Synergistic effect of carboxymethylcellulose and Cryptococcus laurentii on suppressing green mould of postharvest grapefruit and its mechanism.

The synergistic effects of carboxymethylcellulose (CMC) combined with Cryptococcus laurentii FRUC DJ1 were studied on controlling green mould resulting from Penicillium digitatum in grapefruit fruit. The results indicate that both C. laurentii and the CMC treatment suppressed P. digitatum conidia germination. In addition, C. laurentii growth in vitro was not affected by low CMC concentrations, nevertheless, the biofilm of C. laurentii was enhanced. Compared with the control fruit, the grapefruit had a lower green mould in all treatments. Significantly synergistic effects were caused by combining C. laurentii and CMC on minimum decay incidence and lesion diameter. Combined treatment induced defence enzyme activities, including chitinase, ß-1,3-glucanase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase, together with disease tolerance-associated total phenol. Also, this combination inhibited the pathogen growth by adhered to the hyphae and reduced its infection in fruit wounds. Moreover, the commercial quality parameters in the combined treatment of C. laurentii and CMC, including weight loss, total soluble solids, ascorbic acid, and titratable acidity, were superior to single treatment. The combination of C. laurentii and CMC can not only control postharvest decay but also maintain fruit qualities. Thus, it can be used in grapefruit for commercial purposes.

Performance of macroporous resins for debittering HLB-affected grapefruit juice and its impacts on furanocoumarin and consumer sensory acceptability.

About 90% of grapefruit in Florida are affected by Huanglongbing (HLB). HLB negatively affects the organoleptic properties of grapefruit juice because affected trees overproduce bitter secondary-metabolites, mostly naringin. The objective of this research was to remove naringin from HLB-affected grapefruit juice using microporous-adsorbents and to investigate how debittering affected narirutin, limonoids, bergamottin, and consumer acceptability. The adsorption kinetics of naringin on seven adsorbent resins obeyed pseudo-second order. PAD550 and PAD600 showed better static adsorption/desorption. Adsorption-isotherms on these resins were better fitted on Temkin-Pyzhev-model. On a fixed-bed-column packed with PAD550 resin, a slower loading rate increased its breakthrough volume before naringin in effluent reached its taste-threshold. In addition to naringin being reduced to below its taste-threshold, debittering significantly decreased the content of limonin, nomilin, and bergamottin. A consumer taste panel rated debittered and half-debittered juices higher for overall acceptability than the untreated. The half-debittered juice was ranked the most preferred while untreated was the least preferred.

Overexpressing a NPR1-like gene from Citrus paradisi enhanced Huanglongbing resistance in C. sinensis.

KEY MESSAGE: Overexpression of CiNPR4 enhanced resistance of transgenic citrus plants to Huanglongbing by perceiving the salicylic acid and jasmonic acid signals and up-regulating the transcriptional activities of plant-pathogen interaction genes. Developing transgenic citrus plants with enhanced immunity is an efficient strategy to control citrus Huanglongbing (HLB). Here, a nonexpressor of pathogenesis-related gene 1 (NPR1) like gene from HLB-tolerant 'Jackson' grapefruit (Citrus paradisi Macf.), CiNPR4, was introduced into 'Wanjincheng' orange (Citrus sinensis Obseck). CiNPR4 expression was determined in transgenic citrus plants using quantitative real-time PCR analyses. The Candidatus Liberibacter asiaticus (CLas) pathogen of HLB was successfully transmitted to transgenic citrus plants by grafting infected buds. HLB symptoms developed in transgenic and wild-type (WT) plants by 9 months after inoculation. A CLas population analysis showed that 26.9% of transgenic lines exhibited significantly lower CLas titer levels compared with the CLas-infected WT plants at 21 months after inoculation. Lower starch contents and anatomical aberration levels in the phloem were observed in transgenic lines having enhanced resistance compared with CLas-infected WT plants. CiNPR4 overexpression changed the jasmonic acid, but not salicylic acid, level. Additionally, the jasmonic acid and salicylic acid levels increased after CLas infection. Transcriptome analyses revealed that the enhanced resistance of transgenic plants to HLB resulted from the up-regulated transcriptional activities of plant-pathogen interaction-related genes.

ß-aminobutyric acid induces priming defence against Botrytis cinerea in grapefruit by reducing intercellular redox status that modifies posttranslation of VvNPR1 and its interaction with VvTGA1.

