Reaction of detached leaves from different varieties of sweet orange to inoculation with Xanthomonas citri subsp. citri

ABSTRACT Citrus genotypes show a broad range of resistance to infection with Xanthomonas citri subsp citri (Xcc), the causal agent of Asiatic citrus canker (ACC). The aim of this study was to evaluate a detached leaf inoculation method to compare nine different citrus genotypes that exhibit varied resistance levels to ACC, and to evaluate infection, colonization and symptom development of the varieties by using Scanning Electron Microscopy (SEM). Inoculation of Xcc in the leaves was carried out by producing injuries with needles. Lesion diameters were measured at 7, 10 and 13 days after inoculation (DAI). The variety Pera IAC was most resistant to the pathogen and had lesions with smaller diameters, while the variety Washington was most susceptible and had the largest lesions, indicating low resistance to Xcc. SEM images were taken from the lesion development, and sections were obtained from the disease lesions in the tested varieties. The most resistant variety, Pera IAC, had smaller stomatal openings and produced greater quantity of foliar exudates, compared with the variety Washington. We hypothesize that the exudates may contain Xcc toxic compounds and thus may be a defense mechanism against infection by Xcc, but further research is required to establish any antibiotic effects of the exudates against citrus canker.

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tale-Based Genetic Diversity of Chinese Isolates of the Citrus Canker Pathogen Xanthomonas citri subsp. citri

Plant Disease ◽

10.1094/pdis-12-12-1201-re ◽

2013 ◽

Vol 97 (9) ◽

pp. 1187-1194 ◽

Cited By ~ 10

Author(s):

Gang Ye ◽

Ni Hong ◽

Li-Fang Zou ◽

Hua-Song Zou ◽

Muhammad Zakria ◽

...

Keyword(s):

Genetic Diversity ◽

Sweet Orange ◽

Southern China ◽

Citrus Canker ◽

Jiangxi Province ◽

Xanthomonas Citri ◽

Genotype I ◽

Internal Repeat ◽

Pathogenicity Determinant ◽

Citrus Bacterial Canker

Pathotype A of Xanthomonas citri subsp. citri, the cause of citrus bacterial canker (CBC), is assumed to have originated in southern China. PthA, a type III secreted transcriptional activator-like effector (TALE), is a major pathogenicity determinant in X. citri subsp. citri. To investigate the diversity of X. citri subsp. citri in China, genomic and plasmid DNA of 105 X. citri subsp. citri isolates, collected from nine citrus-growing provinces of China, were digested by BamHI and hybridized with an internal repeat region of pthA. Strains were classified into 14 different genotypes (designated A to N) based on the number and size of pthA homologues. Genotypes B and G represented 19 and 62% of the isolate collection, respectively. Genotypes J and L lacked pthA or a pthA-hybridizing fragment and were less virulent on grapefruit (C. paradisi) and sweet orange (C. sinensis) compared with strains containing pthA or a pthA homologue. The virulence of genotypes J and L was increased when the wild-type pthA was introduced. Genotype I, which was isolated from sweet orange in Jiangxi province, caused typical canker symptoms and may contain a novel pthA-like gene. To our knowledge, this is the first description of genetic diversity in Chinese CBC strains based on tale gene analysis.

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First Report of Xanthomonas citri pv. citri Causing Asiatic Citrus Canker in Burkina Faso

Plant Disease ◽

10.1094/pdis-06-13-0600-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1653-1653 ◽

Cited By ~ 3

Author(s):

C. C. Juhasz ◽

A. Leduc ◽

C. Boyer ◽

F. Guérin ◽

C. Vernière ◽

...

