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Percentage of flowers with fire blight symptoms after inoculation at acidic or neutral pH conditions. Percentage of symptomatic flowers inoculated with E. amylovora suspension adjusted to pH 4 (striped bars) or pH 7 (black bars) in two independent experiments (left and right half of diagram). Bars represent mean percentages of 3 replicate boxes with 11-14 inoculated detached apple flowers. Total flower numbers are indicated above bars. Mean percentages of symptomatic flowers are significantly different between pH treatments on a p,0.05 level (T-test). doi:10.1371/journal.pone.0032583.g002
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Pathogen entry through host blossoms is the predominant infection pathway of the gram-negative bacterium Erwinia amylovora leading to manifestation of the disease fire blight. Like in other economically important plant pathogens, E. amylovora pathogenicity depends on a type III secretion system encoded by hrp genes. However, timing and transcriptio...
Contexts in source publication
Context 1
... contrast, expression of amsG, the first gene in the operon for amylovoran synthesis, remained for the first 72 h basally low without expression peak as observed for hrp genes. Control normalizations for all genes investigated against a second reference gene, gyrA, confirmed the observed transcriptional pattern ( Figure S2). ...
Context 2
... order to test whether molecular expression patterns correlate with visual symptom development, we performed standard infection tests modified after Pusey, 1997 [41] by inoculating detached apple blossoms at the stigmas with 10 4 E. amylovora cells suspended in water buffered to pH 4 or pH 7. The inoculation density in this test system was lower than in greenhouse inoculations (10 8 cells), because the critical cell density necessary for infection in detached flowers is lower [46]. Evaluation at 8 dpi showed in two independent experiments significantly (T-test, p,0.05) less fire blight symptoms in flowers inoculated and wetted with acidic pH compared to neutral pH (Figure 2). ...
Context 3
... a clear upregulation of PR-1 at 48 hpi indicated that acidification disturbs the bacterial infection progress leading to activation of the plant defense. Regarding visible symptoms on inoculated blossoms, acidification of the bacterial suspension with pH 4 buffered water could reduce (significant at p,0.05; T-test) the number of flowers showing symptoms typically for fire blight (Figure 2). Together with our gene expression data, this indicates that acidification leads to slower and reduced infection rates and might well be an effective measure to reduce fire blight. ...
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Citations
... Pusey and colleagues have shown that Pantoea agglomerans strain E325 acidifies the pH of the flowers to a point that could deplete the growth of the fire blight pathogen (25). Furthermore, Pester and colleagues have shown that reducing the pH of apple flowers leads to the down expression of virulence genes in E. amylovora (32). Similar dynamics have been described for other pathogen-host systems. ...
Flowers are colonized by a diverse community of microorganisms that can alter plant health and interact with floral pathogens. Erwinia amylovora is a flower-inhabiting bacterium and a pathogen that infects different plant species, including Malus × domestica (apple). Previously, we showed that the co-inoculation of two bacterial strains, members of the genera Pseudomonas and Pantoea, isolated from apple flowers, reduced disease incidence caused by this floral pathogen. Here, we decipher the ecological interactions between the two flower-associated bacteria and E. amylovora in field experimentation and in vitro co-cultures. The two flower commensal strains did not competitively exclude E. amylovora from the stigma habitat, as both bacteria and the pathogen co-existed on the stigma of apple flowers and in vitro . This suggests that plant protection might be mediated by other mechanisms than competitive niche exclusion. Using a synthetic stigma exudation medium, ternary co-culture of the bacterial strains led to a substantial alteration of gene expression in both the pathogen and the two microbiota members. Importantly, the gene expression profiles for the ternary co-culture were not just additive from binary co-cultures, suggesting that some functions only emerged in multipartite co-culture. Additionally, the ternary co-culture of the strains resulted in a stronger acidification of the growth milieu than mono- or binary co-cultures, pointing to another emergent property of co-inoculation. Our study emphasizes the critical role of emergent properties mediated by inter-species interactions within the plant holobiont and their potential impact on plant health and pathogen behavior.