Either NPR1 or TGA1 serve as master redox-sensitive transcriptional regulators for the transcription of PR genes in plants. The redox modification of the two co-activators involved in BABA-induced priming resistance against Botrytis cinerea in grapes was examined in this study. The results showed that 10 mmol L-1 BABA could effectively trigger a priming defense in grapes as manifested by augmented expression levels of PR genes upon inoculation with B. cinerea. Moreover, transcriptome profiling analysis revealed that all of the sets of key genes in the enzymatic ROS scavenging system, the PPP and AsA-GSH cycle were in harmony and were transcriptionally induced in BABA-primed grapes with pathogenic infection; in addition, this enhanced expression caused the accelerated accumulation of reductive substances, namely, AsA, GSH and NADPH, resulting in reduced intercellular conditions. Under reduced conditions, the interaction of VvTGA1 and VvNPR1 in the Y2H assay implied that VvTGA1 can provide the DNA binding capacity required by VvNPR1 for activation of VvPR genes. Consequently, the transactivation of VvNPR1 by the promoters of VvPR1, VvPR2 and VvPR5 was determined via a DLR assay, and it induced the transcription of the VvPR genes. In parallel, the redox-modified reducing condition achieved with an abundant supply of reductive substances was closely associated with the translocation of NPR1 for interaction with TGA in the nucleus. Thus, the posttranslational modification and subsequent interaction of the two redox-sensitive co-activators of VvNPR1 and VvTGA1 under reduced conditions may be responsible for BABA-induced priming for effective disease resistance in grapes.

Laser-induced fluorescence spectroscopy for early disease detection in grapefruit plants.

Biotic and abiotic stress both cause a considerable decrease in the chlorophyll content in plant leaves, which provides a means for the early diagnosis of diseases in plants. The emergence of diseases affects the fluorescence of phenolic compounds and chlorophyll, which have emissions located at 530, 686 and 735 nm. Herein, it was found that the intensity of the emission band of phenolic compounds at 530 nm increased and that of chlorophyll at 735 nm decreased with the onset of diseases. Statistical analysis through principal component analysis (PCA) and partial least squares regression (PLSR) was performed, which differentiated between apparently healthy leaf sites and diseased leaves, providing a basis for the detection of diseases in the early stages. The PLSR model was validated through the coefficient of determination (R2), standard error of prediction (SEP) and standard error of calibration (SEC) with the values of 0.99, 0.394 and 0.0.401, respectively, which authenticated the model. The prediction accuracy of the model was evaluated through root mean square error in prediction (RMSEP), with a value of 0.14, by predicting 22 unknown emission spectra of different leaf sites. Both the PCA and PLSR models produced similar results, proving that fluorescence spectroscopy is an excellent tool for early disease detection in plants.

The flagella of 'Candidatus Liberibacter asiaticus' and its movement in planta.

Citrus huanglongbing (HLB) is the most devastating citrus disease worldwide. 'Candidatus Liberibacter asiaticus' (Las) is the most prevalent HLB causal agent that is yet to be cultured. Here, we analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin-encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin-encoding genes. Only flgJAtu but not flgJLas restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull-down assays demonstrated that FlgJLas interacts with FlgB but not with FliE. Ectopic expression of flaALas in A. tumefaciens mutants restored the swimming motility of ∆flaA mutant and ∆flaAD mutant, but not that of the null mutant ∆flaABCD. No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta. In addition, western blotting using flagellin-specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.

Changes in the physico-chemical properties of the xanthan produced by Xanthomonas citri subsp. citri in grapefruit leaf extract.

Xanthan is a virulence factor produced by Xanthomonas spp. We previously demonstrated that this exopolysaccharide is not only essential for pathogenicity by contributing with bacterial survival but also its pyruvate substituents interfere with some plant defense responses. Deepening our studies about xanthan properties and structure, the aim of this work was to analyze the characteristics of xanthan produced by Xanthomonas in different culture media. We analyzed the xanthan produced by Xanthomonas citri subsp. citri (Xcc) in leaf extracts from grapefruit (a susceptible host of this bacterium) and compared it with the xanthan produced in a synthetic culture medium. We found that the xanthan produced in the grapefruit extract (Xan-GLE) presented shorter and more disordered molecules than xanthan produced in the synthetic medium (Xan-PYM). Besides, Xan-GLE resulted less viscous than Xan-PYM. The disordered molecular conformation of Xan-GLE could be attributed to its higher pyruvilation degree and lower acetylation degree compared with those detected in Xan-PYM. Meanwhile, the difference in the viscosity of both xanthans could be due to their molecules length. Finally, we cultured Xcc in the presence of the Xan-GLE or Xan-PYM and observed the formation of biofilm-like structures in both cases. We found significant differences in biofilm architecture between the two conditions, being the biofilm produced in presence of Xan-GLE similar to that formed in canker lesions developed in lemon plant leaves. Together, these results show how xanthan structure and properties changed when Xcc grew in a natural substrate and can contribute to better understand the biological role of xanthan.

