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1.
Soft Matter ; 20(12): 2804-2811, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38446076

RESUMO

The peels of lime, lemon, pomelo and citron are investigated at macroscopic and microscopic level. The structural composition of the peels is compared and properties such as peel thickness, proportion of flavedo, density and proportion of intercellular spaces are determined. µCT images are used to visualize vascular bundles and oil glands. SEM images provide information about the appearance of the cellular tissue in the outer flavedo and inner albedo. The proportion of intercellular spaces is quantitatively determined by manual and software-assisted analysis (ilastik). While there are macroscopic differences in the fruits, they differ only slightly in the orientation of the vascular bundles and the arrangement of the oil glands. However, in peel thickness and flavedo thickness, the fruit peels differ significantly from each other. There are no significant differences between the two analysis methods used, although the use of ilastik is preferred due to time reduction of up to 70%. The large amount of intercellular spaces in the albedo but also the denser flavedo both have a mechanical protective function to prevent damage to the fruit. In addition, the entire peel structure is mechanically reinforced by vascular bundles. This combination of penetration protection (flavedo) and energy dissipation (albedo) makes Citrus spp. peels a promising inspiration for technical material systems.


Assuntos
Citrus , Citrus/química , Citrus/ultraestrutura , Microscopia , Frutas/química , Frutas/ultraestrutura
2.
Phytopathology ; 108(11): 1253-1262, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29799309

RESUMO

Citrus fruit usually suffer significant losses during the storage and transportation stages. Green mold, a postharvest rot of citrus fruit caused by Penicillium digitatum, is one of the most serious fungal diseases. In this study, the antagonist strain DH-4 was identified as Bacillus amyloliquefaciens according to morphological observation and 16S ribosomal DNA analysis. In addition, it showed broad antifungal activity, especially the suppression of Penicillium spp. The culture filtrate of strain DH-4 exhibited apparent activity against P. digitatum in vitro and in vivo. In storage, the culture filtrate with DH-4 in it showed a better antiseptic effect. The antifungal substances in the culture filtrate, produced by strain DH-4, displayed stable activity in various extreme conditions. In addition, the antifungal substances in the culture filtrate were identified as macrolactin, bacillaene, iturins, fengycin, and surfactin by ultraperformance liquid chromatography (UPLC) electrospray ionization mass spectrometry analysis. The UPLC fractions containing these antifungal compounds were basically heat tolerant and all responsible for the antagonistic activity against P. digitatum. Transmission electron microscope observation indicated that the antifungal substances might cause abnormalities in the P. digitatum cellular ultrastructure, which could be the possible mode of action of B. amyloliquefaciens against P. digitatum. In addition, it was confirmed via scanning electron microscope analysis that the main way it inhibited P. digitatum was by secreting antimicrobial compounds without direct interaction. This study contributes to the understanding of the mechanism of B. amyloliquefaciens against citrus green mold as well as providing a potential application for the biocontrol of postharvest rot diseases in citrus fruit.


Assuntos
Bacillus amyloliquefaciens/fisiologia , Citrus/microbiologia , Penicillium/patogenicidade , Doenças das Plantas/imunologia , Bacillus amyloliquefaciens/ultraestrutura , Citrus/ultraestrutura , Frutas/microbiologia , Frutas/ultraestrutura , Doenças das Plantas/microbiologia
3.
BMC Plant Biol ; 15: 27, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25644332

RESUMO

BACKGROUND: Carotenoids are indispensable plant secondary metabolites that are involved in photosynthesis, antioxidation, and phytohormone biosynthesis. Carotenoids are likely involved in other biological functions that have yet to be discovered. In this study, we integrated genomic, biochemical, and cellular studies to gain deep insight into carotenoid-related biological processes in citrus calli overexpressing CrtB (phytoene synthase from Pantoea agglomerans). Fortunella hindsii Swingle (a citrus relative) and Malus hupehensis (a wild apple) calli were also utilized as supporting systems to investigate the effect of altered carotenoid accumulation on carotenoid-related biological processes. RESULTS: Transcriptomic analysis provided deep insight into the carotenoid-related biological processes of redox status, starch metabolism, and flavonoid/anthocyanin accumulation. By applying biochemical and cytological analyses, we determined that the altered redox status was associated with variations in O2 (-) and H2O2 levels. We also ascertained a decline in starch accumulation in carotenoid-rich calli. Furthermore, via an extensive cellular investigation of the newly constructed CrtB overexpressing Fortunella hindsii Swingle, we demonstrated that starch level reducation occurred in parallel with significant carotenoid accumulation. Moreover, studying anthocyanin-rich Malus hupehensis calli showed a negative effect of carotenoids on anthocyanin accumulation. CONCLUSIONS: In citrus, altered carotenoid accumulation resulted in dramatic effects on metabolic processes involved in redox modification, starch degradation, and flavonoid/anthocyanin biosynthesis. These findings provided new perspectives to understand the biological importance of carotenogenesis and of the developmental processes associated with the nutritional and sensory qualities of agricultural products that accumulate carotenoids.