IMPORTANCE
Fire blight, caused by Erwinia amylovora , is one of the most important plant diseases of pome fruits. Previous work largely suggested plant microbiota commensals suppressed disease by antagonizing pathogen growth. However, inter-species interactions of multiple flower commensals and their influence on pathogen activity and behavior have not been well studied. Here, we show that co-inoculating two bacterial strains that naturally colonize the apple flowers reduces disease incidence. We further demonstrate that the interactions between these two microbiota commensals and the floral pathogen led to the emergence of new gene expression patterns and a strong alteration of the external pH, factors that may modify the pathogen’s behavior. Our findings emphasize the critical role of emergent properties mediated by inter-species interactions between plant microbiota and plant pathogens and their impact on plant health.
... It was also previously shown that mutation of the T3SS regulator hrpL resulted in a 2-fold reduction in E. amylovora population size on flower stigmas (Johnson et al. 2009). A few other genetic studies have identified or examined specific genes that contribute to virulence during flower infection (Pester et al. 2012;Schachterle et al. 2022). It is also important to note that E. amylovora cells at stigma tips are also interacting with the apple flower microbiome, which could potentially affect disease outcome. ...
Fire blight, caused by the bacterial pathogen Erwinia amylovora , continues to be a devastating disease affecting commercial apple and pear plantings in almost all areas of the world, with recent incursions into Korea and China. During the past two decades, significant gains in knowledge of E. amylovora and fire blight disease have been achieved, in topic areas such as genetic and genomic diversity, host-pathogen interactions, host resistance, and disease management. As we look forward to the next two decades and beyond of fire blight research, we summarize the current research knowledge in topics focused on E. amylovora pathogen and population biology and propose research questions that we hope can guide the field forward to gain the necessary understanding that will lead to sustainable management of this disease.
... Meanwhile, the expression of T3SS genes is found to be closely correlated with the pathogenesis of E. amylovora (Kharadi et al. 2021). Several studies demonstrated that genes encoding the T3SS are rapidly induced during early stages of infection in apple flowers (within 48 h) and are decreased during later infection stages (Pester et al. 2012;Schachterle et al. 2022). Johnson et al. (2009) reported that E. amylovora cells that express the T3SS are important for bacterial growth during the epiphytic phase on stigmas. ...
Fire blight, caused by Erwinia amylovora, is an economically important disease in apples and pears worldwide. This pathogen relies on the type III secretion system (T3SS) to cause disease. Compounds that inhibit the function of the T3SS (T3SS inhibitors) have emerged as alternative strategies for bacterial plant disease management, as they block bacterial virulence without affecting growth, unlike traditional antibiotics. In this study, we investigated the mode of action of a T3SS inhibitor named TS108, a plant phenolic acid derivative, in E. amylovora. We showed that adding TS108 to an in vitro culture of E. amylovora repressed the expression of several T3SS regulon genes, including the master regulator gene hrpL. Further studies demonstrated that TS108 negatively regulates CsrB, a global regulatory small RNA, at the post-transcriptional level, resulting in a repression of hrpS, which encodes a key activator of hrpL. Additionally, TS108 has no impact on the expression of T3SS in Dickeya dadantii or Pseudomonas aeruginosa, suggesting that its inhibition of the E. amylovora T3SS is likely species specific. To better evaluate the performance of T3SS inhibitors in fire blight management, we conducted five independent field experiments in four states (Michigan, New York, Oregon, and Connecticut) from 2015 to 2022 and observed reductions in blossom blight incidence as high as 96.7% compared to untreated trees. In summary, the T3SS inhibitors exhibited good efficacy against fire blight.
... com) showed acidic conditions might affect E. amylovora pathogenicity. Indeed, the expression of T3SS genes is no longer induced in acidic conditions (pH 4), proving pH has a role in E. amylovora virulence (Pester et al. 2012). ...
... In E. amylovora, T3SS gene expression is activated in the first 48 h after flower inoculation (Pester et al. 2012;Schachterle et al. 2022). Particularly, its activation would be strongly induced during the epiphytic colonization of stigmas and reduced when E. amylovora cells reach the hypanthium (Cui et al. 2021a). ...
