Search

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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, β-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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

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.

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., (Copyright © 2020. Published by Elsevier Masson SAS.)

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 (R 2 ), 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.

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 flgJ Atu but not flgJ Las restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull-down assays demonstrated that FlgJ Las interacts with FlgB but not with FliE. Ectopic expression of flaA Las 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., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 3 2 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 N max decreased and the parameters λ and t c 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 (R 2  = 0.815-0.977) but not for yeasts (R 2  = 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 (NaHCO 3 ), at two different pH values (5 and 8.3), on cell-wall-degrading enzyme activity over a period of 72 h., Results: Treatment with NaHCO 3 , 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., (© 2018 Society of Chemical Industry.)

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.

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.

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., (© 2016 BSPP AND JOHN WILEY & SONS LTD.)

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.

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.

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'.

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.

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.

An Agrobacterium tumefaciens -mediated transformation (ATMT) system was successfully developed for Colletotrichum truncatum, the causal agent of chili anthracnose. A. tumefaciens carrying a hygromycin phosphotransferase gene (hph) and a green fluorescent protein (gfp) gene was used to transform the conidiospores of two C. truncatum pathotypes F8-3B and BRIP26974. Optimum transformation efficiency was obtained when equal volumes of A. tumefaciens strain AGL1 carrying either pJF1 or pPK2 binary vector was used to transform C. truncatum conidiospores at 10(6) /ml and co-cultivated at 24 °C for three days. Southern blot analysis indicated that 87.5% of the transformants contained randomly inserted, single copies of the T-DNA. Infection and colonisation of chili fruit at the mature red stage with F8-3B-GFP and BRIP26974-GFP confirmed the maintenance of virulence within these transformed pathotypes. In situ studies of infection and colonisation of the susceptible genotype fruit using fluorescent microscopy and transformed isolates of C. truncatum expressing GFP revealed that the pathogen was able to colonise healthy fruit tissue intercellularly in an endophytic manner without producing secondary biotrophic infection structures. The developed transformation system will be used to study the function of pathogenicity genes in C. truncatum using both forward and reverse genetics approaches., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To gain a better understanding of the molecular changes taking place in citrus fruit tissue following the application of the yeast biocontrol agent Metschnikowia fructicola, microarray analysis was performed on grapefruit surface wounds using an Affymetrix Citrus GeneChip. Using a cut-off of P < 0.05 and a 1.5-fold change difference as biologically significant, the data indicated that 1007 putative unigenes showed significant expression changes following wounding and yeast application relative to wounded controls. Microarray results of selected genes were validated by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The data indicated that yeast application induced the expression of the genes encoding Respiratory burst oxidase (Rbo), mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK), G-proteins, chitinase (CHI), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and 4-coumarate-CoA ligase (4CL). In contrast, three genes, peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), were down-regulated in grapefruit peel tissue treated with yeast cells. Moreover, suppression was correlated with significantly higher levels of hydrogen peroxide, superoxide anion and hydroxyl radical production in yeast-treated surface wounds. Interestingly, large amounts of hydrogen peroxide were detected inside yeast cells recovered from wounded fruit tissue, indicating the ability of the yeast to activate reactive oxygen species when it is in contact with plant tissue. This study provides the first global picture of gene expression changes in grapefruit in response to the yeast antagonist M. fructicola., (© 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.)

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)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Diffusible signal factor (DSF) family signal-mediated quorum sensing (QS) has been identified in many gram-negative bacteria. This QS pathway of Xanthomonas spp. consists of three major QS components: RpfF, RpfC, and RpfG. The rpfF gene encodes a putative enoyl-CoA hydratase that catalyzes the synthesis of the signal molecule. RpfC and RpfG serve as a two-component system for the perception and transduction of the extracellular DSF family signals. In order to further characterize the QS regulatory network in Xanthomonas citri subsp. citri, we investigated the RpfF, RpfC, and RpfG regulons by using transcriptome analyses. Comparison of the transcriptomes of the QS mutants (rpfF, rpfC, and rpfG) with that of the wild-type strain revealed a core group of genes controlled by all three QS components, suggesting that the RpfC-RpfG two-component system is a major and conserved signal perception and transduction system for DSF family signal-mediated QS in X. citri subsp. citri. The unique genes controlled by RpfF alone indicate the complexity of the QS pathway and the involvement of additional sensory mechanisms in X. citri subsp. citri. The unique genes controlled by RpfC and RpfG, respectively, support the possibility that RpfC and RpfG play broader roles in gene regulation other than transduction of DSF signals.