Assuntos
Antocianinas/biossíntese , Carotenoides/metabolismo , Citrus/química , Flavonoides/biossíntese , Amido/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carotenoides/genética , Citrus/enzimologia , Citrus/genética , Citrus/ultraestrutura , Geranil-Geranildifosfato Geranil-Geraniltransferase/genética , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Malus/química , Malus/enzimologia , Malus/genética , Malus/ultraestrutura , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Oxirredução , Pantoea/fisiologia , Rutaceae/química , Rutaceae/enzimologia , Rutaceae/genética , Rutaceae/ultraestrutura , Análise de Sequência de DNA
4.
Phytochem Anal ; 26(2): 105-10, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25376900

RESUMO

INTRODUCTION: Hesperidin, a flavonoid known to have important pharmacological effects, accumulates particularly in the peels of satsuma mandarin (Citrus unshiu). Although histochemical studies have suggested that hesperidin forms crystals in some tissues of the Rutaceae and Umbelliferae, there has been no rigorous in situ detection or identification of hesperidin crystals in C. unshiu. OBJECTIVE: To characterise the chemical component of the crystals found in C. unshiu peels using Raman microscopy. METHODS: Sections of C. unshiu peels were made. The distribution and morphology of crystals in the sections were analysed microscopically. Raman microscopy was used to detect hesperidin in the sections directly. RESULTS: The crystals were more abundant in immature peel and were observed particularly in areas surrounding vascular bundles, around the border between the flavedo and albedo layers and just below the epidermal cells. In the morphological analysis by scanning electron microscopy, needle-shaped crystals aggregated and formed clusters of spherical crystals. Spectra obtained by Raman microscopy of the crystals in the peel sections were consistent with those of the hesperidin standard. CONCLUSION: This study showed the detailed distribution of crystals in C. unshiu peels and their main component was identified using Raman microscopy to be hesperidin for the first time.


Assuntos
Citrus/química , Hesperidina/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Citrus/ultraestrutura , Frutas/química , Frutas/ultraestrutura , Hesperidina/química , Microscopia Eletrônica de Varredura , Extratos Vegetais/química
5.
J Exp Bot ; 65(1): 61-74, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24215076

RESUMO

Auxin-like 2,4-dichlorophenoxyacetic acid (2,4-D), a high-efficiency anti-stalling agent for the post-harvest fresh fruit industry, has had its use restricted due to environmental concerns. However, no other substitutes for 2,4-D are available to the post-harvest industry. Insights into the molecular mechanism underlying the effects of 2,4-D on fruit quality preservation will provide a theoretical basis for exploring new safe and effective anti-stalling agents. This study comprehensively analysed changes in the peel of Olinda Valencia orange [Citrus sinensis (L.) Osbeck] induced by 500 ppm 2,4-D using 'omic'-driven approaches. Transcriptional profiling revealed that transcriptional factor (mainly AP2/ERF, WRKY, and NAC family members), transport, and hormone metabolism genes were over-represented and up-regulated within 24h post-treatment (HPT). Stress defence genes were up-regulated, while cell wall metabolism genes were down-regulated after 48 HPT. However, secondary metabolism genes, especially phenylpropanoid and lignin biosynthesis-related genes, were over-represented at all the time points. Comparative proteomic analysis indicated that the expression of proteins implicated in stress responses (25%), hormone metabolism, and signal transduction (12%) significantly accumulated at the post-transcriptional level. Hormone levels detected by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) showed that abscisic acid, salicylic acid, and 2,4-D significantly increased, while ethylene production (detected by gas chromatography) decreased after 2,4-D treatment. In addition, lignin and water content in the fruit peel also increased and the epicuticle wax ultrastructure was modified. In conclusion, 2,4-D retarded fruit senescence by altering the levels of many endogenous hormones and by improving stress defence capabilities by up-regulating defence-related genes and proteins.


Assuntos
Ácido 2,4-Diclorofenoxiacético/farmacologia , Citrus sinensis/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/metabolismo , Proteômica , Ácido Abscísico/metabolismo , Cromatografia Líquida de Alta Pressão , Citrus/efeitos dos fármacos , Citrus/genética , Citrus/fisiologia , Citrus/ultraestrutura , Citrus sinensis/efeitos dos fármacos , Citrus sinensis/genética , Citrus sinensis/ultraestrutura , Eletroforese em Gel Bidimensional , Etilenos/metabolismo , Ácidos Indolacéticos/metabolismo , Lignina/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ácido Salicílico/metabolismo , Estresse Fisiológico , Espectrometria de Massas em Tandem , Fatores de Tempo , Regulação para Cima , Água/metabolismo
6.
Phytopathology ; 103(6): 545-54, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23676087

