scholarly journals CRISPR genotyping as complementary tool for epidemiological surveillance of Erwinia amylovora outbreaks

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250280
Author(s):  
Rafael J. Mendes ◽  
João Pedro Luz ◽  
Conceição Santos ◽  
Fernando Tavares
Keyword(s):  
Fire Blight ◽  
Erwinia Amylovora ◽  
Blot Hybridization ◽  
Fruit Trees ◽  
Epidemiological Surveillance ◽  
Economic Losses ◽  
Pome Fruit ◽  
Dot Blot ◽  
Virulence Markers ◽  
Clonal Population Structure

Fire blight is a destructive plant disease caused by Erwinia amylovora affecting pome fruit trees, and responsible for large yield declines, long phytosanitary confinements, and high economic losses. In Portugal, the first major fire blight outbreaks occurred in 2010 and 2011, and although later considered eradicated, the emergence of other outbreaks in recent years stressed the need to characterize the E. amylovora populations associated with these outbreaks. In this regard, CRISPR genotyping, assessment of three virulence markers, and semi-quantitative virulence bioassays, were carried out to determine the genotype, and assess the virulence of thirty-six E. amylovora isolates associated with outbreaks occurring between 2010 and 2017 and affecting apple and pear orchards located in the country central-west, known as the main producing region of pome fruits in Portugal. The data gathered reveal that 35 E. amylovora isolates belong to one of the widely-distributed CRISPR genotypes (5-24-38 / D-a-α) regardless the host species, year and region. Ea 680 was the single isolate revealing a new CRISPR genotype due to a novel CR2 spacer located closer to the leader sequence and therefore thought to be recently acquired. Regarding pathogenicity, although dot-blot hybridization assays showed the presence of key virulence factors, namely hrpL (T3SS), hrpN (T3E) and amsG from the amylovoran biosynthesis operon in all E. amylovora isolates studied, pathogenicity bioassays on immature pear slices allowed to distinguish four virulence levels, with most of the isolates revealing an intermediate to severe virulence phenotype. Regardless the clonal population structure of the E. amylovora associated to the outbreaks occurring in Portugal between 2010 and 2017, the different virulence phenotypes, suggests that E. amylovora may have been introduced at different instances into the country. This is the first study regarding E. amylovora in Portugal, and it discloses a novel CRISPR genotype for this bacterium.

From the roots to the stem: unveiling pear root colonization and infection pathways by Erwinia amylovora

2020 ◽  
Vol 96 (12) ◽  
Author(s):  
Ricardo D Santander ◽  
José F Català-Senent ◽  
Àngela Figàs-Segura ◽  
Elena G Biosca
Keyword(s):  
Fire Blight ◽  
Plant Pathogens ◽  
Erwinia Amylovora ◽  
Fluorescent Protein ◽  
Epifluorescence Microscopy ◽  
Economic Losses ◽  
Root Infection ◽  
Pome Fruit ◽  
Infection Pathways ◽  
Biofilm Development

ABSTRACT Fire blight caused by Erwinia amylovora affects pome fruit worldwide, generating serious economic losses. Despite the abundant literature on E. amylovora infection mechanisms of aerial plant organs, root infection routes remain virtually unexplored. Assessing these infection pathways is necessary for a full understanding of the pathogen's ecology. Using the pathosystem Pyrus communis–E. amylovora and different experimental approaches including a green fluorescent protein transformant (GFP1) and epifluorescence microscopy (EFM) and laser confocal scanning microscopy (LCSM), we demonstrated the pathogen's ability to infect, colonize and invade pear roots and cause characteristic fire blight symptoms both in the aerial part and in the root system. Plant infections after soil irrigation with E. amylovora-contaminated water were favored by root damage, which agreed with EFM and LCSM observations. E. amylovora GFP1 cells formed aggregates/biofilms on root surfaces and invaded the cortex through wounds and sites of lateral root emergence. Sugars, sugar-alcohols and amino acids typically secreted by roots, favored the in vitro biofilm development by E. amylovora. Migration of E. amylovora cells to aerial tissues mainly occurred after xylem penetration. Overall, our findings revealed, for the first time, common root infection patterns between E. amylovora and well-known soil borne plant pathogens and endophytes.

scholarly journals The Incessant Battle Against Fire Blight in Pears: 30 Years of Challenges and Successes in Managing the Disease in Israel

Plant Disease ◽  
2015 ◽  
Vol 99 (8) ◽  
pp. 1048-1058 ◽  
Author(s):  
Dani Shtienberg ◽  
Shulamit Manulis-Sasson ◽  
Miriam Zilberstaine ◽  
Dov Oppenheim ◽  
Hagai Shwartz
Keyword(s):  
Crucial Role ◽  
Fire Blight ◽  
Erwinia Amylovora ◽  
Fruit Trees ◽  
Future Perspectives ◽  
Full Understanding ◽  
Pome Fruit ◽  
Local Experience