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.

Huanglongbing is an economically damaging disease of citrus associated with infection by 'Candidatus Liberibacter asiaticus'. Transmission of the organism via infection of seeds has not been demonstrated but is a concern since some citrus varieties, particularly those used as rootstocks in commercial plantings are propagated from seed. We compared the incidence of detection of 'Ca. Liberibacter asiaticus' DNA in individual fruit peduncles, seed coats, seeds, and in germinated seedlings from 'Sanguenelli' sweet orange and 'Conners' grapefruit fruits sampled from infected trees. Using real-time quantitative PCR (qPCR) we detected pathogen DNA in nucleic acid extracts of 36 and 100% of peduncles from 'Sanguenelli' and from 'Conners' fruits, respectively. We also detected pathogen DNA in extracts of 37 and 98% of seed coats and in 1.6 and 4% of extracts from the corresponding seeds of 'Sanguenelli' and 'Conners', respectively. Small amounts of pathogen DNA were detected in 10% of 'Sanguenelli' seedlings grown in the greenhouse, but in none of 204 extracts from 'Conners' seedlings. Pathogen DNA was detected in 4.9% and in 89% of seed coats peeled from seeds of 'Sanguenelli' and 'Conners' which were germinated on agar, and in 5% of 'Sanguenelli' but in none of 164 'Conners' seedlings which grew from these seeds on agar. No pathogen DNA was detected in 'Ridge Pineapple' tissue at 3 months post-grafting onto 'Sanguenelli' seedlings, even when pathogen DNA had been detected initially in the 'Sanguenelli' seedling. Though the apparent colonization of 'Conners' seeds was more extensive and nearly uniform compared with 'Sanguenelli' seeds, no pathogen DNA was detected in 'Conners' seedlings grown from these seeds. For either variety, no association was established between the presence of pathogen DNA in fruit peduncles and seed coats and in seedlings.

Bacterial citrus canker disease, which is caused by Xanthomonas citri subsp. citri, is one of the most devastating diseases of citrus plants. In this study, we characterized the role of the two-component regulatory system ColR/ColS in the pathogenicity of X. citri subsp. citri. colS mutants (256A10 and 421E7), colR mutants (386C6 and 417E10), and a colR colS double mutant (306DSR) all lost pathogenicity and produced no symptoms on grapefruit leaves inoculated by either pressure infiltration or the spray method. The pathogenicity defect of the colS, colR, and colR colS mutants could be complemented using the wild-type colS, colR, and colR colS genes, respectively. Mutation of colS or colR significantly reduced X. citri subsp. citri growth in planta. The ColR/ColS system also played important roles in bacterial biofilm formation in glass tubes and on leaf surfaces, lipopolysaccharide (LPS) production, catalase activity, and tolerance of environmental stress, including phenol, copper, and hydrogen peroxide. Furthermore, quantitative reverse transcription-PCR assays demonstrated that the ColR/ColS system positively regulated the expression of important virulence genes, including hrpD6, hpaF, the O-antigen LPS synthesis gene rfbC, and the catalase gene katE. Overall, our data indicate that the two-component regulatory system ColR/ColS is critical for X. citri subsp. citri virulence, growth in planta, biofilm formation, catalase activity, LPS production, and resistance to environmental stress.

Several foodborne human pathogens, when exposed to harsh conditions, enter viable but nonculturable (VBNC) state; however, still open is the question whether VBNC pathogens could be a risk for public health, because, potentially, they can resuscitate. Moreover, cultural methods for food safety control were not able to detect VBNC forms of foodborne bacteria. Particularly, it has not been established whether food chemophysical characteristics can induce VBNC state in contaminating pathogen bacterial populations, especially in food, such as salads and fresh fruit juices, not subjected to any decontamination treatment. In this preliminary study, we intentionally contaminated grapefruit juice to determine whether pathogen bacteria could enter VNBC state. In fact, grapefruit juice contains natural antimicrobial compounds, has an average pH of about 3 and low content in carbohydrates. Such characteristics make grapefruit juice a harsh environment for microbial survival. For this purpose, Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium ATCC 14028, Listeria monocytogenes ATCC 7644, and Shigella flexneri ATCC 12022, at two different inoculum sizes, have been used. Viability by the LIVE/DEAD BacLight Bacterial Viability kit and culturability by plate counts assay were monitored, whereas "resuscitation" of nonculturable populations was attempted by inoculation in nutrient-rich media. The data showed that L. monocytogenes lost both culturability and viability and did not resuscitate within 24 h independently on inoculum size, whereas E. coli O157:H7 was able to resuscitate after 24 h but did not after 48 h. Salmonella Typhimurium and S. flexneri, depending on inoculum size, lost culturability but maintained viability and were able to resuscitate; moreover, S. flexneri was still able to form colonies after 48 h at high inoculum size. In conclusion, entry into VBNC state differs on the species, depending, in turn, on inoculum size and time of incubation.