Keyword(s):

Burkina Faso ◽

Sweet Orange ◽

Citrus Canker ◽

Negative Control ◽

Citrus Species ◽

Xanthomonas Citri ◽

First Report ◽

Mexican Lime ◽

Volkamer Lemon ◽

Wide Range

Citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of economic importance in tropical and sub-tropical citrus-producing areas (EPPO-PQR online database). X. citri pv. citri causes severe infection in a wide range of citrus species, and induces erumpent, callus-like lesions with water-soaked margins leading to premature fruit drop and twig dieback. It has consequently been subjected to eradication efforts and international regulations. It was first described on the African continent in South Africa at the beginning of the 20th century, from which it was eventually eradicated. Since 2006, several outbreaks caused by phylogenetically diverse strains of X. citri pv. citri have been reported from several African countries (Ethiopia, Mali, Senegal, and Somalia). In July 2011, citrus canker in Burkina Faso was suspected in the area adjacent to the Sikassso Province of Mali where X. citri pv. citri has been confirmed. In November and December 2012, leaves of clementine (Citrus clementina), lemon (C. limon), Volkamer lemon (C. volkameriana), sweet orange (C. sinensis), tangelo (C. paradisi× C. reticulata), and mandarin (C. reticulata) were collected from orchards with trees showing symptoms of citrus canker in the Comoé, Houet, and Kénédougou provinces of Burkina Faso. Isolations performed using KC semi-selective medium (4) recovered 45 Xanthomonas-like strains. All Xanthomonas-like strains were tentatively identified as X. citri pv. citri by PCR (4/7 primers) using IAPAR 306 and sterile distilled water as the positive and negative controls, respectively (3). Among these, two strains (LK4-4 and LK4-5) produced a ‘fuscans’-like brown diffusible pigment, a phenotype never reported previously for X. citri pv. citri. MultiLocus Sequence Analysis targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,2) fully identified seven strains from Burkina Faso (LJ301-1, LJ303-1, LK1-1, LK2-6, LK4-3, LK4-4, and LK4-5) as X. citri pv. citri (and not to any other Xanthomonas pathovars pathogenic to citrus or host range-restricted pathotypes of pathovar citri), and more specifically as sequence type ST2 which is composed mostly of pathotype A strains of X. citri pv. citri (2). The same seven strains were inoculated to at least four leaves of each of grapefruit cv. Henderson, Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58 (C. latifolia), and sweet orange cv. Washington Navel, using a detached leaf assay (2). All strains developed typical erumpent, callus-like tissue at wound sites on all citrus species inoculated. No lesions developed on the negative control (sterile 10 mM tris buffer). Koch's postulate was fulfilled after reisolation of Xanthomonas-like yellow colonies from symptoms on Mexican lime produced by the seven strains. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (3) and produced the expected 468-bp amplicon in contrast with the PCR negative control. To our knowledge, this is the first report of X. citri pv. citri in Burkina Faso. Citrus canker-free nurseries and grove sanitation should be implemented for reducing the prevalence of Asiatic canker in Burkina Faso and a thorough survey of citrus nurseries and groves in the region should be conducted. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) J. S. Hartung et al. Phytopathology 86:95, 1996. (4) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005.

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Design of Antibacterial Agents: Alkyl Dihydroxybenzoates against Xanthomonas citri subsp. citri

International Journal of Molecular Sciences ◽

10.3390/ijms19103050 ◽

2018 ◽

Vol 19 (10) ◽

pp. 3050 ◽

Cited By ~ 4

Author(s):

Ana Nazaré ◽

Carlos Polaquini ◽

Lúcia Cavalca ◽

Daiane Anselmo ◽

Marilia Saiki ◽

...

Keyword(s):

Antibacterial Agents ◽

Inhibitory Concentration ◽

Sweet Orange ◽

Citrus Canker ◽

Positive Control ◽

Exponential Phase ◽

Bacterial Cell Division ◽

Xanthomonas Citri ◽

The World ◽

Control Compound

Xanthomonas citri subsp. citri (Xcc) causes citrus canker, affecting sweet orange-producing areas around the world. The current chemical treatment available for this disease is based on cupric compounds. For this reason, the objective of this study was to design antibacterial agents. In order to do this, we analyzed the anti-Xcc activity of 36 alkyl dihydroxybenzoates and we found 14 active compounds. Among them, three esters with the lowest minimum inhibitory concentration values were selected; compounds 4 (52 μM), 16 (80 μM) and 28 (88 μM). Our study demonstrated that alkyl dihydroxybenzoates cause a delay in the exponential phase. The permeability capacity of alkyl dihydroxybenzoates in a quarter of MIC was compared to nisin (positive control). Compound 28 was the most effective (93.8), compared to compound 16 (41.3) and compound 4 (13.9) by percentage values. Finally, all three compounds showed inhibition of FtsZ GTPase activity, and promoted changes in protofilaments, leading to depolymerization, which prevents bacterial cell division. In conclusion, heptyl dihydroxybenzoates (compounds 4, 16 and 28) are promising anti-Xcc agents which may serve as an alternative for the control of citrus canker.

