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1.
BMC Plant Biol ; 21(1): 397, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433413

RESUMO

BACKGROUND: Mandarin 'Shatangju' is susceptible to Huanglongbing (HLB) and the HLB-infected fruits are small, off-flavor, and stay-green at the maturity period. To understand the relationship between pericarp color and HLB pathogen and the effect mechanism of HLB on fruit pericarp coloration, quantitative analyses of HLB bacterial pathogens and carotenoids and also the integrative analysis of metabolome and transcriptome profiles were performed in the mandarin 'Shatangju' variety with four different color fruits, whole green fruits (WGF), top-yellow and base-green fruits (TYBGF), whole light-yellow fruits (WLYF), and whole dark-yellow fruits (WDYF) that were infected with HLB. RESULTS: the HLB bacterial population followed the order WGF > TYBGF > WLYF > WDYF. And there were significant differences between each group of samples. Regarding the accumulation of chlorophyll and carotenoid, the chlorophyll-a content in WGF was the highest and in WDYF was the lowest. The content of chlorophyll-b in WGF was significantly higher than that in other three pericarps. There were significant differences in the total content of carotenoid between each group. WGF and TYBGF pericarps were low in phytoene, γ-carotene, ß-cryptoxanthin and apocarotenal, while other kinds of carotenoids were significantly higher than those in WDYF. And WLYF was only short of apocarotenal. We comprehensively compared the transcriptome and metabolite profiles of abnormal (WGF, TYBGF and WLYF) and normal (WDYF, control) pericarps. In total, 2,880, 2,782 and 1,053 differentially expressed genes (DEGs), including 121, 117 and 43 transcription factors were identified in the three comparisons, respectively. The qRT-PCR confirmed the expression levels of genes selected from transcriptome. Additionally, a total of 77 flavonoids and other phenylpropanoid-derived metabolites were identified in the three comparisons. Most (76.65 %) showed markedly lower abundances in the three comparisons. The phenylpropanoid biosynthesis pathway was the major enrichment pathway in the integrative analysis of metabolome and transcriptome profiles. CONCLUSIONS: Synthesizing the above analytical results, this study indicated that different color pericarps were associated with the reduced levels of some carotenoids and phenylpropanoids derivatives products and the down-regulation of proteins in flavonoids, phenylpropanoids derivatives biosynthesis pathway and the photosynthesis-antenna proteins.


Assuntos
Clorofila/análise , Citrus/genética , Citrus/microbiologia , Flavonoides/análise , Frutas/microbiologia , Interações Hospedeiro-Patógeno , Liberibacter/patogenicidade , Pigmentos Biológicos , Produtos Agrícolas/genética , Produtos Agrícolas/microbiologia , Produtos Agrícolas/fisiologia , Frutas/genética , Frutas/fisiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Metaboloma , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Transcriptoma
2.
Nat Commun ; 12(1): 4049, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193873

RESUMO

Xyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.


Assuntos
Parede Celular/metabolismo , Citrus/microbiologia , Glucanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Fatores de Virulência/genética , Xanthomonas/metabolismo , Xilanos/metabolismo , Proteínas de Bactérias/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Ativação Transcricional , Sistemas de Secreção Tipo III/metabolismo , Fatores de Virulência/metabolismo , Xanthomonas/genética , Xanthomonas/patogenicidade
3.
World J Microbiol Biotechnol ; 37(6): 95, 2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-33963452

RESUMO

Huanglongbing (HLB), also known as 'citrus greening', is an extremely destructive disease of citrus worldwide. HLB is associated with three species of the fastidious proteobacterium, Candidatus Liberibacter asiaticus (CaLas), Ca. L. africanus and Ca. L. americanus with CaLas being the most widely distributed around the world and the only species detected and described so far in India, one of the major global citrus fruit producers. Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. Three types of prophages, Type 1, Type 2 and Type 3 have been identified and described in CaLas so far. In the present study, 441 CaLas isolates sampled across 18 Indian states were used for prophage typing. Based on detection of three prophage types by PCR, all the eight probable combinations of CaLas prophages were identified, including single Type 1 (26.5%), single Type 2 (18.8%), single Type 3 (1.4%), Type 1 + Type 2 (20.4%), Type 1 + Type 3 (12.5%), Type 2 + Type 3 (4.8%), Type 1 + Type 2 + Type 3 (11.3%) and None type (4.3%). Prophage types were confirmed by PCR amplicon sequencing and subsequent phylogenetic analysis. By discovery of all 3 prophages and based on genetic identity and genetic distance, CaLas populations from eighteen citrus growing states were separated into two major Prophage Typing Groups (PTGs): PTG1 and PTG2. The PTG1 comprised of CaLas from North-West India and PTG2 from rest of the country (North-East, Central and South India), and both major groups were further divided into two (PTG1-A, PTG1-B) and three (PTG2-A, PTG2-B and PTG2-C) subgroups respectively. The findings of CaLas population patterns provide evidence for independent origins of HLB-associated CaLas. CRISPR (clustered regularly interspaced short palindromic repeats) array was also detected in CaLas isolates. This is the first report evaluating the genetic variation of a large population of CaLas bacterium in India using the PCR markers from the prophage regions which would certainly assist the ongoing HLB management efforts in India.


