Exploring Rain as Source of Biological Control Agents for Fire Blight on Apple.
ABSTRACT: Poor survival on plants can limit the efficacy of Biological Control Agents (BCAs) in the field. Yet bacteria survive in the atmosphere, despite their exposure to high solar radiation and extreme temperatures. If conditions in the atmosphere are similar to, or more extreme than, the environmental conditions on the plant surface, then precipitation may serve as a reservoir of robust BCAs. To test this hypothesis, two hundred and fifty-four rain-borne isolates were screened for in vitro inhibition of Erwinia amylovora, the causal agent of fire blight, as well as of other plant pathogenic bacteria, fungi and oomycetes. Two isolates showed strong activity against E. amylovora and other plant pathogenic bacteria, while other isolates showed activity against fungal and oomycete pathogens. Survival assays suggested that the two isolates that inhibited E. amylovora were able to survive on apple blossoms and branches similarly to E. amylovora. Pathogen population size and associated fire blight symptoms were significantly reduced when detached apple blossoms were treated with the two isolates before pathogen inoculation, however, disease reduction on attached blossoms within an orchard was inconsistent. Using whole genome sequencing, the isolates were identified as Pantoea agglomerans and P. ananatis, respectively. A UV-mutagenesis screen pointed to a phenazine antibiotic D-alanylgriseoluteic acid synthesis gene cluster as being at the base of the antimicrobial activity of the P. agglomerans isolate. Our work reveals the potential of precipitation as an under-explored source of BCAs, whole genome sequencing as an effective approach to precisely identify BCAs, and UV-mutagenesis as a technically simple screen to investigate the genetic basis of BCAs. More field trials are needed to determine the efficacy of the identified BCAs in fire blight control.
Project description:Antibiotics are used extensively to control animal, plant, and human pathogens. They are sprayed on apple and pear orchards to control the bacterium Erwinia amylovora, the causative agent of fire blight. This phytopathogen is developing antibiotic resistance and alternatives either have less efficacy, are phytotoxic, or more management intensive. The objective of our study was to develop an effective biological control agent colonizing the host plant and competing with Erwinia amylovora. It must not be phytotoxic, have a wide spectrum of activity, and be unlikely to induce resistance in the pathogen. To this end, several bacterial isolates from various environmental samples were screened to identify suitable candidates that are antagonistic to E. amylovora. We sampled bacteria from the flowers, leaves, and soil from apple and pear orchards from the springtime bloom period until the summer. The most effective bacteria, including isolates of Pseudomonas poae, Paenibacillus polymyxa, Bacillus amyloliquefaciens and Pantoea agglomerans, were tested in vitro and in vivo and formulated into products containing both the live strains and their metabolites that were stable for at least 9 months. Trees treated with the product based on P. agglomerans NY60 had significantly less fire blight than the untreated control and were statistically not different from streptomycin-treated control trees. With P. agglomerans NY60, fire blight never extended beyond the central vein of the inoculated leaf. The fire blight median disease severity score, 10 days after inoculation, was up to 70% less severe on trees treated with P. agglomerans NY60 as compared to untreated controls.
Project description:Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants. Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community. Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria. Here, we report that a multidrug efflux transporter, designated NorM, of E. amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms. According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family. The corresponding gene is widely distributed among E. amylovora strains and related plant-associated bacteria. NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine. A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106. However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms. The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined. The promoter activity of norM was twofold greater at 18 degrees C than at 28 degrees C. The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites. Thus, E. amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature.
Project description:Pantoea vagans is a Gram-negative enterobacterial plant epiphyte of a broad range of plants. Here we report the 4.89-Mb genome sequence of P. vagans strain C9-1 (formerly Pantoea agglomerans), which is commercially registered for biological control of fire blight, a disease of pear and apple trees caused by Erwinia amylovora.