RESUMO

'Candidatus Liberibacter asiaticus' is the bacterium implicated as a causal agent of the economically damaging disease of citrus called huanglongbing (HLB). Vertical transmission of the organism through seed to the seedling has not been demonstrated. Previous studies using real-time polymerase chain reaction assays indicated abundant bacterial 16S rRNA sequences in seed coats of citrus seed but the presence of intact bacterial cells was not demonstrated. We used microscopy to verify that intact bacterial cells were present in citrus seed coats. Bacterial cells with the morphology and physical dimensions appropriate for 'Ca. L. asiaticus' were seen in phloem sieve elements in the vascular bundle of grapefruit seed coats using transmission electron microscopy (TEM). Fluorescence in situ hybridization (FISH) analyses utilizing probes complementary to the 'Ca. L. asiaticus' 16S rRNA gene revealed bacterial cells in the vascular tissue of intact seed coats of grapefruit and pummelo and in fragmented vascular bundles excised from grapefruit seed coats. The physical measurements and the morphology of individual bacterial cells were consistent with those ascribed in the literature to 'Ca. L. asiaticus'. No bacterial cells were observed in preparations of seed from fruit from noninfected trees. A small library of clones amplified from seed coats from a noninfected tree using degenerate primers targeting prokaryote 16S rRNA gene sequences contained no 'Ca. L. asiaticus' sequences, whereas 95% of the sequences in a similar library from DNA from seed coats from an infected tree were identified as 'Ca. L. asiaticus', providing molecular genetic corroboration that the bacterial cells observed by TEM and FISH in seed coats from infected trees were 'Ca. L. asiaticus'.


Assuntos
Citrus/microbiologia , Hibridização in Situ Fluorescente/métodos , Rhizobiaceae/isolamento & purificação , Rhizobiaceae/ultraestrutura , Sementes/microbiologia , Sementes/ultraestrutura , Citrus/ultraestrutura , DNA Bacteriano , RNA Bacteriano/genética , RNA Ribossômico 16S , Rhizobiaceae/genética
7.
Phytopathology ; 103(5): 488-500, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23268581

RESUMO

Citrus leprosis in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR)-based diagnostic methods failed to identify CiLV-C from citrus samples with symptoms similar to citrus leprosis; however, virions similar to CiLV-C were observed in the cytoplasm of the symptomatic leaves by transmission electron microscopy. Furthermore, the causal organism was transmitted by the false spider mite, Brevipalpus phoenicis, to healthy citrus seedlings. A library of small RNAs was constructed from symptomatic leaves and used as the template for Illumina high-throughput parallel sequencing. The complete genome sequence and structure of a new bipartite RNA virus was determined. RNA1 (8,717 nucleotides [nt]) contained two open reading frames (ORFs). ORF1 encoded the replication module, consisting of five domains: namely, methyltransferase (MTR), cysteine protease-like, FtsJ-MTR, helicase (Hel), and RNA-dependent RNA polymerase (RdRp); whereas ORF2 encoded the putative coat protein. RNA2 (4,989 nt) contained five ORFs that encode the movement protein (MP) and four hypothetical proteins (p7, p15, p24, and p61). The structure of this virus genome resembled that of CiLV-C except that it contained a long 3' untranslated terminal region and an extra ORF (p7) in RNA2. Both the RNA1 and RNA2 of the new virus had only 58 and 50% nucleotide identities, respectively, with known CiLV-C sequences and, thus, it appears to be a novel virus infecting citrus. Phylogenetic analyses of the MTR, Hel, RdRp, and MP domains also indicated that the new virus was closely related to CiLV-C. We suggest that the virus be called Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) and it should be unambiguously classified as a definitive member of the genus Cilevirus. A pair of CiLV-C2 genome-specific RT-PCR primers was designed and validated to detect its presence in citrus leprosis samples collected from the Casanare and Meta states in Colombia.


Assuntos
Vetores Aracnídeos/virologia , Citrus/virologia , Ácaros/virologia , Doenças das Plantas/virologia , Vírus de RNA/isolamento & purificação , Sequência de Aminoácidos , Animais , Citrus/ultraestrutura , Colômbia , Frutas , Biblioteca Gênica , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Filogenia , Folhas de Planta/virologia , Vírus de RNA/classificação , Vírus de RNA/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Plântula/ultraestrutura , Plântula/virologia , Alinhamento de Sequência , Análise de Sequência de DNA
8.
J Exp Bot ; 63(12): 4403-17, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22611233