Assuntos
Citrus/microbiologia , Liberibacter/classificação , Prófagos/genética , Análise de Sequência de DNA/métodos , Sistemas CRISPR-Cas , DNA Viral/genética , Variação Genética , Índia , Liberibacter/isolamento & purificação , Liberibacter/virologia , Tipagem Molecular , Filogenia , Doenças das Plantas/microbiologia , Prófagos/classificação
4.
Plant Mol Biol ; 106(4-5): 349-366, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33871796

RESUMO

KEY MESSAGE: The temporal expression profiles of citrus leaves explain the sink-source transition of immature leaves to mature leaves and provide knowledge regarding the differential responses of mature and immature leaves to biotic stress such as citrus canker and Asian citrus psyllid (Diaphorina citri). Citrus is an important fruit crop worldwide. Different developmental stages of citrus leaves are associated with distinct features, such as differences in susceptibilities to pathogens and insects, as well as photosynthetic capacity. Here, we investigated the mechanisms underlying these distinctions by comparing the gene expression profiles of mature and immature citrus leaves. Immature (stages V3 and V4), transition (stage V5), and mature (stage V6) Citrus sinensis leaves were chosen for RNA-seq analyses. Carbohydrate biosynthesis, photosynthesis, starch biosynthesis, and disaccharide metabolic processes were enriched among the upregulated differentially expressed genes (DEGs) in the V5 and V6 stages compared with that in the V3 and V4 stages. Glucose level was found to be higher in V5 and V6 than in V3 and V4. Among the four stages, the largest number of DEGs between contiguous stages were identified between V5 and V4, consistent with a change from sink to source, as well as with the sucrose and starch quantification data. The differential expression profiles related to cell wall synthesis, secondary metabolites such as flavonoids and terpenoids, amino acid biosynthesis, and immunity between immature and mature leaves may contribute to their different responses to Asian citrus psyllid infestation. The expression data suggested that both the constitutive and induced gene expression of immunity-related genes plays important roles in the greater resistance of mature leaves against Xanthomonas citri compared with immature leaves. The gene expression profiles in the different stages can help identify stage-specific promoters for the manipulation of the expression of citrus traits according to the stage. The temporal expression profiles explain the sink-source transition of immature leaves to mature leaves and provide knowledge regarding the differential responses to biotic stress.


Assuntos
Citrus/crescimento & desenvolvimento , Citrus/genética , Folhas de Planta/genética , Transcriptoma , Citrus/imunologia , Citrus/microbiologia , Resistência à Doença/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Desenvolvimento Vegetal , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo
5.
Molecules ; 26(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800893

RESUMO

In order to replace the huge amounts of copper salts used in citrus orchards, alternatives have been sought in the form of organic compounds of natural origin with activity against the causative agent of citrus canker, the phytopathogen Xanthomonas citri subsp. Citri. We synthesized a series of 4-alkoxy-1,2-benzene diols (alkyl-BDOs) using 1,2,4-benzenetriol (BTO) as a starting material through a three-step synthesis route and evaluated their suitability as antibacterial compounds. Our results show that alkyl ethers derived from 1,2,4-benzenetriol have bactericidal activity against X. citri, disrupting the bacterial cell membrane within 15 min. Alkyl-BDOs were also shown to remain active against the bacteria while in solution, and presented low toxicity to (human) MRC-5 cells. Therefore, we have demonstrated that 1,2,4-benzenetriol-a molecule that can be obtained from agricultural residues-is an adequate precursor for the synthesis of new compounds with activity against X. citri.