Reduction of Escherichia coli, as a surrogate for Salmonella spp., on the surface of grapefruit during various packingline processes.

The US Produce Safety Rule allows for use of water that does not meet its microbial standards if corrective measures are employed. This research was initiated to determine the suitability of nonpathogenic Escherichia coli as a surrogate for Salmonella during citrus washing, and to evaluate the removal of E. coli from grapefruit on two pilot packinglines (CREC and IRREC) as corrective measures. Whole grapefruit were inoculated with either E. coli or Salmonella and dried, and exposed to a variety of treatments on a lab-scale brush wash system. Individual processes were evaluated on the pilot packinglines with E. coli only. In all lab-scale brush wash system treatments, bacterial population reductions between E. coli and Salmonella were not significantly different (P ≤ 0.05). On pilot packinglines, E. coli populations were reduced by 3.59 to >5.11 log CFU/grapefruit at the CREC packingline, and by 3.30 to >5.13 log CFU/grapefruit at the IRREC packingline. Treatment of fruit through complete packingline processing at both locations reduced E. coli populations to levels below the detection limit (<1 log CFU/grapefruit). The studies indicate E. coli is an appropriate surrogate for Salmonella under tested conditions, and that standard citrus packingline processes can be used as a corrective measure.

Use of microbial models to evaluate the effect of UV-C light and trans-cinnamaldehyde on the native microbial load of grapefruit (Citrus × paradisi) juice.

The aim of this research was to evaluate the storage stability (5 °C), and microbial modeling, of Rubi red grapefruit (Citrus × paradisi) juice treated with ultraviolet-C (UV-C) light (0, 10 and 20 min), alone or in combination with trans-cinnamaldehyde (trans-CAH) (0, 25 and 50 µg/mL). A 32 factorial design was used and data modeled with the Weibull, Modified Gompertz and Logistic models. A response surface model was used to evaluate the effect of modeling parameters for suggesting the optimum treatment conditions. Treated and some untreated juice lasted up to 9 days without physicochemical and microbial changes. At the higher combination of UV-C light and trans-CAH, the microbial load of grapefruit juice was maintained below 100 CFU/mL up to 15 days. For mesophiles, the three predictive models indicated that the parameters n and Nmax decreased and the parameters λ and tc increased as the combination of UV-C light and trans-CAH increased. The response surface modeling of the parameters obtained by the predictive models showed acceptable correlation for mesophiles (R2 = 0.815-0.977) but not for yeasts (R2 = 0.618-0.815). The three predictive models showed that, the concentration of trans-CAH had more effect on stopping the microbial growth than the UV-C light treatment.

Induced Systemic Resistance Against Citrus Canker Disease by Rhizobacteria.

Citrus canker, caused by Xanthomonas citri subsp. citri, is an important citrus disease that causes significant economic losses worldwide. All commercial citrus varieties are susceptible to citrus canker. Currently, chemical control with copper based products is the main approach to control X. citri subsp. citri dispersal and plant colonization. However, extensive use of copper compounds can result in copper-resistant strains and cause adverse effects on the environment. Alternatives to chemical control involve the activation of citrus immunity to control the disease. Here, we investigated the ability of multiple rhizobacteria to induce a systemic defense response in cultivar Duncan grapefruit. Burkholderia territorii strain A63, Burkholderia metallica strain A53, and Pseudomonas geniculata strain 95 were found to effectively activate plant defense and significantly reduce symptom development in leaves challenged with X. citri subsp. citri. In the priming phase, root application of P. geniculata induced the expression of salicylic acid (SA)-signaling pathway marker genes (PR1, PR2, PR5, and salicylic acid carboxyl methyltransferase [SAM-SACM]). Gene expression analyses after X. citri subsp. citri challenge showed that root inoculation with P. geniculata strain 95 increased the relative levels of phenylalanine ammonia lyase 1 and SAM-SACM, two genes involved in the phenylpropanoid pathway as well as the biosynthesis of SA and methyl salicylate (MeSA), respectively. However, hormone analyses by UPLC-MS/MS showed no significant difference between SA in P. geniculata-treated plants and control plants at 8 days post-beneficial bacteria root inoculation. Moreover, P. geniculata root-treated plants contained higher reactive oxygen species levels in aerial tissues than control plants 8 days post-treatment application. This study demonstrates that rhizobacteria can modulate citrus immunity resulting in a systemic defense response against X. citri subsp. citri under greenhouse conditions.