RESUMO

Genetic manipulation of carotenoid biosynthesis has become a recent focus for the alleviation of vitamin A deficiency. However, the genetically modified phenotypes often challenge the expectation, suggesting the incomplete comprehension of carotenogenesis. Here, embryogenic calli were engineered from four citrus genotypes as engineered cell models (ECMs) by over-expressing a bacterial phytoene synthase gene (CrtB). Ripe flavedos (the coloured outer layer of citrus fruits), which exhibit diverse natural carotenoid patterns, were offered as a comparative system to the ECMs. In the ECMs, carotenoid patterns showed diversity depending on the genotypes and produced additional carotenoids, such as lycopene, that were absent from the wild-type lines. Especially in the ECMs from dark-grown culture, there emerged a favoured ß,ß-pathway characterized by a striking accumulation of ß-carotene, which was dramatically different from those in the wild-type calli and ripe flavedos. Unlike flavedos that contained a typical chromoplast development, the ECMs sequestered most carotenoids in the amyloplasts in crystal form, which led the amyloplast morphology to show a chromoplast-like profile. Transcriptional analysis revealed a markedly flavedo-specific expression of the ß-carotene hydroxylase gene (HYD), which was suppressed in the calli. Co-expression of CrtB and HYD in the ECMs confirmed that HYD predominantly mediated the preferred carotenoid patterns between the ECMs and flavedos, and also revealed that the carotenoid crystals in the ECMs were mainly composed of ß-carotene. In addition, a model is proposed to interpret the common appearance of a favoured ß,ß-pathway and the likelihood of carotenoid degradation potentially mediated by photo-oxidation and vacuolar phagocytosis in the ECMs is discussed.


Assuntos
Citrus/química , Plastídeos/química , Vitaminas/metabolismo , beta Caroteno/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carotenoides/análise , Carotenoides/genética , Carotenoides/metabolismo , Citrus/enzimologia , Citrus/genética , Citrus/ultraestrutura , Erwinia/enzimologia , Erwinia/genética , Erwinia/imunologia , Frutas/química , Frutas/enzimologia , Frutas/genética , Frutas/ultraestrutura , Regulação da Expressão Gênica de Plantas , Genótipo , Geranil-Geranildifosfato Geranil-Geraniltransferase , Microscopia Eletrônica de Transmissão , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Modelos Biológicos , Oryza/enzimologia , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Plastídeos/enzimologia , Plastídeos/genética , Plastídeos/ultraestrutura , Coelhos , Técnicas de Cultura de Tecidos , Vitaminas/genética , beta Caroteno/análise , beta Caroteno/genética
9.
Phytopathology ; 102(1): 122-7, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21916557

RESUMO

A Citrus volkameriana tree displaying symptoms similar to citrus leprosis on its leaves and bark was found in Hawaii. Citrus leprosis virus C (CiLV-C)-specific detection assays, however, were negative for all tissues tested. Short, bacilliform virus-like particles were observed by transmission electron microscopy in the cytoplasm of symptomatic leaves but not in healthy controls. Double-stranded (ds) RNAs ≈8 and 3 kbp in size were present in symptomatic leaf tissue but not in healthy controls. Excluding poly(A) tails, the largest molecule, RNA1, was 8,354 bp in length. The ≈3 kbp dsRNA band was found to be composed of two distinct molecules, RNA2 and RNA3, which were 3,169 and 3,113 bp, respectively. Phylogenetic analyses indicated that the RNA-dependent RNA polymerase (RdRp) domain located in RNA1 was most closely related to the RdRp domain of CiLV-C. A reverse-transcription polymerase chain reaction assay developed for the detection of this virus was used to screen nearby citrus trees as well as Hibiscus arnottianus plants with symptoms of hibiscus green spot, a disease associated with infection by Hibiscus green spot virus (HGSV). All nearby citrus trees tested negative with the assay; however, symptomatic H. arnottianus plants were positive. All three RNAs were present in symptomatic H. arnottianus and were >98% identical to the RNAs isolated from C. volkameriana. We contend that the virus described in this study is HGSV, and propose that it be the type member of a new virus genus, Higrevirus.


Assuntos
Citrus/virologia , Doenças das Plantas/virologia , Vírus de Plantas/isolamento & purificação , Vírus de RNA/isolamento & purificação , RNA Polimerase Dependente de RNA/genética , Sequência de Aminoácidos , Sequência de Bases , Citrus/ultraestrutura , DNA Complementar/química , DNA Complementar/genética , Genoma Viral/genética , Havaí , Hibiscus/virologia , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Filogenia , Casca de Planta/virologia , Folhas de Planta/virologia , Vírus de Plantas/classificação , Vírus de Plantas/genética , Vírus de Plantas/ultraestrutura , Estrutura Terciária de Proteína/genética , Vírus de RNA/classificação , Vírus de RNA/genética , Vírus de RNA/ultraestrutura , RNA de Cadeia Dupla/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Vírion/ultraestrutura
10.
Cells ; 10(11)2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34831444