Assuntos
Antibacterianos/farmacologia , Derivados de Benzeno/farmacologia , Citrus/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Doenças das Plantas/microbiologia , Folhas de Planta/efeitos dos fármacos , Xanthomonas/patogenicidade , Antibacterianos/química , Derivados de Benzeno/química , Proliferação de Células , Citrus/microbiologia , Fibroblastos/citologia , Humanos , Folhas de Planta/microbiologia
6.
PLoS One ; 16(3): e0242392, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33730040

RESUMO

"Candidatus Liberibacter asiaticus" (CLas) and Spiroplasma citri are phloem-limited bacteria that infect citrus and are transmitted by insect vectors. S. citri causes citrus stubborn disease (CSD) and is vectored by the beet leafhopper in California. CLas is associated with the devastating citrus disease, Huanglongbing (HLB), and is vectored by the Asian citrus psyllid. CLas is a regulatory pathogen spreading in citrus on residential properties in southern California and is an imminent threat to spread to commercial citrus plantings. CSD is endemic in California and has symptoms in citrus that can be easily confused with HLB. Therefore, the objective of this study was to develop a multiplex qPCR and duplex droplet digital PCR (ddPCR) assay for simultaneous detection of CLas and S. citri to be used where both pathogens can co-exist. The multiplex qPCR assay was designed to detect multicopy genes of CLas-RNR (5 copies) and S. citri-SPV1 ORF1 (13 copies), respectively, and citrus cytochrome oxidase (COX) as internal positive control. Absolute quantitation of these pathogens was achieved by duplex ddPCR as a supplement for marginal qPCR results. Duplex ddPCR allowed higher sensitivity than qPCR for detection of CLas and S. citri. ddPCR showed higher tolerance to inhibitors and yielded highly reproducible results. The multiplex qPCR assay has the benefit of testing both pathogens at reduced cost and can serve to augment the official regulatory protocol for CLas detection in California. Moreover, the ddPCR provided unambiguous absolute detection of CLas and S. citri at very low concentrations without any standards for pathogen titer.


Assuntos
DNA Bacteriano/análise , Liberibacter/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Spiroplasma citri/genética , Citrus/microbiologia , DNA Bacteriano/metabolismo , Liberibacter/isolamento & purificação , Doenças das Plantas/microbiologia , Reprodutibilidade dos Testes , Spiroplasma citri/isolamento & purificação
7.
Cells ; 10(2)2021 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-33672559

RESUMO

(1) Background: This study was aimed at identifying the Colletotrichum species associated with twig and shoot dieback of citrus, a new syndrome occurring in the Mediterranean region and also reported as emerging in California. (2) Methods: Overall, 119 Colletotrichum isolates were characterized. They were recovered from symptomatic trees of sweet orange, mandarin and mandarin-like fruits during a survey of citrus groves in Albania and Sicily (southern Italy). (3) Results: The isolates were grouped into two distinct morphotypes. The grouping of isolates was supported by phylogenetic sequence analysis of two genetic markers, the internal transcribed spacer regions of rDNA (ITS) and ß-tubulin (TUB2). The groups were identified as Colletotrichum gloeosporioides and C. karstii, respectively. The former accounted for more than 91% of isolates, while the latter was retrieved only occasionally in Sicily. Both species induced symptoms on artificially wound inoculated twigs. C. gloeosporioides was more aggressive than of C. karstii. Winds and prolonged drought were the factor predisposing to Colletotrichum twig and shoot dieback. (4) Conclusions: This is the first report of C. gloeosporioides and C. karstii as causal agents of twig and shoot dieback disease in the Mediterranean region and the first report of C. gloeosporioides as a citrus pathogen in Albania.


Assuntos
Citrus/microbiologia , Colletotrichum/fisiologia , Doenças das Plantas/microbiologia , Colletotrichum/crescimento & desenvolvimento , Colletotrichum/isolamento & purificação , DNA Intergênico/genética , Micélio/fisiologia , Necrose , Filogenia , Folhas de Planta/microbiologia
8.
Microbiol Res ; 248: 126747, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33740671