Fire blight represents a great threat to apple and pear production worldwide. The ability of its causal agent, Erwinia amylovora , to spread rapidly in the host plants makes this devastating disease difficult to manage. Copper and antibiotics are still the most effective solutions to control fire blight, although their application contribute to environmental pollution and to the development of E. amylovora resistant populations. Thus, there is an urgent need to find new alternatives to such plant protection products. In this review, we summarized what is known on E. amylovora biology, as the knowledge of the plant pathogen biology is essential to develop eco-friendly management strategies. Notably, the presence of E. amylovora alone does not necessarily result in the disease development as it is the final outcome of multiple interactions established between E. amylovora cells, flower microbiota, plant host, insect vectors and environment. For instance, specific humidity and temperature create the suitable conditions for E. amylovora to grow and reach the specific cell density needed for plant infection. Once fire blight develops, insects act as potential vectors of E. amylovora , playing a role in the dispersal of the disease. The host plant represents an important factor as its susceptibility varies among the species belonging to the Rosaceae family. Recent studies showed apple flower microbiota might promote or hinder the infection progress, thus representing a possible source of new biocontrol agents effective in controlling E. amylovora .
... Pester and colleagues have shown that reduction of pH on apple flowers lead to the down expression of 293 virulence genes in E. amylovora (34). Similar dynamics have been described for other pathogen-host 294 systems. ...
Flowers are colonized by a diverse community of microorganisms that alter plant health and interact with floral pathogens. Erwinia amylovora is a flower-inhabiting bacterium and a pathogen that infects different plant species, including Malus × domestica (apple). Previously, we showed that the co-inoculation of two bacterial strains, Pseudomonas and Pantoea natural inhabitants of apple flowers, reduced disease incidence caused by this floral pathogen. Here, we decipher the ecological interactions between these two flower-associated bacteria and E. amylovora in field experimentation and in vitro co-cultures. The two flower microbiota strains did not competitively exclude E. amylovora from the stigma habitat, as both commensal bacteria and the pathogen co-existed on the apple flowers, proscribing microbial antibiosis or niche exclusion as predominant mechanisms of host protection. Inspection of the total and the active microbiota populations on flowers revealed that strain co-inoculations altered microbiota activity. Using synthetic stigma exudation medium, tripartite co-culture of the bacterial strains strongly acidified the growth milieu and led to a substantial alteration of gene expression in both the pathogen and the two microbiota members. Our study emphasizes the critical role of emergent properties mediated by inter-species interactions within the plant holobiont and their impact on plant health and pathogen behavior.
Importance
Fire blight, caused by Erwinia amylovora , is one of the most important plant diseases of pome fruits. Plant-associated microbiota can influence plant disease occurrence through inter-species interactions. Previous studies have shown that plant microbiota commensals could suppress disease mainly by antagonizing of the pathogen growth, however, whether plant-associated microbiota could alter pathogen activity and behavior have not been well studied. Here, we show that the co-inoculation of two bacterial strains that naturally colonize the apple flowers reduce disease pressure. We further demonstrate that the interactions between these two microbiota commensals and the floral pathogen lead to a strong alteration of the pH and to the emergence of new gene expression patterns that may alter the pathogen behavior. Our findings emphasize the critical role of emergent properties mediated by inter-species interactions between plant microbiota and plant pathogens and their impact on plant health.
... Although several studies have been conducted to understand E. amylovora transcriptional regulatory networks using in vitro growth conditions (Ancona et al., 2016;Li et al., 2014;McNally et al., 2012;Wang et al., 2009), studies of gene expression during infection are limited. Expression of T3SS genes was assessed by reverse transcription quantitative real-time PCR (RT-qPCR) during infection of apple flowers, and it was found that these genes were rapidly induced, and expression peaked between 24 and 48 h postinoculation (hpi) (Pester et al., 2012). A recent study conducted transcriptomic analysis of E. amylovora in shoots of two apple cultivars, one with low susceptibility and the other highly susceptible (Puławska et al., 2017). ...
... While these in vivo expression studies provide important groundwork, the studies were conducted under controlled greenhouse (Pester et al., 2012;Puławska et al., 2017) or laboratory conditions (Zhao et al., 2005). In this study, we inoculated a native strain of E. amylovora to apple flowers on trees in a research orchard, and analysed E. amylovora gene expression at the transcriptome level. ...