RESUMO

Zn2+- and Ca2+-dependent nucleases exhibit activity toward dsDNA in the four classes of cation-dependent nucleases in plants. Programmed cell death (PCD) is involved in the degradation of cells during schizolysigenous secretory cavity formation in Citrus fruits. Recently, the Ca2+-dependent DNase CgCAN was proven to play a key role in nuclear DNA degradation during the PCD of secretory cavity formation in Citrus grandis 'Tomentosa' fruits. However, whether Zn2+-dependent nuclease plays a role in the PCD of secretory cells remains poorly understood. Here, we identified a Zn2+-dependent nuclease gene, CgENDO1, from Citrus grandis 'Tomentosa', the function of which was studied using Zn2+ ions cytochemical localization, DNase activity assays, in situ hybridization, and protein immunolocalization. The full-length cDNA of CgENDO1 contains an open reading frame of 906 bp that encodes a protein 301 amino acids in length with a S1/P1-like functional domain. CgENDO1 degrades linear double-stranded DNA at acidic and neutral pH. CgENDO1 is mainly expressed in the late stage of nuclear degradation of secretory cells. Further spatiotemporal expression patterns of CgENDO1 showed that CgENDO1 is initially located on the endoplasmic reticulum and then moves into intracellular vesicles and nuclei. During the late stage of nuclear degradation, it was concentrated in the area of nuclear degradation involved in nuclear DNA degradation. Our results suggest that the Zn2+-dependent nuclease CgENDO1 plays a direct role in the late degradation stage of the nuclear DNA in the PCD of secretory cavity cells of Citrus grandis 'Tomentosa' fruits.


Assuntos
Apoptose , Núcleo Celular/metabolismo , Citrus/citologia , Endonucleases/metabolismo , Frutas/citologia , Proteínas de Plantas/metabolismo , Zinco/metabolismo , Núcleo Celular/ultraestrutura , Citrus/genética , Citrus/ultraestrutura , Fragmentação do DNA , Desoxirribonucleases/metabolismo , Frutas/ultraestrutura , Regulação da Expressão Gênica de Plantas , Modelos Biológicos , Frações Subcelulares/metabolismo
11.
Viruses ; 12(12)2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33256049

RESUMO

"Cross-protection", a nearly 100 years-old virological term, is suggested to be changed to "close protection". Evidence for the need of such change has accumulated over the past six decades from the laboratory experiments and field tests conducted by plant pathologists and plant virologists working with different plant viruses, and, in particular, from research on Citrus tristeza virus (CTV). A direct confirmation of such close protection came with the finding that "pre-immunization" of citrus plants with the variants of the T36 strain of CTV but not with variants of other virus strains was providing protection against a fluorescent protein-tagged T36-based recombinant virus variant. Under natural conditions close protection is functional and is closely associated both with the conservation of the CTV genome sequence and prevention of superinfection by closely similar isolates. It is suggested that the mechanism is primarily directed to prevent the danger of virus population collapse that could be expected to result through quasispecies divergence of large RNA genomes of the CTV variants continuously replicating within long-living and highly voluminous fruit trees. This review article provides an overview of the CTV cross-protection research, along with a discussion of the phenomenon in the context of the CTV biology and genetics.


Assuntos
Citrus/imunologia , Citrus/virologia , Closterovirus/fisiologia , Proteção Cruzada/imunologia , Genoma Viral , Doenças das Plantas/prevenção & controle , Doenças das Plantas/virologia , Replicação Viral , Citrus/ultraestrutura , Evolução Molecular , Genômica/métodos , Interações Hospedeiro-Patógeno , Fenótipo , Superinfecção
12.
Phytopathology ; 99(5): 480-6, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19351243

RESUMO

A new medium designated Liber A has been designed and used to successfully cultivate all three 'Candidatus Liberibacter spp.,' the suspect causative agents of huanglongbing (HLB) in citrus. The medium containing citrus vein extract and a growth factor sustained growth of 'Ca. Liberibacter spp.' for four or five single-colony transfers before viability declined. Colonies, positive for 'Ca. L. asiaticus' by a 16s-based rDNA real-time polymerase chain reaction (RT-PCR) assay and sequencing, were irregular-shaped, convex, and 0.1 to 0.3 mm after 3 to 4 days. Suspect 'Ca. L. asiaticus' and 'Ca. L. americanus' cells were observed in infected tissue and on agar culture by scanning electron microscopy. The cells were ovoid to rod shaped, 0.3 to 0.4 by 0.5 to 2.0 microm, often with fimbriae-like appendages. Two strains of 'Ca. L. asiaticus' and one of 'Ca. L. americanus' grown on Liber A medium were pathogenic on citrus and could be isolated from noninoculated tissues of inoculated trees and seedlings 9 and 2 months later, respectively. The identity was confirmed by RT-PCR and 16s rDNA sequencing. This is the first report of the cultivation and pathogenicity of 'Ca. L. asiaticus' and 'Ca. L. americanus' associated with symptoms of HLB.