RESUMO

The host-selective ACT toxin is essential for the pathogenesis of the citrus fungal pathogen Alternaria alternata. However, the mechanism of ACT-toxin gene clusters ACT-toxin biosynthesis regulated by is still poorly understood. The biosynthesis of ACT toxin is mainly regulated by multiple ACT toxin genes located in the secondary metabolite gene cluster. In this study, we reported a transcription regulator ACTR contributes ACT toxin biosynthesis through mediating ACT toxin synthesis gene ACTS4 in Alternaria alternata. We generated ACTR-disrupted and -silenced mutants in the tangerine pathotype of A. alternata. Phenotype analysis showed that the ACTR mutants displayed a significant loss of ACT toxin production and a decreased virulence on citrus leaves whereas the vegetative growth and sporulation were not affected, indicating an essential role of ACTR in both ACT toxin biosynthesis and pathogenicity. To elucidate the transcription network of ACTR, we performed RNA-Seq experiments on wild-type and ACTR null mutant and identified genes that were differentially expressed between two genotypes. Transcriptome profiling and RT-qPCR analysis demonstrated that the ACT toxin biosynthetic gene ACTS4 is down-regulated in ACTR mutant. We generated ACTS4 knock-down mutant and found that the pathogenicity of ACTS4 mutant was severely impaired. Interestingly, both ACTR and ACTS4 are not involved in the response to different abiotic stresses including oxidative stress, salt stress, cell-wall disrupting regents, and metal ion stress, indicating the function of these two genes is highly specific. In conclusion, our results highlight the important regulatory role of ACTR in ACT toxin biosynthesis through mediating ACT toxin synthesis gene ACTS4 and underline the essential role of in the tangerine pathotype of A. alternata.


Assuntos
Alternaria/metabolismo , Micotoxinas/biossíntese , Fatores de Transcrição/metabolismo , Alternaria/genética , Alternaria/patogenicidade , Vias Biossintéticas , Citrus/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Família Multigênica , Doenças das Plantas/microbiologia , Metabolismo Secundário , Fatores de Transcrição/genética , Virulência
9.
Sci Rep ; 11(1): 6536, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33753809

RESUMO

Sharpshooter leafhoppers (Hemiptera: Cicadellidae: Cicadellinae) are important vectors of the plant pathogenic bacterium Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae). This pathogen causes economically significant diseases in olive, citrus, and grapes on multiple continents. Bacterial acquisition and inoculation mechanisms are linked to X. fastidiosa biofilm formation and fluid dynamics in the functional foregut of sharpshooters, which together result in egestion (expulsion) of fluids likely carrying bacteria. One key X. fastidiosa vector is the blue-green sharpshooter, Graphocephala atropunctata (Signoret, 1854). Herein, a 3D model of the blue-green sharpshooter functional foregut is derived from a meta-analysis of published microscopy images. The model is used to illustrate preexisting and newly defined anatomical terminology that is relevant for investigating fluid dynamics in the functional foregut of sharpshooters. The vivid 3D illustrations herein and supplementary interactive 3D figures are suitable resources for multidisciplinary researchers who may be unfamiliar with insect anatomy. The 3D model can also be used in future fluid dynamic simulations to better understand acquisition, retention, and inoculation of X. fastidiosa. Improved understanding of these processes could lead to new targets for preventing diseases caused by X. fastidiosa.


Assuntos
Sistema Digestório/ultraestrutura , Hemípteros/ultraestrutura , Insetos Vetores/ultraestrutura , Doenças das Plantas/microbiologia , Animais , Citrus/microbiologia , Sistema Digestório/anatomia & histologia , Hemípteros/anatomia & histologia , Hemípteros/microbiologia , Insetos Vetores/anatomia & histologia , Insetos Vetores/microbiologia , Olea/microbiologia , Vitis/microbiologia , Xylella/patogenicidade
10.
mBio ; 12(1)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563828