Effect of NaHCO3 treatments on the activity of cell-wall-degrading enzymes produced by Penicillium digitatum during the pathogenesis process on grapefruit.

BACKGROUND: This study was performed to clarify the strategies of Penicillium digitatum during pathogenesis on citrus, assessing, on albedo plugs, the effects of treatment with sodium bicarbonate (NaHCO3 ), at two different pH values (5 and 8.3), on cell-wall-degrading enzyme activity over a period of 72 h. RESULTS: Treatment with NaHCO3 , under alkaline pH, delayed the polygalacturonase activity for 72 h, or 48 h in the case of the pectin lyase, compared with the control or the same treatment at pH 5. In contrast, pectin methyl esterase activity rapidly increased after 24 h, in plugs dipped in the same solution. In this case, the activity remained higher than untreated or pH 5-treated plugs up to 72 h. CONCLUSION: The rapid increase in pectin methyl esterase activity under alkaline conditions is presumably the strategy of the pathogen to lower the pH, soon after the initiation of infection, in order to restore an optimal environment for the subsequent polygalacturonase and pectin lyase action. In fact, at the same time, a low pH delayed the enzymatic activity of polygalacturonase and pectin lyase, the two enzymes that actually cleave the α-1,4-linkages between the galacturonic acid residues. © 2018 Society of Chemical Industry.

Metabolic Analysis Reveals Altered Long-Chain Fatty Acid Metabolism in the Host by Huanglongbing Disease.

Candidatus Liberibacter asiaticus (CLas) is the presumed causal agent of Huanglongbing, one of the most destructive diseases in citrus. However, the lipid metabolism component of host response to this pathogen has not been investigated well. Here, metabolic profiling of a variety of long-chain fatty acids and their oxidation products was first performed to elucidate altered host metabolic responses of disease. Fatty acid signals were found to decrease obviously in response to disease regardless of cultivar. Several lipid oxidation products strongly correlated with those fatty acids were also consistently reduced in the diseased group. Using a series of statistical methods and metabolic pathway mapping, we found significant markers contributing to the pathological symptoms and identified their internal relationships and metabolic network. Our findings suggest that the infection of CLas may cause the altered metabolism of long-chain fatty acids, possibly leading to manipulation of the host's defense derived from fatty acids.

Molecular characterization of XopAG effector AvrGf2 from Xanthomonas fuscans ssp. aurantifolii in grapefruit.

Xanthomonas fuscans ssp. aurantifolii group C strains exhibit host specificity on different citrus species. The strains possess a type III effector, AvrGf2, belonging to the XopAG effector gene family, which restricts host range on citrus. We dissected the modular nature and mode of action of AvrGf2 in grapefruit resistance. XopAG effectors possess characteristic features, such as a chloroplast localization signal, a cyclophilin-binding domain characteristic amino acid sequence motif (GPLL) and a C-terminal domain-containing CLNAxYD. Mutation of GPLL to AASL in AvrGf2 abolished the elicitation of the hypersensitive response (HR), whereas mutation of only the first amino acid to SPLL delayed the HR in grapefruit. Yeast two-hybrid experiments showed strong interaction of AvrGf2 with grapefruit cyclophilin (GfCyp), whereas AvrGf2-SPLL and AvrGf2-AASL mutants showed weak and no interaction, respectively. Molecular modelling and in silico docking studies for the cyclophilin-AvrGf2 interaction predicted the binding of citrus cyclophilins (CsCyp, GfCyp) to hexameric peptides spanning the cyclophilin-binding domain of AvrGf2 and AvrGf2 mutants (VAGPLL, VASPLL and VAAASL) with affinities equivalent to or better than a positive control peptide (YSPSA) previously demonstrated to bind CsCyp. In addition, the C-terminal domain of XopAG family effectors contains a highly conserved motif, CLNAxYD, which was identified to be crucial for the induction of HR based on site-directed mutagenesis (CLNAxYD to CASAxYD). Our results suggest a model in which grapefruit cyclophilin promotes a conformational change in AvrGf2, thereby triggering the resistance response.