... Because of the complexity of different host tissues and environments, rather than sample whole flowers we sampled and sequenced RNA from the stigma, flower base, or pedicel of inoculated flowers at four sampling time points postinoculation. Our sequencing results matched previously reported expression patterns (Pester et al., 2012) for T3SS genes. We furthermore confirmed several hypothesized gene expression patterns across time as infection progressed. ...
The enterobacterial pathogen Erwinia amylovora uses multiple virulence‐associated traits to cause fire blight, a devastating disease of apple and pear trees. Many virulence‐associated phenotypes have been studied that are critical for virulence and pathogenicity. Despite the in vitro testing that has revealed how these systems are transcriptionally regulated, information on when and where in infected tissues these genes are being expressed is lacking. Here, we used a high‐throughput sequencing approach to characterize the transcriptome of E. amylovora during disease progression on apple flowers under field infection conditions. We report that type III secretion system genes and flagellar genes are strongly co‐expressed. Likewise, genes involved in biosynthesis of the exopolysaccharide amylovoran and sorbitol utilization had similar expression patterns. We further identified a group of 16 genes whose expression is increased and maintained at high levels throughout disease progression across time and tissues. We chose five of these genes for mutational analysis and observed that deletion mutants lacking these genes all display reduced symptom development on apple shoots. Furthermore, these induced genes were over‐represented for genes involved in sulphur metabolism and cycling, suggesting the possibility of an important role for maintenance of oxidative homeostasis during apple flower infection. We used RNA sequencing to characterize the transcriptome of Erwinia amylovora during disease progression on apple flowers in the field, and report expression for several known virulence genes and identify novel virulence genes.
... Either blossoms, shoots, or the rootstock can show blight symptoms leading to severe economic losses to varying extents. Unintended trade of latently infected plants is responsible for its spread to distant ecological areas, while wind, rain, and pollinating insects disseminate the pathogen to nearby regions (Pester et al., 2012). ...
Fire blight, affecting more than one hundred and thirty species in the Rosaceae, is probably the most destructive disease affecting pear and apple cultivars in many countries. Currently, there are no effective synthetic compounds with systemic properties. Another major problem is the occurrence and spread of strains of Erwinia amylovora with resistance to streptomycin and copper. Taking into consideration human and environmental health, the use of biocontrol agents either as an alternative or as a supplement within an integrated fire blight management strategy has attracted worldwide attention. In this study, E. amylovora solution of 107 CFU ml-1 was treated with bio-control agents, Bacillus subtilis str. QST 713, B. amyloliquefaciens str. MBI 600 and their mixture (at solution densities of 106, 107, and 108 CFU ml-1 for each one) on Petri dishes, containing King’s B medium and, compared with positive (streptomycin sulphate) and negative (sterile distilled water) controls. In vivo studies were performed on two-year-old apple cv. Gala seedlings were grown in 45-cm-diameter pots containing a sterilized mix of soil–sand–peat under controlled greenhouse conditions (85% relative humidity, 25°C temperature, and 16h of daylight). The plants were irrigated as needed by drip irrigation and each pot received a mineral solution (NPK: 20–20–20) at 2 g l-1 twice. When plant shoots reached a length of 30-35 cm, bio-control agents, individually and their mixture, were applied to the plants by a hand-sprayer. Obtaining the data, 108 CFU ml-1 of Bacillus spp. suspension mixture showed the strongest in vitro antibacterial effect (26mm) among the tested treatments after positive control streptomycin (28.6mm). Parallel to in vitro findings, the mixture was most effective against the pathogen on cv. Gala (66.03%). Findings show that the use of a mixture of beneficial microorganisms with individual antagonistic properties against the pathogen can be an effective strategy as a natural alternative to agrochemicals in the scope of good agriculture practices.
... Ea growth in host leaf tissue is correlated with strong and early expression of its T3SS-encoding hrp genes and particularly of DspA/E, the major Ea T3E (Pester et al., 2012). In vitro, the nature and the quantity of N available for Ea are known to affect expression of its hrp genes (Wei et al., 1992). ...