RESUMO

Penicillium digitatum is the most aggressive pathogen of citrus fruits. Tryptoquialanines are major indole alkaloids produced by P. digitatum It is unknown if tryptoquialanines are involved in the damage of citrus fruits caused by P. digitatum. To investigate the pathogenic roles of tryptoquialanines, we initially asked if tryptoquialanines could affect the germination of Citrus sinensis seeds. Exposure of the citrus seeds to tryptoquialanine A resulted in a complete inhibition of germination and an altered metabolic response. Since this phytotoxic effect requires the extracellular export of tryptoquialanine A, we investigated the mechanisms of extracellular delivery of this alkaloid in P. digitatum We detected extracellular vesicles (EVs) released by P. digitatum both in culture and during infection of citrus fruits. Compositional analysis of EVs produced during infection revealed the presence of a complex cargo, which included tryptoquialanines and the mycotoxin fungisporin. The EVs also presented phytotoxicity activity in vitro and caused damage to the tissues of citrus seeds. Through molecular networking, it was observed that the metabolites present in the P. digitatum EVs are produced in all of its possible hosts. Our results reveal a novel phytopathogenic role of P. digitatum EVs and tryptoquialanine A, implying that this alkaloid is exported in EVs during plant infection.IMPORTANCE During the postharvest period, citrus fruits can be affected by phytopathogens such as Penicillium digitatum, which causes green mold disease and is responsible for up to 90% of total citrus losses. Chemical fungicides are widely used to prevent green mold disease, leading to concerns about environmental and health risks. To develop safer alternatives to control phytopathogens, it is necessary to understand the molecular basis of infection during the host-pathogen interaction. In the P. digitatum model, the virulence strategies are poorly known. Here, we describe the production of phytotoxic extracellular vesicles (EVs) by P. digitatum during the infection of citrus fruits. We also characterized the secondary metabolites in the cargo of EVs and found in this set of molecules an inhibitor of seed germination. Since EVs and secondary metabolites have been related to virulence mechanisms in other host-pathogen interactions, our data are important for the comprehension of how P. digitatum causes damage to its primary hosts.


Assuntos
Alcaloides/metabolismo , Alcaloides/farmacologia , Citrus/microbiologia , Vesículas Extracelulares/química , Penicillium/patogenicidade , Sementes/crescimento & desenvolvimento , Alcaloides/biossíntese , Frutas/microbiologia , Fungicidas Industriais/farmacologia , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Metabolismo Secundário , Sementes/efeitos dos fármacos , Sementes/metabolismo , Sementes/microbiologia
11.
Micron ; 143: 103024, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33549851

RESUMO

The organelle-like structures of Xanthomonas citri, a bacterial pathogen that causes citrus canker, were investigated using an analytical transmission electron microscope. After high-pressure freezing, the bacteria were then freeze-substituted for imaging and element analysis. Miniscule electron-dense structures of varying shapes without a membrane enclosure were frequently observed near the cell poles in a 3-day culture. The bacteria formed cytoplasmic electron-dense spherical structures measuring approximately 50 nm in diameter. Furthermore, X. citri produced electron-dense or translucent ellipsoidal intracellular or extracellular granules. Single- or double-membrane-bound vesicles, including outer-inner membrane vesicles, were observed both inside and outside the cells. Most cells had been lysed in the 3-week X. citri culture, but they harbored one or two electron-dense spherical structures. Contrast-inverted scanning transmission electron microscopy images revealed distinct white spherical structures within the cytoplasm of X. citri. Likewise, energy-dispersive X-ray spectrometry showed the spatial heterogeneity and co-localization of phosphorus, oxygen, calcium, and iron only in the cytoplasmic electron-dense spherical structures, thus corroborating the nature of polyphosphate granules.


Assuntos
Grânulos Citoplasmáticos/ultraestrutura , Vacúolos/ultraestrutura , Xanthomonas/química , Xanthomonas/ultraestrutura , Cálcio/química , Citrus/microbiologia , Grânulos Citoplasmáticos/química , Ferro/química , Microscopia Eletrônica de Transmissão , Fósforo/química , Doenças das Plantas/microbiologia
12.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33526689

RESUMO

Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrus australiasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application.


Assuntos
Citrus/efeitos dos fármacos , Doenças das Plantas/prevenção & controle , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Rutaceae/química , Citrus/microbiologia , Resistência à Doença/genética , Liberibacter/efeitos dos fármacos , Liberibacter/patogenicidade , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética
13.
J Sci Food Agric ; 101(10): 4229-4240, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-33426638