Identification of seven novel virulence genes from Xanthomonas citri subsp. citri by Tn5-based random mutagenesis.

To identify novel virulence genes, a mutant library of Xanthomonas citri subsp. citri 29-1 was produced using EZ-Tn5 transposon and the mutants were inoculated into susceptible grapefruit. Forty mutants with altered virulence phenotypes were identified. Nine of the mutants showed a complete loss of citrus canker induction, and the other 31 mutants resulted in attenuated canker symptoms. Southern blot analysis revealed that each of the mutants carried a single copy of Tn5. The flanking sequence was identified by plasmid rescue and 18 different ORFs were identified in the genome sequence. Of these 18 ORFs, seven had not been previously associated with the virulence of X. citri subsp. citri and were therefore confirmed by complementation analysis. Real-time PCR analysis showed that the seven genes were upregulated when the bacteria were grown in citrus plants, suggesting that the expression of these genes was essential for canker development.

Spatial imaging of Zn and other elements in Huanglongbing-affected grapefruit by synchrotron-based micro X-ray fluorescence investigation.

Huanglongbing (HLB) is a highly destructive, fast-spreading disease of citrus, causing substantial economic losses to the citrus industry worldwide. Nutrient levels and their cellular distribution patterns in stems and leaves of grapefruit were analysed after graft-inoculation with lemon scions containing 'Candidatus Liberibacter asiaticus' (Las), the heat-tolerant Asian type of the HLB bacterium. After 12 months, affected plants showed typical HLB symptoms and significantly reduced Zn concentrations in leaves. Micro-XRF imaging of Zn and other nutrients showed that preferential localization of Zn to phloem tissues was observed in the stems and leaves collected from healthy grapefruit plants, but was absent from HLB-affected samples. Quantitative analysis by using standard references revealed that Zn concentration in the phloem of veins in healthy leaves was more than 10 times higher than that in HLB-affected leaves. No significant variation was observed in the distribution patterns of other elements such as Ca in stems and leaves of grapefruit plants with or without graft-inoculation of infected lemon scions. These results suggest that reduced phloem transport of Zn is an important factor contributing to HLB-induced Zn deficiency in grapefruit. Our report provides the first in situ, cellular level visualization of elemental variations within the tissues of HLB-affected citrus.

Viability of 'Candidatus Liberibacter asiaticus' prolonged by addition of citrus juice to culture medium.

Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium 'Candidatus Liberibacter asiaticus'. Infection with 'Ca. L. asiaticus' is incurable; therefore, knowledge regarding 'Ca. L. asiaticus' biology and pathogenesis is essential to develop a treatment. However, 'Ca. L. asiaticus' cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of 'Ca. L. asiaticus' in vitro, 'Ca. L. asiaticus' inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima 'Mato Buntan') was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air-liquid interface of juice cultures contained 'Ca. L. asiaticus' cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining 'Ca. L. asiaticus' viability in vitro, which will contribute to future development of a culture medium for 'Ca. L. asiaticus'.

Comparative genomic and transcriptome analyses of pathotypes of Xanthomonas citri subsp. citri provide insights into mechanisms of bacterial virulence and host range.