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A Genomic and Transcriptomic Overview of MATE, ABC, and MFS Transporters in Citrus sinensis Interaction with Xanthomonas citri subsp. citri

Plants ◽

10.3390/plants9060794 ◽

2020 ◽

Vol 9 (6) ◽

pp. 794 ◽

Cited By ~ 1

Author(s):

Maria H. M. Julião ◽

Saura R. Silva ◽

Jesus A. Ferro ◽

Alessandro M. Varani

Keyword(s):

Plant Defense ◽

Membrane Trafficking ◽

Citrus Sinensis ◽

Defense Mechanism ◽

In Silico Analysis ◽

Citrus Canker ◽

Membrane Transporters ◽

Major Facilitator Superfamily ◽

Xanthomonas Citri ◽

Mfs Transporters

The multi-antimicrobial extrusion (MATE), ATP-binding cassette (ABC), and major facilitator superfamily (MFS) are the main plant transporters families, playing an essential role in the membrane-trafficking network and plant-defense mechanism. The citrus canker type A (CC), is a devastating disease caused by Xanthomonas citri subsp. citri (Xac), affecting all citrus species. In this work, we performed an in silico analysis of genes and transcripts from MATE, ABC, and MFS families to infer the role of membrane transporters in Citrus-Xac interaction. Using as reference, the available Citrus sinensis genome and the citrus reference transcriptome from CitrusKB database, 67 MATE, 91 MFS, and 143 ABC genes and 82 MATE, 139 MFS, and 226 ABC transcripts were identified and classified into subfamilies. Duplications, alternative-splicing, and potentially non-transcribed transporters’ genes were revealed. Interestingly, MATE I and ABC G subfamilies appear differently regulated during Xac infection. Furthermore, Citrus spp. showing distinct levels of CC susceptibility exhibited different sets of transporters transcripts, supporting dissimilar molecular patterns of membrane transporters in Citrus-Xac interaction. According to our findings, 4 MATE, 10 ABC, and 3 MFS are potentially related to plant-defense mechanisms. Overall, this work provides an extensive analysis of MATE, ABC, and MFS transporters’ in Citrus-Xac interaction, bringing new insights on membrane transporters in plant-pathogen interactions.

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Detection of Phenylpropanoids in Citrus Leaves Produced in Response to Xanthomonas citri subsp. citri

Phytopathology ◽

10.1094/phyto-06-19-0219-r ◽

2020 ◽

Vol 110 (2) ◽

pp. 287-296 ◽

Cited By ~ 2

Author(s):

Christopher M. Ference ◽

John A. Manthey ◽

Jan A. Narciso ◽

Jeffrey B. Jones ◽

Elizabeth A. Baldwin

Keyword(s):

