pairs for detection and differentiation of Ca Liberibacter spp. is discussed.

Development of rapid, sensitive and non-radioactive tissue-blot diagnostic method for the detection of citrus greening.

Citrus huanglongbing (HLB or citrus greening) is one of the most devastating diseases of citrus worldwide. The disease is caused by Gram-negative, phloem-limited α-proteobacterium, 'Candidatus Liberibacter asiaticus', vectored by the psyllid, Diaphorina citri Kuwayama. Citrus plants infected by the HLB bacterium may not show visible symptoms sometimes for years following infection and non-uniform distribution within the tree makes the detection of the pathogen very difficult. Efficient management of HLB disease requires rapid and sensitive detection early in the infection followed by eradication of the source of pathogen and the vector. The polymerase chain reaction (PCR) based method is most commonly employed for screening the infected/suspected HLB plants and psyllids. This is time consuming, cumbersome and not practical for screening large number of samples in the field. To overcome this, we developed a simple, sensitive, non-radioactive, tissue-blot diagnostic method for early detection and screening of HLB disease. Digoxigenin labeled molecular probes specific to 'Ca. L. asiaticus' nucleotide sequences have been developed and used for the detection of the pathogen of the HLB disease. The copy number of the target genes was also assessed using real-time PCR experiments and the optimized real-time PCR protocol allowed positive 'Ca. L. asiaticus' detection in citrus samples infected with 'Ca. L. asiaticus' bacterium.

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.