BACKGROUND: Citrus bacterial canker is a disease that has severe economic impact on citrus industries worldwide and is caused by a few species and pathotypes of Xanthomonas. X. citri subsp. citri strain 306 (XccA306) is a type A (Asiatic) strain with a wide host range, whereas its variant X. citri subsp. citri strain A(w)12879 (Xcaw12879, Wellington strain) is restricted to Mexican lime. RESULTS: To characterize the mechanism for the differences in host range of XccA and Xcaw, the genome of Xcaw12879 that was completed recently was compared with XccA306 genome. Effectors xopAF and avrGf1 are present in Xcaw12879, but were absent in XccA306. AvrGf1 was shown previously for Xcaw to cause hypersensitive response in Duncan grapefruit. Mutation analysis of xopAF indicates that the gene contributes to Xcaw growth in Mexican lime but does not contribute to the limited host range of Xcaw. RNA-Seq analysis was conducted to compare the expression profiles of Xcaw12879 and XccA306 in Nutrient Broth (NB) medium and XVM2 medium, which induces hrp gene expression. Two hundred ninety two and 281 genes showed differential expression in XVM2 compared to in NB for XccA306 and Xcaw12879, respectively. Twenty-five type 3 secretion system genes were up-regulated in XVM2 for both XccA and Xcaw. Among the 4,370 common genes of Xcaw12879 compared to XccA306, 603 genes in NB and 450 genes in XVM2 conditions were differentially regulated. Xcaw12879 showed higher protease activity than XccA306 whereas Xcaw12879 showed lower pectate lyase activity in comparison to XccA306. CONCLUSIONS: Comparative genomic analysis of XccA306 and Xcaw12879 identified strain specific genes. Our study indicated that AvrGf1 contributes to the host range limitation of Xcaw12879 whereas XopAF contributes to virulence. Transcriptome analyses of XccA306 and Xcaw12879 presented insights into the expression of the two closely related strains of X. citri subsp. citri. Virulence genes including genes encoding T3SS components and effectors are induced in XVM2 medium. Numerous genes with differential expression in Xcaw12879 and XccA306 were identified. This study provided the foundation to further characterize the mechanisms for virulence and host range of pathotypes of X. citri subsp. citri.

The effect of 'Candidatus Liberibacter asiaticus' infection on the proteomic profiles and nutritional status of pre-symptomatic and symptomatic grapefruit (Citrus paradisi) plants.

BACKGROUND: Huanglongbing (HLB) is a highly destructive citrus disease which threatens citrus production worldwide and 'Candidatus Liberibacter asiaticus' (Las), a non-culturable phloem-limited bacterium, is an associated causal agent of the disease. To better understand the physiological and molecular processes involved in host responses to Las, 2-DE and mass spectrometry analyses, as well as ICP spectroscopy analysis were employed to elucidate the global protein expression profiles and nutrient concentrations in leaves of Las-infected grapefruit plants at pre-symptomatic or symptomatic stages for HLB. RESULTS: This study identified 123 protein spots out of 191 spots that showed significant changes in the leaves of grapefruit plants in response to Las infection and all identified spots matched to 69 unique proteins/peptides. A down-regulation of 56 proteins including those associated with photosynthesis, protein synthesis, and metabolism was correlated with significant reductions in the concentrations of Ca, Mg, Fe, Zn, Mn, and Cu in leaves of grapefruit plants in response to Las infection, particularly in symptomatic plants. Oxygen-evolving enhancer (OEE) proteins, a PSI 9 kDa protein, and a Btf3-like protein were among a small group of proteins that were down-regulated in both pre-symptomatic and symptomatic plants in response to Las infection. Furthermore, a Las-mediated up-regulation of 13 grapefruit proteins was detected, which included Cu/Zn superoxide dismutase, chitinases, lectin-related proteins, miraculin-like proteins, peroxiredoxins and a CAP 160 protein. Interestingly, a Las-mediated up-regulation of granule-bound starch synthase was correlated with an increase in the K concentrations of pre-symptomatic and symptomatic plants. CONCLUSIONS: This study constitutes the first attempt to characterize the interrelationships between protein expression and nutritional status of Las-infected pre-symptomatic or symptomatic grapefruit plants and sheds light on the physiological and molecular mechanisms associated with HLB disease development.

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.

Biotransformations of 6',7'-dihydroxybergamottin and 6',7'-epoxybergamottin by the citrus-pathogenic fungi diminish cytochrome P450 3A4 inhibitory activity.

Penicillium digitatum, as well as five other citrus pathogenic species, (Penicillium ulaiense Link, Geotrichum citri Link, Botrytis cinerea P. Micheli ex Pers., Lasiodiplodia theobromae (Pat.) Griffon & Maubl., and Phomopsis citri (teleomorph Diaporthe citri)) were observed to convert 6',7'-epoxybergamottin (1) into 6',7'-dihydroxybergamottin (2), bergaptol (3), and an opened lactone ring metabolite 6,7-furano-5-(6',7'-dihydroxy geranyloxy)-2-hydroxy-hydrocoumaric acid (4). Metabolism of 2 by these fungi also proceeded to 4. The structure of 4 was established by high resolution mass spectrometry and (1)H and (13)C NMR techniques. The inhibitory activity of 4 towards human intestinal cytochrome P450 3A4 (CYP3A4) was greatly decreased (IC(50) >172.0 µM) compared to 2 (IC(50)=0.81 µM).