Fire blight, caused by the bacterium Erwinia amylovora, is the most destructive disease of pears and other pome fruit trees worldwide. The disease was first detected in Israel in 1985, and in the 30 years since, the intensity of fire blight epidemics has varied markedly. During this time, there were two national pandemics: the first between 1994 and 1996 and the second in 2010. In both cases, it was feared that the Israeli pear industry would not recover. National efforts were devoted to combat the problem and after both pandemics the industry survived. In this paper we indicate some unique characteristics that play a crucial role in the epidemiology of the disease under Israeli conditions. We then describe the continual struggle of the Israeli pear industry with fire blight over the last 30 years, elaborating on the two national pandemics and the efforts devoted to cope with them. Finally, we summarize the conclusions derived from our local experience and present our future perspectives regarding fire blight management. The take-home message of the Israeli fire blight story is that the battle against this hazardous disease is neverending. In some years, growers are able to adequately suppress the disease; in others, the pathogen overcomes management efforts and severe outbreaks occur. The latter could be minimized if growers have a full understanding of the management protocols suitable for the conditions and applied them rigorously.

Induction of Antimicrobial 3-Deoxyflavonoids in Pome Fruit Trees Controls Fire Blight

2003 ◽  
Vol 58 (11-12) ◽  
pp. 765-770 ◽  
Author(s):  
Heidrun Halbwirth ◽  
Thilo C. Fischer ◽  
Susanne Roemmelt ◽  
Francesco Spinelli ◽  
Karin Schlangen ◽  
...  
Keyword(s):  
Fire Blight ◽  
Fruit Trees ◽  
Effective Control ◽  
Economic Losses ◽  
Bacterial Disease ◽  
Flavonoid Pathway ◽  
Pome Fruit ◽  
New Approach ◽  
Pome Fruits ◽  
Potential Risks

Abstract Fire blight, a devastating bacterial disease in pome fruits, causes severe economic losses worldwide. Hitherto, an effective control could only be achieved by using antibiotics, but this implies potential risks for human health, livestock and environment. A new approach allows transient inhibition of a step in the flavonoid pathway, thereby inducing the formation of a novel antimicrobial 3-deoxyflavonoid controlling fire blight in apple and pear leaves. This compound is closely related to natural phytoalexins in sorghum. The approach does not only provide a safe method to control fire blight: Resistance against different pathogens is also induced in other crop plants.

scholarly journals Fire Blight Symptomatic Shoots and the Presence of Erwinia amylovora in Asymptomatic Apple Budwood

Plant Disease ◽  
2017 ◽  
Vol 101 (1) ◽  
pp. 186-191 ◽  
Author(s):  
K. A. Tancos ◽  
E. Borejsza-Wysocka ◽  
S. Kuehne ◽  
D. Breth ◽  
Kerik D. Cox
Keyword(s):  
New York ◽  
Fire Blight ◽  
Erwinia Amylovora ◽  
Causal Agent ◽  
Economic Losses ◽  
Apple Trees ◽  
The Mean ◽  
Collection Practices ◽  
Time Of Collection ◽  
Young Apple

Erwinia amylovora, the causal agent of fire blight, causes considerable economic losses in young apple plantings in New York on a yearly basis. Nurseries make efforts to only use clean budwood for propagation, which is essential, but E. amylovora may be present in trees that appear to have no apparent fire blight symptoms at the time of collection. We hypothesized that the use of infected budwood, especially by commercial nursery operations, could be the cause, in part, of fire blight outbreaks that often occur in young apple plantings in New York. Our goal was to investigate the presence of E. amylovora in asymptomatic budwood from nursery source plantings as it relates to trees with fire blight symptoms. From 2012 to 2015, apple budwood was collected from two commercial budwood source plantings of ‘Gala’ and ‘Topaz’ at increasing distances from visually symptomatic trees. From these collections, internal contents of apple buds were analyzed for the presence of E. amylovora. E. amylovora was detected in asymptomatic budwood in trees more than 20 m from trees with fire blight symptoms. In some seasons, there were significant (P ≤ 0.05) differences in the incidence of E. amylovora in asymptomatic budwood collected from symptomatic trees and those up to 20 m from them. In 2014 and 2015, the mean E. amylovora CFU per gram recovered from budwood in both the Gala and Topaz plantings were significantly lower in budwood collected 20 m from symptomatic trees. Further investigation of individual bud dissections revealed that E. amylovora was within the tissue beneath the bud scales containing the meristem. Results from the study highlight the shortcomings of current budwood collection practices and the need to better understand the factors that lead to the presence of E. amylovora in bud tissues to ensure the production of pathogen-free apple trees.

scholarly journals In Vitro Evaluation of Five Antimicrobial Peptides against the Plant Pathogen Erwinia amylovora

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 554
Author(s):  
Rafael J. Mendes ◽  
Laura Regalado ◽  
João P. Luz ◽  
Natália Tassi ◽  
Cátia Teixeira ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Antimicrobial Peptides ◽  
Erwinia Amylovora ◽  
Growth Curves ◽  
Fruit Trees ◽  
Membrane Permeabilization ◽  
Control Measures ◽  
Minimal Bactericidal Concentration ◽  
Pome Fruit