Assuntos
Citrus/microbiologia , Meios de Cultura/farmacologia , Doenças das Plantas/microbiologia , Rhizobiaceae/efeitos dos fármacos , Rhizobiaceae/crescimento & desenvolvimento , Citrus/ultraestrutura , Meios de Cultura/química , Técnicas de Cultura , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , RNA Ribossômico 16S/genética , Rhizobiaceae/isolamento & purificação , Rhizobiaceae/patogenicidade , Rhizobiaceae/ultraestrutura
13.
Commun Agric Appl Biol Sci ; 74(3): 715-21, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20222555

RESUMO

In order to improve the treatment efficiency of conventional synthetic fungicides we investigated the relationship between the decay control efficacy and the concentration of imazalil (IMZ) when the fungicide was co-applied with NaHCO3 (SBC) for 2 min. Experiments were carried out using lemon fruit (Citrus limon (L.) Burm .f. cv Verna), either wounded or not before the treatment with 0, 50, 100, 250 or 1000 microg mL(-1) IMZ or the co-application of 3% SBC with 0, 50, 100 and 250 microg mL(-1) IMZ. Wounded lemons were used to simulate and study, the host-fungicide interaction in a potential infection court as affected by the SBC-IMZ coapplication. The occurrence of natural decay was monitored after 7, 14 and 30 days of storage at 8 degrees C and after a 6 day simulated marketing period (SMP) at 20 degrees C and 75% RH. During storage and SMP a comparable efficacy to 1000 microg mL(-1) IMZ, in controlling natural decay development, was obtained with a ten times lesser amount of IMZ when co-applied with SBC. Studies performed following the SBC-IMZ co-application evidenced the induction of natural resistance along with structural changes and an increase of IMZ allocated in the potential infection courts.


Assuntos
Citrus/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Imidazóis/farmacologia , Doenças das Plantas/prevenção & controle , Bicarbonato de Sódio/farmacologia , Citrus/ultraestrutura , Quimioterapia Combinada , Microscopia Eletrônica de Varredura , Doenças das Plantas/microbiologia
14.
Commun Agric Appl Biol Sci ; 74(3): 659-65, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20222548

RESUMO

The feasibility to restrain citrus green mould (Penicillium digitatum Sacc.) during storage, by means of induced natural resistance, was investigated following combined heat and ultravioLet light C (254 nm, UV-C) treatments. Heat treatment (HT) was performed by keeping Citrus fruits in a humid saturated room at 36 degrees C for 36 h while, the UV-C light treatment (254 nm) was carried out by illuminating the fruits with 3 or 6 kJm(-2). Physical treatments were sequential and reversely applied to un-inoculated or to 24 h-old inoculated lemons or oranges. Then, fruits were stored for 30 days at 8 or 5 degrees C with 95% relative humidity (RH) followed by a 6 day simulated marketing period at 20 degrees C and 75% RH. Constitutive (cuticle and epicuticular wax) and induced resistance (scoparone biosynthesis) were monitored and the variation in natural resistance was correlated to the decay development. HT influenced positively the constitutive factors of resistance by reducing or delaying cuticular cracking and by remodelling the epicuticular wax layer. Following HT the synthesis of scoparone took place only in the albedo of wounds and was greater when fruits were inoculated. Following UV-C illumination, scoparone accumulated rapidly in the outer rind (flavedo) but, particularly with 6 kJm(-2), favoured cuticular fracturing during storage. Sequential treatments resulted significantly more effective in controlling decay on either, inoculated and non inoculated fruit. The best control of green mould during storage of lemons and oranges was obtained when HT preceded 6 kJm(-2) UV-C illumination with 52 and 45% reduction of natural decay in fruit stored at 8 degrees C compared to the control, respectively.


Assuntos
Citrus/microbiologia , Conservação de Alimentos/métodos , Imunidade Inata , Penicillium/patogenicidade , Doenças das Plantas/microbiologia , Citrus/ultraestrutura , Citrus sinensis/microbiologia , Citrus sinensis/ultraestrutura , Frutas/microbiologia , Frutas/ultraestrutura , Umidade , Microscopia Eletrônica de Varredura , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Sesquiterpenos , Temperatura , Terpenos/metabolismo , Fatores de Tempo , Fitoalexinas
15.
Plant Physiol Biochem ; 135: 372-384, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30616112

RESUMO

Nutrient deficiency has economic and ecological repercussions for citrus fruit crops worldwide. Citrus crops rely on fertilization to maintain good fruit output and quality, whereas new crop management policy aims to reduce fertilizers input. New rootstocks are needed to meet to this constraint, and the use of new tetraploid rootstocks better adapted to lower nutrient intake could offer a promising way forward. Here we compared physiological, biochemical and anatomic traits of leaves in diploid (2x) and doubled-diploid (4x) Citrumelo 4475 (Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) and Volkamer lemon (Citrus limonia Osb.) seedlings over 7 months of nutrient deficiency. Photosynthetic parameters (Pnet, Gs and Fv/Fm) decreased, but to a lesser extent in 4x genotypes than 2x. Degradation of the ultrastructural organelles (chloroplasts and mitochondria) and compound cells (thylakoids and starches) was also lower in 4x genotypes, suggesting that tetraploidy may enhance tolerance to nutrient deficiency. However, leaf surface (stomata, stomatal density and epithelial cells) showed no nutrient deficiency-induced change. In 4x Citrumelo 4475, the higher tolerance to nutrient deficiency was associated with a lower MDA and H2O2 accumulation than in the 2x, suggesting a more efficient antioxidant system in the 4x genotype. However, few differences in antioxidant system and oxidative status were observed between 2x and 4x Volkamer lemons.