RESUMO

BACKGROUND: In the postharvest handling of horticultural commodities, plant extracts with fungicidal activity are a valid alternative to synthetic fungicides. The fungicidal activity of myrtle leaf extracts from eight cultivars was studied in vitro against Penicillium digitatum, Penicillium italicum, and Penicillium expansum and on artificially inoculated mandarins with green and blue molds during storage for 12 days at 20 °C and 90% RH. RESULTS: Hydroxybenzoic acids, hydrolysable tannins, and flavonols were identified by high-performance liquid chromatography (HPLC). Despite sharing the same phenolic profile, extracts of eight myrtle cultivars significantly differed in the concentration of phenolics. Hydrolysable tannins are the principal subclass representing nearly 44.9% of the total polyphenols, whereas myricitrin was the most abundant flavonol in all cultivars. Myrtle extracts strongly inhibited conidial germination of the pathogens tested, although the greatest efficacy was observed against P. digitatum. At a concentration of 20 g L-1 , all the extracts completely inhibited fungi growth; only 'Angela', 'Tonina' and 'Grazia' extracts were effective at lower concentrations (15 g L-1 ). On inoculated fruit, myrtle extracts significantly controlled rot development. As a preventive treatment, 'Ilaria' and 'Maria Rita' extracts significantly reduced the rate of fruit with green mold decay lesions. When applied as a curative treatment, all the exacts decreased the incidence of decay. Against P. italicum, all the extracts applied as preventive treatments controlled decay effectively, while as curative treatment some of the extracts were not effective. All the extracts reduced the size of the infected areas. CONCLUSION: The results propose myrtle extracts as a possible natural alternative to synthetic fungicides. © 2021 Society of Chemical Industry.


Assuntos
Citrus/microbiologia , Conservação de Alimentos/métodos , Conservantes de Alimentos/farmacologia , Fungicidas Industriais/farmacologia , Myrtus/química , Penicillium/efeitos dos fármacos , Doenças das Plantas/prevenção & controle , Extratos Vegetais/farmacologia , Conservação de Alimentos/instrumentação , Conservantes de Alimentos/química , Armazenamento de Alimentos , Frutas/microbiologia , Penicillium/classificação , Penicillium/crescimento & desenvolvimento , Doenças das Plantas/microbiologia , Extratos Vegetais/química , Folhas de Planta/química
14.
J Agric Food Chem ; 69(3): 945-954, 2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33438400

RESUMO

A novel chemical conjugate between chitosan (CH) and riboflavin (RF) has been synthesized and characterized via Fourier transform infrared, NMR, and other spectroscopic methods. Photophysical and photochemical properties such as absorption spectra, fluorescence emission, fluorescence anisotropy, and singlet oxygen generation were characterized as well. This new biopolymer-based conjugate was designed to have an antifungal effect enhanced through antimicrobial photodynamic therapy. The antifungal effect of this conjugate (CH-RF) was compared with CH and RF against Penicillium digitatum in vitro. The conjugate showed the highest fungal growth inhibition of all systems tested at a dose of 0.5% w/v. This new biopolymer-based compound could be a promising alternative to fungicides used in citrus fruits postharvest.


Assuntos
Quitosana/química , Quitosana/farmacologia , Fungicidas Industriais/farmacologia , Penicillium/efeitos dos fármacos , Riboflavina/química , Riboflavina/farmacologia , Citrus/microbiologia , Fungicidas Industriais/síntese química , Fungicidas Industriais/química , Luz , Penicillium/crescimento & desenvolvimento , Doenças das Plantas/microbiologia
15.
PLoS Genet ; 17(1): e1009310, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33465093

RESUMO

Transcription activator-like effectors (TALEs) are virulence factors of Xanthomonas that induce the expression of host susceptibility (S) genes by specifically binding to effector binding elements (EBEs) in their promoter regions. The DNA binding specificity of TALEs is dictated by their tandem repeat regions, which are highly variable between different TALEs. Mutation of the EBEs of S genes is being utilized as a key strategy to generate resistant crops against TALE-dependent pathogens. However, TALE adaptations through rearrangement of their repeat regions is a potential obstacle for successful implementation of this strategy. We investigated the consequences of TALE adaptations in the citrus pathogen Xanthomonas citri subsp. citri (Xcc), in which PthA4 is the TALE required for pathogenicity, whereas CsLOB1 is the corresponding susceptibility gene, on host resistance. Seven TALEs, containing two-to-nine mismatching-repeats to the EBEPthA4 that were unable to induce CsLOB1 expression, were introduced into Xcc pthA4:Tn5 and adaptation was simulated by repeated inoculations into and isolations from sweet orange for a duration of 30 cycles. While initially all strains failed to promote disease, symptoms started to appear between 9-28 passages in four TALEs, which originally harbored two-to-five mismatches. Sequence analysis of adapted TALEs identified deletions and mutations within the TALE repeat regions which enhanced putative affinity to the CsLOB1 promoter. Sequence analyses suggest that TALEs adaptations result from recombinations between repeats of the TALEs. Reintroduction of these adapted TALEs into Xcc pthA4:Tn5 restored the ability to induce the expression of CsLOB1, promote disease symptoms and colonize host plants. TALEs harboring seven-to-nine mismatches were unable to adapt to overcome the incompatible interaction. Our study experimentally documented TALE adaptations to incompatible EBE and provided strategic guidance for generation of disease resistant crops against TALE-dependent pathogens.