... For the Ea virulence genes expression in planta, all the steps were carried out similarly as for plant gene expression, except that the reverse transcription was performed from 5 µg total RNA using random primers (SuperScript) instead of oligo(dT). For qPCR, each Ea target gene was normalized with the recA (Pester et al., 2012) and rplU (Khokhani et al., 2013) references genes. The normalized expression patterns obtained using both reference genes were similar, so only the data normalized with recA are shown. ...
We showed previously that nitrogen (N) limitation decreases Arabidopsis resistance to Erwinia amylovora (Ea). We show that decreased resistance to bacteria in low N is correlated with lower apoplastic reactive oxygen species (ROS) accumulation and lower jasmonic acid (JA) pathway expression. Consistently, pretreatment with methyl jasmonate (Me‐JA) increased the resistance of plants grown under low N. In parallel, we show that in planta titres of a nonvirulent type III secretion system (T3SS)‐deficient Ea mutant were lower than those of wildtype Ea in low N, as expected, but surprisingly not in high N. This lack of difference in high N was consistent with the low expression of the T3SS‐encoding hrp virulence genes by wildtype Ea in plants grown in high N compared to plants grown in low N. This suggests that expressing its virulence factors in planta could be a major limiting factor for Ea in the nonhost Arabidopsis. To test this hypothesis, we preincubated Ea in an inducing medium that triggers expression of hrp genes in vitro, prior to inoculation. This preincubation strongly enhanced Ea titres in planta, independently of the plant N status, and was correlated to a significant repression of JA‐dependent genes. Finally, we identify two clusters of metabolites associated with resistance or with susceptibility to Ea. Altogether, our data showed that high susceptibility of Arabidopsis to Ea, under low N or following preincubation in hrp‐inducing medium, is correlated with high expression of the Ea hrp genes in planta and low expression of the JA signalling pathway, and is correlated with the accumulation of specific metabolites. Under low nitrate, infection of Arabidopsis thaliana plants with the phytopathogenic bacterium Erwinia amylovora leads to reduced nonhost resistance and increased virulence gene expression.
... Using quantitative real-time PCR analyses, Pester et al. (91) showed that several T3SS regulon genes were expressed in E. amylovora during the initial infection of apple flowers. In addition, Venisse et al. (125) demonstrated that wild-type E. amylovora induces a host defense response with the associated genesis of reactive oxygen species (ROS) during flower infection, whereas a hrp mutant defective in production of the T3SS does not. ...
Fire blight, caused by the bacterial phytopathogen Erwinia amylovora, is an economically important and mechanistically complex disease that affects apple and pear production in most geographic production hubs worldwide. We compile, assess, and present a genetic outlook on the progression of an E. amylovora infection in the host. We discuss the key aspects of type III secretion–mediated infection and systemic movement, biofilm formation in xylem, and pathogen dispersal via ooze droplets, a concentrated suspension of bacteria and exopolysaccharide components. We present an overall outlook on the genetic elements contributing to E. amylovora pathogenesis, including an exploration of the impact of floral microbiomes on E. amylovora colonization, and summarize the current knowledge of host responses to an incursion and how this response stimulates further infection and systemic spread. We hope to facilitate the identification of new, unexplored areas of research in this pathosystem that can help identify evolutionarily susceptible genetic targets to ultimately aid in the design of sustainable strategies for fire blight disease mitigation.
Expected final online publication date for the Annual Review of Phytopathology, Volume 59 is August 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Pathogenicity potential of the 36 E. amylovora isolates were evaluated by dot blot hybridization of three virulence markers designated by hrpN M , hrpL M , and amsG M , targeting respectively the hrpL gene which is a transcriptional switch of the T3SS hrp operon [38,65]; the hrpN gene coding for a translocator protein [66,67]; and amsG, a gene coding the amylovoran biosynthesis protein AmsG, involved in the ams gene cluster [38,68]. DNA probes for dot blot hybridization were prepared by PCR amplification of the three virulence specific markers on E. amylovora type strain LMG 2024, using primers previously described by Pester et al. [64] for each gene, and by designing their respective complementary primers (Table 2), resorting to the full genome of the type strain LMG 2024 (AN: CAPB00000000.1), to obtain DNA probes with the following sizes: 368bp for amsG M , 378bp for hrpL M , and 410bp for hrpN M . ...
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.