Assuntos
Citrus/genética , Diploide , Plântula/genética , Tetraploidia , Clorofila A/metabolismo , Cloroplastos/ultraestrutura , Citrus/metabolismo , Citrus/fisiologia , Citrus/ultraestrutura , Citrus paradisi/genética , Citrus paradisi/metabolismo , Citrus paradisi/fisiologia , Citrus paradisi/ultraestrutura , Microscopia Eletrônica de Varredura , Mitocôndrias/ultraestrutura , Nutrientes/deficiência , Fotossíntese , Poncirus/genética , Poncirus/metabolismo , Poncirus/fisiologia , Poncirus/ultraestrutura , Plântula/metabolismo , Plântula/fisiologia , Plântula/ultraestrutura , Estresse Fisiológico
16.
J Plant Physiol ; 164(1): 100-3, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16781796

RESUMO

Seedlings of Citrus volkameriana (L.) were grown hydroponically for 43 days in order to study the effect of Mn concentration (0, 2, 14, 98 and 686 microM) in the nutrient solution on leaf anatomy and mesophyll chloroplast ultrastructure. Increasing Mn concentration stimulated leaf lamina thickness. The size of mesophyll chloroplasts decreased and increased under 0 and 686 microM Mn, respectively, compared to the intermediate Mn concentrations, similar with regard to the number of chloroplasts per mesophyll cell area. Thylakoid membranes of plants grown under 0 microM Mn were somewhat swelled, while those in other Mn treatments did not present any visible malformation. The relative volume of starch grains per chloroplast was significantly smaller under 0-98 microM Mn (12.8-16.0%) than in the treatment with 686 microM Mn (67.6%). Further, under 686 microM Mn, dark deposits were found in vacuoles. The existence of a cell adaptation mechanism to excessive Mn availability (686 microM Mn) by increasing the size of chloroplasts as well as their number per cellular area, is discussed.


Assuntos
Cloroplastos/efeitos dos fármacos , Citrus/efeitos dos fármacos , Manganês/farmacologia , Cloroplastos/ultraestrutura , Citrus/anatomia & histologia , Citrus/ultraestrutura , Folhas de Planta/anatomia & histologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/ultraestrutura , Tilacoides/efeitos dos fármacos , Tilacoides/ultraestrutura
17.
Bioinspir Biomim ; 11(4): 045002, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27433857

RESUMO

Fruit walls as well as nut and seed shells typically perform a multitude of functions. One of the biologically most important functions consists in the direct or indirect protection of the seeds from mechanical damage or other negative environmental influences. This qualifies such biological structures as role models for the development of new materials and components that protect commodities and/or persons from damage caused for example by impacts due to rough handling or crashes. We were able to show how the mechanical properties of metal foam based components can be improved by altering their structure on various hierarchical levels inspired by features and principles important for the impact and/or puncture resistance of the biological role models, rather than by tuning the properties of the bulk material. For this various investigation methods have been established which combine mechanical testing with different imaging methods, as well as with in situ and ex situ mechanical testing methods. Different structural hierarchies especially important for the mechanical deformation and failure behaviour of the biological role models, pomelo fruit (Citrus maxima) and Macadamia integrifolia, were identified. They were abstracted and transferred into corresponding structural principles and thus hierarchically structured bio-inspired metal foams have been designed. A production route for metal based bio-inspired structures by investment casting was successfully established. This allows the production of complex and reliable structures, by implementing and combining different hierarchical structural elements found in the biological concept generators, such as strut design and integration of fibres, as well as by minimising casting defects. To evaluate the structural effects, similar investigation methods and mechanical tests were applied to both the biological role models and the metallic foams. As a result an even deeper quantitative understanding of the form-structure-function relationship of the biological concept generators as well as the bio-inspired metal foams was achieved, on deeper hierarchical levels and overarching different levels.


Assuntos
Absorção Fisico-Química , Materiais Biomiméticos , Citrus/ultraestrutura , Frutas/ultraestrutura , Macadamia/ultraestrutura , Metais , Sementes/ultraestrutura , Substâncias Viscoelásticas , Biomimética , Frutas/fisiologia , Macadamia/fisiologia , Sementes/fisiologia
18.
Plant Sci ; 247: 13-24, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27095396