Assuntos
Interações Hospedeiro-Patógeno/genética , Efetores Semelhantes a Ativadores de Transcrição/genética , Fatores de Virulência/genética , Xanthomonas/genética , Proteínas de Bactérias/genética , Citrus/genética , Citrus/microbiologia , Regulação da Expressão Gênica de Plantas/genética , Mutação/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Xanthomonas/patogenicidade
16.
PLoS Genet ; 17(1): e1009316, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33493197

RESUMO

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases in citrus industry worldwide. Most citrus cultivars such as sweet orange are susceptible to canker disease. Here, we utilized wild citrus to identify canker-resistant germplasms, and found that Atalantia buxifolia, a primitive (distant-wild) citrus, exhibited remarkable resistance to canker disease. Although the susceptibility gene LATERAL ORGAN BOUNDARIES 1 (LOB1) could also be induced in Atalantia after canker infection, the induction extent was far lower than that in sweet orange. In addition, three of amino acids encoded by transcription factor TFIIAγ in Atalantia (AbTFIIAγ) exhibited difference from those in sweet orange (CsTFIIAγ) which could stabilize the interaction between effector PthA4 and effector binding element (EBE) of LOB1 promoter. The mutation of AbTFIIAγ did not change its interaction with transcription factor binding motifs (TFBs). However, the AbTFIIAγ could hardly support the LOB1 expression induced by the PthA4. In addition, the activity of AbLOB1 promoter was significantly lower than that of CsLOB1 under the induction by PthA4. Our results demonstrate that natural variations of AbTFIIAγ and effector binding element (EBE) in the AbLOB1 promoter are crucial for the canker disease resistance of Atalantia. The natural mutations of AbTFIIAγ gene and AbLOB1 promoter in Atalantia provide candidate targets for improving the resistance to citrus canker disease.


Assuntos
Resistência à Doença/genética , Doenças das Plantas/genética , Rutaceae/genética , Fator de Transcrição TFIIA/genética , Citrus/genética , Citrus/crescimento & desenvolvimento , Citrus/microbiologia , Regulação da Expressão Gênica de Plantas , Mutação/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Rutaceae/crescimento & desenvolvimento , Rutaceae/microbiologia , Xanthomonas/genética , Xanthomonas/patogenicidade
17.
J Appl Microbiol ; 131(3): 1378-1390, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33484589

RESUMO

AIMS: The present study evaluated, for the first time, the inhibitory effects of the filtrate of Purpureocillium lilacinum against Penicillium digitatum. METHODS AND RESULTS: No direct contact between P. lilacinum and P. digitatum was observed during the dual culture test and the inhibition zone was 6·1 mm. The filtrate of P. lilacinum completely inhibited P. digitatum growth and spore germination at the concentration of 64%. The filtrate increased the permeability of the cell membrane and the content of MDA in P. digitatum. The ergosterol content in P. digitatum was strongly inhibited at 32% by 81·1%. The green mould incidence and severity in filtrate-treated fruit at 64% were 71·7 and 80·7% lower than in the control, respectively. The filtrate enhanced the activity of PAL, PPO and POD enzymes in orange fruit. The POD and PAL gene expression levels were significantly upregulated in the fruit treated with the filtrate. CONCLUSIONS: This study indicated that the antifungal mechanism of P. lilacinum filtrate against P. digitatum is mainly by the damage of the fungal cell membrane and its components. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the pioneer evidence on the application of P. lilacinum filtrate as a novel biocontrol agent for orange green mould.


Assuntos
Agentes de Controle Biológico , Citrus , Contaminação de Alimentos/prevenção & controle , Hypocreales/fisiologia , Penicillium , Citrus/microbiologia , Microbiologia de Alimentos , Frutas/microbiologia , Penicillium/patogenicidade
18.
J Sci Food Agric ; 101(6): 2584-2591, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33063337