Xanthomonas citri subsp. citri is the causal agent of citrus canker, which has a significant impact on citrus production. In this study, we characterized the galU gene of X. citri subsp. citri. Two galU mutants (F6 and D12) were identified in an X. citri subsp. citri EZ-Tn5 Tnp transposon library. Rescue cloning, sequence analysis, and Southern blot analysis indicated that both of these mutants had a single copy of the EZ-Tn5 transposon inserted in galU in the chromosome. Further study showed that galU was required for biosynthesis of extracellular polysaccharides (EPS; xanthan gum) and capsular polysaccharide (CPS) and biofilm formation. Mutation of galU resulted in a loss of pathogenicity for grapefruit. The loss of pathogenicity of a galU mutant resulted from its inability to grow in planta rather than from the effect on virulence genes. Quantitative reverse transcription-PCR assays indicated that mutation of galU did not impair the expression of key virulence genes, such as pthA of X. citri subsp. citri. Although D12 had a growth rate similar to that of the wild-type strain in nutrient broth, no D12 population became established in the intercellular spaces of citrus leaves. Coinoculation of a galU mutant with the wild-type strain did not promote growth of the galU mutant in planta. Defects in EPS and CPS production, pathogenicity, and growth in planta of the galU mutant were complemented to the wild-type level using plasmid pCGU2.1 containing an intact galU gene. These data indicate that the galU gene contributes to X. citri subsp. citri growth in intercellular spaces and is involved in EPS and CPS synthesis and biofilm formation.

The most severe form of citrus canker disease is caused by Xanthomonas axonopodis pv. citri (Xac) and affects all types of important citrus crops, reducing fruit yield and quality. Copper-based products are routinely used as a standard control measure for citrus canker. In this work we demonstrate that copper treatment induces the viable but nonculturable (VBNC) state in Xac but does not prevent the development of symptoms in susceptible plants. Short-term exposures to different concentrations of copper solutions were assayed to determine which treatment resulted in Xac nonculturability. Treatment of 10(6) mL(-1) Xac cells for 10 min in a 135-muM CuSO(4) solution (equivalent to three times the free soluble copper concentration applied in one field treatment) resulted in nonculturability. However, 16% of cells were viable based on 5-cyano-2,3-ditolyl tetrazolium chloride staining and 1% were capable of producing canker lesions after infiltrating grapefruit plants. If induction of the VBNC state in Xac cells were to occur under field conditions, this would have to be taken into consideration for an effective control of canker disease.

Red grapefruit juice was treated with continuous dense phase carbon dioxide (DPCD) equipment to inactivate yeasts and molds and total aerobic microorganisms. A central composite design was used with pressure (13.8, 24.1, and 34.5 MPa) and residence time (5, 7, and 9 min) as variables at constant temperature (40 degrees C), and CO(2) level (5.7%) after experimentally measuring CO(2) solubility in the juice. Five log reduction for yeasts and molds and total aerobic microorganisms occurred at 34.5 MPa and 7 min of treatment. A storage study was performed on the fresh juice DPCD treated at these conditions. degrees Brix, pH, titratable acidity (TA), pectinesterase (PE) inactivation, cloud, color, hue tint and color density, total phenolics, antioxidant capacity, and ascorbic acid were measured after the treatment and during 6 wk storage at 4 degrees C. During storage, the DPCD-treated juice showed no growth of total aerobic microorganisms and yeasts and molds. Cloud increased (91%) while percent PE inactivation was partial (69.17%). No significant (alpha= 0.05) differences were detected between treated and untreated samples for degrees Brix, pH, and TA. Treated juice had higher lightness and redness and lower yellowness. No significant differences (alpha= 0.05) were detected for the hue tint values while the color density value was higher for the treated samples compared to the untreated. The treatment and the storage did not affect the total phenolic content of the juice. Slight differences were detected for the ascorbic acid content and the antioxidant capacity. The experimental results showed evidence that the treatment can maintain the physical and quality attributes of the juice, extending its shelf life and safety.