RESUMO

Citrus is a wide genus in which most of the cultivated species and cultivars are natural parthenocarpic mutants or hybrids (i.e. orange, mandarin, tangerine, grapefruit). The autonomous increase in GA1 ovary concentration during anthesis was suggested as being the stimulus responsible for parthenocarpy in Citrus regardless of the species. To determine the exact GA-role in parthenocarpic fruit set, the following hypothesis was tested: GA triggers and maintains cell division in ovary walls causing fruit set. Obligate and facultative parthenocarpic Citrus species were used as a model system because obligate parthenocarpic Citrus sp (i.e. Citrus unshiu) have higher GA levels and better natural parthenocarpic fruit set compared to other facultative parthenocarpic Citrus (i.e. Citrus clementina). The autonomous activation of GA synthesis in C. unshiu ovary preceded cell division and CYCA1.1 up-regulation (a G2-stage cell cycle regulator) at anthesis setting a high proportion of fruits, whereas C. clementina lacked this GA-biosynthesis and CYCA1.1 up-regulation failing in fruit set. In situ hybridization experiments revealed a tissue-specific expression of GA20ox2 only in the dividing tissues of the pericarp. Furthermore, CYCA1.1 expression correlated endogenous GA1 content with GA3 treatment, which stimulated cell division and ovary growth, mostly in C. clementina. Instead, paclobutrazol (GA biosynthesis inhibitor) negated cell division and reduced fruit set. Results suggest that in parthenocarpic citrus the specific GA synthesis in the ovary walls at anthesis triggers cell division and, thus, the necessary ovary growth rate to set fruit.


Assuntos
Citrus/fisiologia , Frutas/fisiologia , Giberelinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Divisão Celular , Parede Celular/metabolismo , Citrus/genética , Citrus/crescimento & desenvolvimento , Citrus/ultraestrutura , Flores/genética , Flores/crescimento & desenvolvimento , Flores/fisiologia , Flores/ultraestrutura , Frutas/genética , Frutas/crescimento & desenvolvimento , Frutas/ultraestrutura , Regulação da Expressão Gênica de Plantas , Microscopia Eletrônica de Varredura , Filogenia , Análise de Sequência de DNA , Regulação para Cima
19.
J Agric Food Chem ; 53(11): 4517-23, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15913319

RESUMO

Three diploid citrus somatic hybrids (cybrids) were produced by fusions combining nucellar callus-derived protoplasts of Willow Leaf mandarin (Citrus deliciosa Ten.) and Commune clementine (Citrus clementina Hort. ex Tan.) with, respectively, leaf protoplasts of Eureka lemon [Citrus limon (L.) Burm.] and Marumi kumquat [Fortunella japonica (Thunb.) Swing.] and leaf protoplasts of Marumi kumquat. Ploidy and origins of the nuclear, chloroplastic, and mitochondrial genomes were investigated by flow cytometry and nuclear and cytoplasmic simple sequence repeat analyses. Volatile compounds were extracted from the leaves of the three cybrids by a pentane/ether (1:1) mixture, analyzed by GC-MS, and compared to those of their parents. The cybrids were found to be very close to their nucleus-giving parent, suggesting that the main information for volatile compounds biosynthesis is contained in the nucleus. However, nucleo-cytoplasmic interactions occurred: the (mandarin + lemon) cybrid, possessing nucleus and chloroplasts of lemon and mitochondria from mandarin, synthesizes more monoterpene alcohols and esters than its nucleus-giving parent; the (clementine + kumquat) cybrid, possessing nucleus from kumquat and organelles from mandarin, synthesizes more monoterpene and sesquiterpene hydrocarbons and sesquiterpene alcohols than its nucleus-giving parent.


Assuntos
Núcleo Celular/química , Citrus/genética , Citoplasma/química , Hibridização Genética , Folhas de Planta/química , Folhas de Planta/ultraestrutura , Citrus/química , Citrus/ultraestrutura , DNA Mitocondrial/análise , DNA de Plantas/análise , Citometria de Fluxo , Monoterpenos/análise , Rutaceae/química , Rutaceae/genética , Rutaceae/ultraestrutura , Sesquiterpenos/análise , Volatilização
20.
J Food Prot ; 64(3): 310-4, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11252472

RESUMO

Studies were conducted to evaluate the infiltration of dye and bacteria into the interior of orange fruit and the impact of possible infiltration on achieving a 5-log microbial reduction during fresh juice processing. Fresh orange fruit were treated at the stem end area with dye and either Salmonella Rubislaw or Escherichia coli strains expressing green fluorescent protein. Microscopic images showed that bacterial contaminants localized at the surface or near surface areas that may be sanitized by surface treatments. Dye infiltration was not a reliable indicator of bacterial penetration in citrus fruit. To quantify the reduction of bacterial contamination, orange fruit were inoculated with E. coli and processed with and without hot water treatments. Greater than 5-log reductions were achieved in juice extracted from fruit immersed in hot water for 1 or 2 min at 80 degrees C, in comparison to the E. coli level detected in the control juice obtained by homogenization of inoculated fruit.


Assuntos
Bebidas/microbiologia , Citrus/microbiologia , Desinfecção/métodos , Manipulação de Alimentos , Citrus/ultraestrutura , Contagem de Colônia Microbiana , Qualidade de Produtos para o Consumidor , Escherichia coli , Proteínas de Fluorescência Verde , Histocitoquímica , Temperatura Alta , Indicadores e Reagentes/química , Proteínas Luminescentes/química , Microscopia Eletrônica de Varredura , Salmonella , Propriedades de Superfície , Água
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