Sweet Orange ◽

Bacterial Pathogen ◽

Leaf Tissue ◽

Citrus Canker ◽

Xanthomonas Citri ◽

Tree Health ◽

Murraya Paniculata ◽

The World ◽

Citrus Production ◽

Citrus Leaves

Citrus canker (CC), caused by the bacterial pathogen Xanthomonas citri subsp. citri, impacts citrus production in many areas of the world by reducing yields, degrading tree health, and severely blemishing the outer peels of fresh fruit. The relative susceptibility to CC among different species of Citrus varies from the highly susceptible lime (Citrus × aurantifolia), sweet orange (C. × sinensis), and grapefruit (C. × paradisi) to the much less susceptible calamondin (C. × microcarpa) and kumquat (C. japonica). This investigation compares the responses to infection with X. citri subsp. citri of these five genotypes with respect to phenylpropanoid compound profiles and relative increases or decreases of specific compounds postinoculation. In response to X. citri subsp. citri infection, all hosts possessed increased concentrations of phenylpropanoids in leaf tissue, whereas the similarly treated nonhost orange jessamine (Murraya paniculata) did not. Several of the tested genotypes exhibited notably increased production of fluorescent phenylpropanoids, including umbelliferone, herniarin, auraptene, scoparone, and others. The profiles of these compounds and their levels of production varied among the tested species yet all investigated Citrus genotypes exhibited increased concentrations of phenylpropanoids regardless of their degree of susceptibility to X. citri subsp. citri. Kumquat and calamondin, the tested genotypes least susceptible to X. citri subsp. citri, also exhibited the highest levels of the dihydrochalcone 3′,5′-di-C-glucosyl phloretin, the aglycone portion of which, phloretin, is a known antibiotic, although levels of this compound were not affected by inoculation with X. citri subsp. citri.

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Temporal Transcription Profiling of Sweet Orange in Response to PthA4-Mediated Xanthomonas citri subsp. citri Infection

Phytopathology ◽

10.1094/phyto-09-15-0201-r ◽

2016 ◽

Vol 106 (5) ◽

pp. 442-451 ◽

Cited By ~ 4

Author(s):

Yang Hu ◽

Shuo Duan ◽

Yunzeng Zhang ◽

Deepak Shantharaj ◽

Jeffrey B. Jones ◽

...

Keyword(s):

Gene Expression ◽

Cell Wall ◽

Sweet Orange ◽

Expression Patterns ◽

Susceptibility Gene ◽

Transcriptional Response ◽

Leaf Tissue ◽

Citrus Canker ◽

Xanthomonas Citri ◽

Promoter Regions

Citrus canker, caused by Xanthomonas citri subsp. citri, is a devastating disease of most commercial citrus varieties. In our previous study, we analyzed the transcriptional response of ‘Valencia’ sweet orange to X. citri subsp. citri wild-type and pthA4 mutant infection at 48 h postinoculation (hpi). Using microarray analysis, two PthA4 targets, CsLOB1 and CsSWEET1, were identified. We have shown that PthA4 binds to the effector binding element (EBE) of CsLOB1 and activates gene expression of this susceptibility gene. However, how PthA4 modulates host genes at different stages of infection remains to be determined. In this study, we compared the transcriptional profiles between citrus leaf tissue inoculated with Xcc306 and those inoculated with a pthA4-deletion mutant strain (Xcc306∆pthA4) at 6, 48, and 120 hpi. At both 48 and 120 hpi, the PthA4-mediated infection significantly upregulated expression of a variety of genes involved in cell-wall degradation and modification, DNA packaging, G-protein, protein synthesis, sucrose metabolism, and cell division functions, while the downregulated genes were mainly enriched in photosynthesis, transport, secondary metabolism, cytochrome P450, and various plant defense-associated mechanisms. To validate microarray results, gene expression of 26 genes representing genes associated with cell-wall-associated, immunity system, and carbohydrate metabolism was confirmed using quantitative reverse-transcription polymerase chain reaction. Expression patterns of these genes at 48 and 120 hpi were consistent with the microarray results. We also identified putative EBE for PthA4 (EBEPthA4) in the promoter regions of multiple genes upregulated by PthA4, to which PthA4 might bind directly to control their gene expression. Our study provided a dynamic picture of citrus genes regulated by PthA4 during the X. citri subsp. citri infection of citrus leaves at different stages. This study will be useful in further understanding the virulence mechanism of X. citri subsp. citri and identifying potential targets of PthA4.

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Chlorine dioxide, peroxyacetic acid, and calcium oxychloride for post-harvest decontamination of citrus fruit against Xanthomonas citri subsp. citri, causal agent of citrus canker

Crop Protection ◽

10.1016/j.cropro.2021.105679 ◽

2021 ◽

Vol 146 ◽

pp. 105679

Author(s):

Franklin Behlau ◽

Alexandre Paloschi ◽

Tamiris G.S. Marin ◽

Talita A. Santos ◽

Henrique Ferreira ◽

...