Trophic interactions involving predators, herbivores, and plants have been described in terrestrial systems. However, there is almost no information on the effect of trophic interactions on microbial phyllosphere community abundance, diversity, or structure. In this study, the interaction between a parasitoid, an insect herbivore, and the fungal phyllosphere community is examined. Parasitoid wasps have an indirect negative impact on fungal community diversity. On the citrus phyllosphere, the exotic wasp species, Amitus hesperidum and Encarsia opulenta, may parasitize the citrus blackfly (Aleurocanthus woglumi). If parasitism levels are low, the blackfly may produce significant amounts of honeydew secretions on the surface of the leaf. Honeydew deposition provides a carbon-rich substrate for the development of fungal growth persisting as sooty mold on the leaves. Leaves from sooty mold-infested grapefruit (Citrus paradisi) trees were collected from multiple orchards in south Texas. The effect of different levels of exotic parasite activity, citrus blackfly, and sooty mold infestation on phyllosphere mycobiota community structure and diversity was examined. Our results suggest the presence of the parasitoid may lead to a top-down trophic cascade affecting phyllosphere fungal community diversity and structure. Additionally, persistent sooty mold deposits that have classically been referred to as Capnodium citri (and related asexual morphological forms) actually comprise a myriad of fungal species including many saprophytes and potential fruit and foliar pathogens of citrus.

We provide the first conclusive evidence that Xanthomonas axonopodis pv. citri Asiatic strain (Xac-A) and, in particular, Xac-A(w), a unique citrus canker A strain isolated from Key lime in Wellington, Florida, induces a hypersensitive reaction (HR) in grapefruit leaves. Using the heterologous tomato pathogen X. perforans, as a recipient of the Xac-A(w) genomic library, we identified a 1599-bp open reading frame responsible for HR in grapefruit, but not Key lime, and designated it avrGf1. Xac-A(w)DeltaavrGf1 produced typical, although visibly reduced, citrus canker symptoms (i.e. raised pustules) in grapefruit and typical canker symptoms in Key lime. We also determined that the X. perforans transconjugant carrying an Xac-A(w) hrpG elicited HR in grapefruit and Key lime leaves, and that xopA in X. perforans was partly responsible for HR. Xac-A transconjugants carrying the X. perforans xopA were reduced in ability to grow in grapefruit leaves relative to wild-type Xac-A. The X. perforans xopA appears to be a host-limiting factor. An avrBs3 homologue, which contained 18.5 repeats and induced HR in tomato, was designated avrTaw. This gene, when expressed in a pustule-minus Xac-A(w), did not complement pustule formation; however, pthA(w), a functional pthA homologue, complemented the mutant strain to produce typical pustules in Key lime, but markedly reduced pustules in grapefruit. Both avrBs3 homologues, when expressed in a typical Xac-A strain, resulted in typical citrus canker pustules in grapefruit, indicating that neither homologue suppressed pustule size in grapefruit. Xac-A(w) contains other unidentified factors that suppress development in grapefruit.

Furanocoumarins (FCs) in grapefruit are involved in the "grapefruit/drug interactions" in humans, in which the FCs inhibit the intestinal cytochrome P450 3A4 (CYP 3A4) activity responsible for metabolizing certain prescribed medications. These interactions have adversely affected the grapefruit industry and have led a need to develop a process to remove the FCs from grapefruit juice (GFJ) in a manner that retains much of the original juice sensory attributes. In our experiments, grapefruit juice was incubated with Aspergillus niger, and the compositional changes in hydroxycinnamates, flavonoid glycosides, and the FCs were monitored. Many of the FCs and 7-geranyloxycoumarin were efficiently taken up by the fungal tissue, whereas no uptake occurred with the polar hydroxycinnamates, flavonoid glycosides, and a few of the polar FCs. This biosorption was also observed with autoclaved A. niger, indicating that the uptake of non-polar FCs by the fungal hyphae was due to adsorption rather than metabolism. The binding of the FCs to autoclaved fungus was complete within 4 h, and the level of binding was proportional to the amount of autoclaved fungal hyphae used. This removal of the FCs from GFJ led to a reduced inhibition of CYP 3A4 activity in in vitro assays by both GFJ and GFJ extracts.