Project description:The Gram-negative bacterium Erwinia amylovora causes fire blight disease of apples and pears. While the virulence systems of E. amylovora have been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophic E. amylovora mutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-type E. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenic E. amylovora auxotrophs could have utility as fire blight biocontrol agents.IMPORTANCE This study has revealed the availability of a range of host metabolites to E. amylovora cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial de novo synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic E. amylovora mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent E. amylovora on flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenic E. amylovora auxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.
Project description:To identify genes involved in the response to the fire blight pathogen Erwinia amylovora in apple (Malus×domestica), expression profiles were investigated using an apple oligo (70-mer) array representing 40, 000 genes. Blossoms of a fire blight-susceptible apple cultivar Gala were collected from trees growing in the orchard, placed on a tray in the laboratory, and spray-inoculated with a suspension of E. amylovora at a concentration of 10(8) cfu ml(-1). Uninoculated detached flowers served as controls at each time point. Expression profiles were captured at three different time points post-inoculation at 2, 8, and 24 h, together with those at 0 h (uninoculated). A total of about 3500 genes were found to be significantly modulated in response to at least one of the three time points. Among those, a total of 770, 855, and 1002 genes were up-regulated, by 2-fold, at 2, 8, and 24 h following inoculation, respectively; while, 748, 1024, and 1455 genes were down-regulated, by 2-fold, at 2, 8, and 24 h following inoculation, respectively. Over the three time points post-inoculation, 365 genes were commonly up-regulated and 374 genes were commonly down-regulated. Both sets of genes were classified based on their functional categories. The majority of up-regulated genes were involved in metabolism, signal transduction, signalling, transport, and stress response. A number of transcripts encoding proteins/enzymes known to be up-regulated under particular biotic and abiotic stress were also up-regulated following E. amylovora treatment. Those up- or down-regulated genes encode transcription factors, signaling components, defense-related, transporter, and metabolism, all of which have been associated with disease responses in Arabidopsis and rice, suggesting similar response pathways are involved in apple blossoms.
Project description:The epiphyte Pantoea agglomerans 48b/90 (Pa48b) is a promising biocontrol strain against economically important bacterial pathogens such as Erwinia amylovora. Strain Pa48b produces the broad-spectrum antibiotic 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine (APV) in a temperature-dependent manner. An APV-negative mutant still suppressed the E. amylovora population and fire blight disease symptoms in apple blossom experiments under greenhouse conditions, but was inferior to the Pa48b wild-type indicating the influence of APV in the antagonism. In plant experiments with the soybean pathogen Pseudomonas syringae pv. glycinea both, Pa48b and the APV-negative mutant, successfully suppressed the pathogen. Our results demonstrate that the P. agglomerans strain Pa48b is an efficient biocontrol organism against plant pathogens, and we prove its ability for fast colonization of plant surfaces over a wide temperature range.
Project description:The molecular basis of resistance and susceptibility of host plants to fire blight, a major disease threat to pome fruit production globally, is largely unknown. RNA-sequencing data from challenged and mock-inoculated flowers were analyzed to assess the susceptible response of apple to the fire blight pathogen Erwinia amylovora. In presence of the pathogen 1,080 transcripts were differentially expressed at 48 h post inoculation. These included putative disease resistance, stress, pathogen related, general metabolic, and phytohormone related genes. Reads, mapped to regions on the apple genome where no genes were assigned, were used to identify potential novel genes and open reading frames. To identify transcripts specifically expressed in response to E. amylovora, RT-PCRs were conducted and compared to the expression patterns of the fire blight biocontrol agent Pantoea vagans strain C9-1, another apple pathogen Pseudomonas syringae pv. papulans, and mock inoculated apple flowers. This led to the identification of a peroxidase superfamily gene that was lower expressed in response to E. amylovora suggesting a potential role in the susceptibility response. Overall, this study provides the first transcriptional profile by RNA-seq of the host plant during fire blight disease and insights into the response of susceptible apple plants to E. amylovora.