Fire blight is a major pome fruit trees disease that is caused by the quarantine phytopathogenic Erwinia amylovora, leading to major losses, namely, in pear and apple productions. Nevertheless, no effective sustainable control treatments and measures have yet been disclosed. In that regard, antimicrobial peptides (AMPs) have been proposed as an alternative biomolecule against pathogens but some of those AMPs have yet to be tested against E. amylovora. In this study, the potential of five AMPs (RW-BP100, CA-M, 3.1, D4E1, and Dhvar-5) together with BP100, were assessed to control E. amylovora. Antibiograms, minimal inhibitory, and bactericidal concentrations (minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), growth and IC50 were determined and membrane permeabilization capacity was evaluated by flow cytometry analysis and colony-forming units (CFUs) plate counting. For the tested AMPs, the higher inhibitory and bactericidal capacity was observed for RW-BP100 and CA-M (5 and 5–8 µM, respectively for both MIC and MBC), whilst for IC50 RW-BP100 presented higher efficiency (2.8 to 3.5 µM). Growth curves for the first concentrations bellow MIC showed that these AMPs delayed E. amylovora growth. Flow cytometry disclosed faster membrane permeabilization for CA-M. These results highlight the potential of RW-BP100 and CA-M AMPs as sustainable control measures against E. amylovora.

scholarly journals Activation of metabolic and stress responses during subtoxic expression of the type I toxin hok in Erwinia amylovora

2021 ◽  
Author(s):  
Jingyu Peng ◽  
Lindsay R. Triplett ◽  
George Sundin
Keyword(s):  
Cell Death ◽  
Stress Responses ◽  
Erwinia Amylovora ◽  
Physiological State ◽  
Fruit Trees ◽  
Toxin Gene ◽  
Type I ◽  
Pome Fruit ◽  
Membrane Rupture ◽  
High Level

Abstract Background: Toxin-antitoxin (TA) systems, abundant in prokaryotes, are composed of a toxin gene and its cognate antitoxin. Several toxins are implied to affect the physiological state and stress tolerance of bacteria in a population. We previously identified a chromosomally encoded hok-sok type I TA system in Erwinia amylovora, the causative agent of fire blight disease on pome fruit trees. A high-level induction of the hok gene was lethal to E. amylovora cells through unknown mechanisms. The molecular targets or regulatory roles of Hok were unknown.Results: Here, we examined the physiological and transcriptomic changes of Erwinia amylovora cells expressing hok at subtoxic levels that were confirmed to confer no cell death, and at toxic levels that resulted in killing of cells. In both conditions, hok caused membrane rupture and collapse of the proton motive force in a subpopulation of E. amylovora cells. We demonstrated that induction of hok resulted in upregulation of ATP biosynthesis genes, and caused leakage of ATP from cells only at toxic levels. We showed that overexpression of the phage shock protein gene pspA largely reversed the cell death phenotype caused by high levels of hok induction. We also showed that induction of hok at a subtoxic level rendered a greater proportion of stationary phase E. amylovora cells tolerant to the antibiotic streptomycin. Conclusions: We characterized the molecular mechanism of toxicity by high-level of hok induction and demonstrated that low-level expression of hok primes the stress responses of E. amylovora against further membrane and antibiotic stressors.

scholarly journals Distinct patterns of natural selection determine sub-population structure in the fire blight pathogen, Erwinia amylovora

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jugpreet Singh ◽  
Awais Khan
Keyword(s):  
Genetic Diversity ◽  
Population Structure ◽  
Population Differentiation ◽  
Selection Pressure ◽  
Fire Blight ◽  
Erwinia Amylovora ◽  
Balancing Selection ◽  
Genomic Diversity ◽  
Economic Losses ◽  
Population Structure Analysis

Abstract The fire blight pathogen, Erwinia amylovora (EA), causes significant economic losses in rosaceae fruit crops. Recent genome sequencing efforts have explored genetic variation, population structure, and virulence levels in EA strains. However, the genomic aspects of population bottlenecks and selection pressure from geographical isolation, host range, and management practices are yet unexplored. We conducted a comprehensive analysis of whole genome sequences of 41 strains to study genetic diversity, population structure, and the nature of selection affecting sub-population differentiation in EA. We detected 72,741 SNPs and 2,500 Indels, representing about six-fold more diversity than previous reports. Moreover, nonsynonymous substitutions were identified across the effector regions, suggesting a role in defining virulence of specific strains. EA plasmids had more diversity than the chromosome sequence. Population structure analysis identified three distinct sub-groups in EA strains, with North American strains displaying highest genetic diversity. A five kilobase genomic window scan showed differences in genomic diversity and selection pressure between these three sub-groups. This analysis also highlighted the role of purifying and balancing selection in shaping EA genome structure. Our analysis provides novel insights into the genomic diversity and selection forces accompanying EA population differentiation.

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