Keyword(s):

Chlorine Dioxide ◽

Citrus Fruit ◽

Citrus Canker ◽

Causal Agent ◽

Peroxyacetic Acid ◽

Xanthomonas Citri ◽

Post Harvest

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Modified Monosaccharides Content of Xanthan Gum Impairs Citrus Canker Disease by Affecting the Epiphytic Lifestyle of Xanthomonas citri subsp. citri

Microorganisms ◽

10.3390/microorganisms9061176 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1176

Author(s):

Simone Cristina Picchi ◽

Laís Moreira Granato ◽

Maria Júlia Festa Franzini ◽

Maxuel Oliveira Andrade ◽

Marco Aurélio Takita ◽

...

Keyword(s):

Biofilm Formation ◽

Xanthan Gum ◽

Wild Type Strain ◽

Natural Infection ◽

Citrus Canker ◽

Wild Type ◽

Xanthomonas Citri ◽

Canker Disease ◽

Key Enzyme ◽

Citrus Canker Disease

Xanthomonas citri subsp. citri (X. citri) is a plant pathogenic bacterium causing citrus canker disease. The xanA gene encodes a phosphoglucomutase/phosphomannomutase protein that is a key enzyme required for the synthesis of lipopolysaccharides and exopolysaccharides in Xanthomonads. In this work, firstly we isolated a xanA transposon mutant (xanA::Tn5) and analyzed its phenotypes as biofilm formation, xanthan gum production, and pathogenesis on the sweet orange host. Moreover, to confirm the xanA role in the impaired phenotypes we further produced a non-polar deletion mutant (ΔxanA) and performed the complementation of both xanA mutants. In addition, we analyzed the percentages of the xanthan gum monosaccharides produced by X. citri wild-type and xanA mutant. The mutant strain had higher ratios of mannose, galactose, and xylose and lower ratios of rhamnose, glucuronic acid, and glucose than the wild-type strain. Such changes in the saccharide composition led to the reduction of xanthan yield in the xanA deficient strain, affecting also other important features in X. citri, such as biofilm formation and sliding motility. Moreover, we showed that xanA::Tn5 caused no symptoms on host leaves after spraying, a method that mimetics the natural infection condition. These results suggest that xanA plays an important role in the epiphytical stage on the leaves that is essential for the successful interaction with the host, including adaptive advantage for bacterial X. citri survival and host invasion, which culminates in pathogenicity.

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GC-TOF/MS-based metabolomics analysis to investigate the changes driven by N-Acetylcysteine in the plant-pathogen Xanthomonas citri subsp. citri

Scientific Reports ◽

10.1038/s41598-021-95113-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Simone Cristina Picchi ◽

Mariana de Souza e Silva ◽

Luiz Leonardo Saldanha ◽

Henrique Ferreira ◽

Marco Aurélio Takita ◽

...

Keyword(s):

Amino Acids ◽

Cell Proliferation ◽

Plant Pathogen ◽

Model Organism ◽

Citrus Canker ◽

Bacterial Cells ◽

Xanthomonas Citri ◽

Canker Disease ◽

Potential Use

AbstractN-Acetylcysteine (NAC) is an antioxidant, anti-adhesive, and antimicrobial compound. Even though there is much information regarding the role of NAC as an antioxidant and anti-adhesive agent, little is known about its antimicrobial activity. In order to assess its mode of action in bacterial cells, we investigated the metabolic responses triggered by NAC at neutral pH. As a model organism, we chose the Gram-negative plant pathogen Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker disease, due to the potential use of NAC as a sustainable molecule against phytopathogens dissemination in citrus cultivated areas. In presence of NAC, cell proliferation was affected after 4 h, but damages to the cell membrane were observed only after 24 h. Targeted metabolite profiling analysis using GC–MS/TOF unravelled that NAC seems to be metabolized by the cells affecting cysteine metabolism. Intriguingly, glutamine, a marker for nitrogen status, was not detected among the cells treated with NAC. The absence of glutamine was followed by a decrease in the levels of the majority of the proteinogenic amino acids, suggesting that the reduced availability of amino acids affect protein synthesis and consequently cell proliferation.

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