RESUMO

BACKGROUND: The largest and most profitable market for citrus is the production of fresh fruit. Xanthomonas citri subsp. citri is a Gram-negative plant pathogen and the etiological agent of citrus canker, one of the major threats to citrus production worldwide. In the early stages of infection, X. citri can attach to plant surfaces by means of biofilms. Biofilm is considered an essential virulence factor, which helps tissue colonization in plants. Thus, sanitization of citrus fruit is mandatory in packinghouses before any logistic operation as packing and shipment to the market. The aim of this study was to evaluate electrolysed water (EW) as a sanitizer for the disinfection of citrus fruit in packinghouses. RESULTS: Using a protocol to monitor cell respiration we show that EW, obtained after 8 and 9 min of electrolysis, sufficed to kill X. citri when applied at a concentration of 500 µL mL-1 . Furthermore, microscopy analysis, combined with time-response growth curves, confirmed that EW affects the bacterial cytoplasmatic membrane and it leads to cell death in the first few minutes of contact. Pathogenicity tests using limes to simulate packinghouse treatment showed that EW, produced with 9 min of electrolysis, was a very effective sanitizer capable of eliminating X. citri from contaminated fruit. CONCLUSION: It was possible to conclude that EW is significantly effective as sodium hypochlorite (NaClO) at 200 ppm. Therefore, EW could be an alternative for citrus sanitization in packinghouses. © 2020 Society of Chemical Industry.


Assuntos
Citrus/microbiologia , Desinfetantes/química , Desinfetantes/farmacologia , Desinfecção/métodos , Água/química , Água/farmacologia , Biofilmes/efeitos dos fármacos , Citrus/efeitos dos fármacos , Desinfecção/instrumentação , Eletrólise , Frutas/efeitos dos fármacos , Frutas/microbiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Xanthomonas/efeitos dos fármacos , Xanthomonas/crescimento & desenvolvimento
19.
Int J Food Microbiol ; 337: 108918, 2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33126075

RESUMO

Major facilitator superfamily (MFS) comprises a large family of fungal transporters. In this work four Penicillium digitatum MFS transporters named PdMFS2-5 were identified and functionally characterized through gene elimination and gene overexpression with aim of unveil the similarities and differences among members of the same family during pathogen-fruit interaction. Fungal mutants in which each of the MFS transporters were individually deleted, displayed a clear effect on their infective capacity during citrus fruit infection especially in two of them. In contrast, the observed effect on fungicide sensitivity limits PdMFS2 and PdMFS3 as transporters underlying fungicide resistance. Moreover, overexpression transformants confirmed P. digitatum MFS transporters function and PdMFS2 and PdMFS3 were able to confer fungicide resistance to P. digitatum strains originally fungicide sensitive. Gene transcription rate depended on each MFS transporter being PdMFS4 the one with higher gene expression. Transcriptional profiling was similar regardless the P. digitatum strain. The gene expression analysis showed an increase of PdMFSs transcription in all overexpression transformants, particularly in Pd27 strain. Expression analysis carried out during P. digitatum-citrus fruit interaction confirmed the contribution of all PdMFSs, excepting PdMFS5, in fungal virulence. These results indicate that MFS fungal transporters might be part of different processes and can replace other genes functions giving them a very high degree of versatility.


Assuntos
Citrus/microbiologia , Microbiologia de Alimentos , Interações Hospedeiro-Patógeno/genética , Penicillium/metabolismo , Fungicidas Industriais , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Penicillium/genética , Virulência/genética
20.
Food Chem ; 336: 127590, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32763742

RESUMO

This work has been aimed at studying the effect of red thyme oil (RTO, Thymus vulgaris L.) on the shelf-life and Penicillium decay of oranges during cold storage. RTO vapours significantly reduced (P ≤ 0.05) the percentage of infected wounds, the external growth area and the production of spores in inoculated orange fruit stored for 12 days at 7 °C in a polypropylene film selected for its appropriate permeability. Among the RTO compounds, p-cymene and thymol were the most abundant in packed boxes at the end of cold storage. The RTO vapours did not affect the main quality parameters of the oranges, or the taste and odour of the juice. The results have shown that an active packaging, using RTO vapours, could be employed, by the citrus industry, to extend the shelf-life of oranges for fresh market use and juice processing.


Assuntos
Qualidade dos Alimentos , Armazenamento de Alimentos/métodos , Óleos Voláteis/farmacologia , Penicillium/efeitos dos fármacos , Thymus (Planta)/metabolismo , Antioxidantes/química , Citrus/química , Citrus/metabolismo , Citrus/microbiologia , Temperatura Baixa , Sucos de Frutas e Vegetais/análise , Cromatografia Gasosa-Espectrometria de Massas , Concentração de Íons de Hidrogênio , Óleos Voláteis/análise , Penicillium/fisiologia
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