Herbaria are important resources for the study of the origins and dispersal of plant pathogens, particularly bacterial plant pathogens that incite local lesions in which large numbers of pathogen genomes are concentrated. Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus bacterial canker disease, is a notable example of such a pathogen. The appearance of novel strains of the pathogen in Florida and elsewhere make it increasingly important to understand the relationships among strains of this pathogen. USDA-ARS at Beltsville, Maryland maintains approximately 700 herbarium specimens with citrus canker disease lesions up to 90 years old, originally collected from all over the world, and so is an important resource for phytogeographic studies of this bacterium. Unfortunately, DNA in herbarium specimens is degraded and may contain high levels of inhibitors of PCR. In this study, we compared a total of 23 DNA isolation techniques in combination with 31 novel primer pairs in order to develop an efficient protocol for the analysis of Xac DNA in herbarium specimens. We identified the most reliable extraction method, identified in terms of successful amplification by our panel of 31 primer pairs. We also identified the most robust primer pairs, identified as successful in the largest number of extracts prepared by different methods. We amplified Xac genomic sequences up to 542 bp long from herbarium samples up to 89 years old. Primers varied in effectiveness, with some primer pairs amplifying Xac DNA from a 1/10,000 dilution of extract from a single lesion from a citrus canker herbarium specimen. Our methodology will be useful to identify pathogens and perform molecular analyses of bacterial and possibly fungal genomes from herbarium specimens.

An aldehyde oxime O-methyl ether, phenylacetaldehyde O-methyloxime, was detected using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) in the headspace above grapefruit leaves infected with Xanthomonas axonopodis pv. citri, the causal agent of citrus bacterial canker disease (CBCD). This disease is a major phytosanitary concern, and an eradication campaign against it is currently underway in Florida. Phenylacetaldehyde O-methyloxime has been reported to be produced by other plants and fragrant flowers, but it was not observed in the headspace above uninfected grapefruit leaves, the pathogenic bacterium X. axonopodis pv. citri itself, or grapefruit leaves infected with another closely related bacterial pathogen, X. axonopodis pv. citrumelo, which causes citrus bacterial spot, a disease of no phytosanitary significance. It was also not detected from CBCD infected fruits, including orange, lemon, grapefruit, and lime. We conclude that phenylacetaldehyde O-methyloxime may potentially be used to identify CBCD infestations. However, more intensive studies will be required to fully evaluate the potential of phenylacetaldehyde O-methyloxime as a diagnostic compound for CBCD. Using SPME and GC-MS to measure phenylacetaldehyde O-methyloxime may provide an easy and feasible tool to complement current methods used to detect X. axonopodis pv. citri in environmental samples.

The yeast Candida oleophila, the base of the commercial product Aspire, is recommended for the control of postharvest decay of citrus and pome fruit. Competition for nutrients and space is believed to be the major mode of action. Involvement of fungal cell wall-degrading enzymes is also suggested to play a role in the mechanism of action of yeast antagonists. The present study showed that the yeast C. oleophila is capable of producing and secreting various cell wall-degrading enzymes, including exo-beta-1,3-glucanase, chitinase and protease. Exo-beta-1,3-glucanase and chitinase were produced and maximized in the early stages of growth, whereas protease reached a maximum level only after 6-8 days. Production of exo-beta-1,3-glucanase, chitinase and protease was stimulated by the presence of cell wall fragments of Penicillium digitatum in the growth medium, in addition to glucose. This study also provided evidence that C. oleophila is capable of secreting exo-beta-1,3-glucanase into the wounded surface of grapefruit. The role of exo-beta-1,3-glucanase ( CoEXG1) in the biocontrol activity of C. oleophila was tested using CoEXG1-knockouts and double- CoEXG1 over-producing transformants. In vitro bioassays showed that wild-type C. oleophila and exo-beta-1,3-glucanase over-expressing transformants had similar inhibitory effects on spore germination and germ-tube elongation; and both were more inhibitory to the fungus than the knockout transformant. In experiments conducted on fruit to test the biocontrol activity against infection by P. digitatum, no significant difference in inhibition was observed between transformants and untransformed C. oleophila cells at the high concentrations of cells used, whereas at a lower concentration of yeast cells the knockout transformants appeared to be less effective.