Project description:Lytic bacteriophages are in development as biological control agents for the prevention of fire blight disease caused by Erwinia amylovora. Temperate phages should be excluded as biologicals since lysogeny produces the dual risks of host resistance to phage attack and the transduction of virulence determinants between bacteria. The extent of lysogeny was estimated in wild populations of E.?amylovora and Pantoea agglomerans with real-time polymerase chain reaction primers developed to detect E.?amylovora phages belonging to the Myoviridae and Podoviridae families. Pantoea agglomerans, an orchard epiphyte, is easily infected by Erwinia spp. phages, and it serves as a carrier in the development of the phage-mediated biological control agent. Screening of 161 E.?amylovora isolates from 16 distinct geographical areas in North America, Europe, North Africa and New Zealand and 82 P.?agglomerans isolates from southern Ontario, Canada showed that none possessed prophage. Unstable phage resistant clones or lysogens were produced under laboratory conditions. Additionally, a stable lysogen was recovered from infection of bacterial isolate Ea110R with Podoviridae phage ?Ea35-20. These laboratory observations suggested that while lysogeny is possible in E.?amylovora, it is rare or absent in natural populations, and there is a minimal risk associated with lysogenic conversion and transduction by Erwinia spp. phages.
Project description:BACKGROUND: 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 transcriptional order of hrp gene expression during flower infections are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using quantitative real-time PCR analyses, we addressed the questions of how fast, strong and uniform key hrp virulence genes and the effector dspA/E are expressed when bacteria enter flowers provided with the full defense mechanism of the apple plant. In non-invasive bacterial inoculations of apple flowers still attached to the tree, E. amylovora activated expression of key type III secretion genes in a narrow time window, mounting in a single expression peak of all investigated hrp/dspA/E genes around 24-48 h post inoculation (hpi). This single expression peak coincided with a single depression in the plant PR-1 expression at 24 hpi indicating transient manipulation of the salicylic acid pathway as one target of E. amylovora type III effectors. Expression of hrp/dspA/E genes was highly correlated to expression of the regulator hrpL and relative transcript abundances followed the ratio: hrpA>hrpN>hrpL>dspA/E. Acidic conditions (pH 4) in flower infections led to reduced virulence/effector gene expression without the typical expression peak observed under natural conditions (pH 7). CONCLUSION/SIGNIFICANCE: The simultaneous expression of hrpL, hrpA, hrpN, and the effector dspA/E during early floral infection indicates that speed and immediate effector transmission is important for successful plant invasion. When this delicate balance is disturbed, e.g., by acidic pH during infection, virulence gene expression is reduced, thus partly explaining the efficacy of acidification in fire blight control on a molecular level.
Project description:Fire blight remains a serious threat to commercial apple production in the USA and worldwide. Other diseases and spray damage can result in fire blight-like symptoms that can lead to misdiagnosis and affect disease management strategies. Accurate and timely detection of the fire blight pathogen, Erwinia amylovora, is extremely important to deploy appropriate and timely measures to reduce fire blight epidemics in commercial apple orchards. We tested two commercial lateral flow immunoassays (AgriStrip®, and Pocket Diagnostics kit), Loop mediated isothermal amplification (LAMP), and quantitative PCR (qPCR) to diagnose E. amylovora infected samples in lab and field settings. The AgriStrip® and Pocket Diagnostics kits were able to detect actively growing bacteria up to ×106 cfu/ml bacterial concentration. Pocket Diagnostics kit had less specificity and showed positive tests for E. pyrifolia in addition to E. amylovora. The LAMP assay showed high specificity for E. amylovora and was able to detect up to ×103 cfu/ml bacterial concentrations. The qPCR assay was also able to detect bacterial cells up to ×10-3 cfu/ml bacterial concentration with highly specific E. amylovora detection. Grower surveys and comparative cost-benefit analysis indicated that immunoassay kits are less expensive, easier to use, and require less technical expertise for on-site fire blight diagnosis than LAMP and qPCR. However, the choice of a specific diagnostic assay depends on the time, sensitivity, and specificity required for the detection of fire blight and its management.