Comparative evaluation of lateral flow immunoassays, LAMP, and quantitative PCR for diagnosis of fire blight in apple orchards.
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ABSTRACT:
SUBMITTER: Singh J
PROVIDER: S-EPMC7456764 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Fire blight (FB) is a bacterial disease affecting plants from Rosaceae family, including apple and pear. FB develops after the infection of Erwinia amylovora, gram-negative enterobacterium, and results in burnt-like damages and wilting, which can affect all organs of the plant. Although the mechanisms underlying disease response in apples are not elucidated yet, it has been well described that FB resistance depends on the rootstock type. The main objective of this work was to identify miRNAs involved in response to bacterial infection in order to better explain apple defense mechanisms against fire blight disease. We performed deep sequencing of eighteen small RNA libraries obtained from inoculated and non-inoculated Gala apple leaves. 233 novel plant mature miRNAs were identified together with their targets and potential role in response to bacterial infection. We identify three apple miRNAs responding to inoculation (mdm-miR168a,b, mdm-miR194C and mdm-miR1392C) as well as miRNAs reacting to bacterial infection in a rootstock-specific manner (miR395 family). Our results provide insights into the mechanisms of fire blight resistance in apple.
2016-08-28 | GSE72346 | GEO
Project description:miRNAs are key players in multiple biological processes, therefore analysis and characterization of these small regulatory RNAs is a critical step towards better understanding of animal and plant biology. In apple (Malus domestica) two hundred microRNAs are known, which most probably represents only a fraction of miRNAome diversity. As a result, more effort is required to better annotate miRNAs and their functions in this economically important species. We performed deep sequencing of twelve small RNA libraries obtained for fire blight resistant and fire blight sensitive trees. In the sequencing results we identified 116 novel microRNAs and confirmed a majority of previously reported apple miRNAs. We then experimentally verified selected candidates with RT-PCR and stem-loop qPCR and performed differential expression analysis. Finally, we identified and characterized putative targets of all known apple miRNAs. In this study we considerably expand the apple miRNAome by identifying and characterizing dozens of novel microRNAs. Moreover, our data suggests that apple microRNAs might be considered as regulators and markers of fire blight resistance.
2015-01-09 | GSE59482 | GEO
Project description:BACKGROUND:Although it is known that resistant rootstocks facilitate management of fire blight disease, incited by Erwinia amylovora, the role of rootstock root traits in providing systemic defense against E. amylovora is unclear. In this study, the hypothesis that rootstocks of higher root vigor provide higher tolerance to fire blight infection in apples is tested. Several apple scion genotypes grafted onto a single rootstock genotype and non-grafted 'M.7' rootstocks of varying root vigor are used to assess phenotypic and molecular relationships between root traits of rootstocks and fire blight susceptibility of apple scion cultivars. RESULTS:It is observed that different root traits display significant (p?<?0.05) negative correlations with fire blight susceptibility. In fact, root surface area partially dictates differential levels of fire blight susceptibility of 'M.7' rootstocks. Furthermore, contrasting changes in gene expression patterns of diverse molecular pathways accompany observed differences in levels of root-driven fire blight susceptibility. It is noted that a singular co-expression gene network consisting of genes from defense, carbohydrate metabolism, protein kinase activity, oxidation-reduction, and stress response pathways modulates root-dependent fire blight susceptibility in apple. In particular, WRKY75 and UDP-glycotransferase are singled-out as hub genes deserving of further detailed analysis. CONCLUSIONS:It is proposed that low root mass may incite resource-limiting conditions to activate carbohydrate metabolic pathways, which reciprocally interact with plant immune system genes to elicit differential levels of fire blight susceptibility.
| S-EPMC6929320 | biostudies-literature
Project description:BACKGROUND: Desirable apple varieties are clonally propagated by grafting vegetative scions onto rootstocks. Rootstocks influence many phenotypic traits of the scion, including resistance to pathogens such as Erwinia amylovora, which causes fire blight, the most serious bacterial disease of apple. The purpose of the present study was to quantify rootstock-mediated differences in scion fire blight susceptibility and to identify transcripts in the scion whose expression levels correlated with this response. RESULTS: Rootstock influence on scion fire blight resistance was quantified by inoculating three-year old, orchard-grown apple trees, consisting of 'Gala' scions grafted to a range of rootstocks, with E. amylovora. Disease severity was measured by the extent of shoot necrosis over time. 'Gala' scions grafted to G.30 or MM.111 rootstocks showed the lowest rates of necrosis, while 'Gala' on M.27 and B.9 showed the highest rates of necrosis. 'Gala' scions on M.7, S.4 or M.9F56 had intermediate necrosis rates. Using an apple DNA microarray representing 55,230 unique transcripts, gene expression patterns were compared in healthy, un-inoculated, greenhouse-grown 'Gala' scions on the same series of rootstocks. We identified 690 transcripts whose steady-state expression levels correlated with the degree of fire blight susceptibility of the scion/rootstock combinations. Transcripts known to be differentially expressed during E. amylovora infection were disproportionately represented among these transcripts. A second-generation apple microarray representing 26,000 transcripts was developed and was used to test these correlations in an orchard-grown population of trees segregating for fire blight resistance. Of the 690 transcripts originally identified using the first-generation array, 39 had expression levels that correlated with fire blight resistance in the breeding population. CONCLUSIONS: Rootstocks had significant effects on the fire blight susceptibility of 'Gala' scions, and rootstock-regulated gene expression patterns could be correlated with differences in susceptibility. The results suggest a relationship between rootstock-regulated fire blight susceptibility and sorbitol dehydrogenase, phenylpropanoid metabolism, protein processing in the endoplasmic reticulum, and endocytosis, among others. This study illustrates the utility of our rootstock-regulated gene expression data sets for candidate trait-associated gene data mining.
| S-EPMC3277459 | biostudies-literature
Project description:The generation and selection of novel fire blight resistant apple genotypes would greatly improve the management of this devastating disease, caused by Erwinia amylovora. Such resistant genotypes are currently developed by conventional breeding, but novel breeding technologies including cisgenesis could be an alternative approach. A cisgenic apple line C44.4.146 was regenerated using the cisgene FB_MR5 from wild apple Malus ×robusta 5 (Mr5), and the previously established method involving A. tumefaciens-mediated transformation of the fire blight susceptible cultivar 'Gala Galaxy' using the binary vector p9-Dao-FLPi. The line C44.4.146 was shown to carry only the cisgene FB_MR5, controlled by its native regulatory sequences and no transgenes were detected by PCR or Southern blot following heat induced recombinase-mediated elimination of the selectable markers. Although this line contains up to 452 bp of vector sequences, it still matches the original definition of cisgenesis. A single insertion of T-DNA into the genome of 'Gala Galaxy' in chromosome 16 was identified. Transcription of FB_MR5 in line C44.4.146 was similar to the transcription in classically bred descendants of Mr5. Three independent shoot inoculation experiments with a Mr5 avirulent strain of Erwinia amylovora were performed using scissors or syringe. Significantly lower disease symptoms were detected on shoots of the cisgenic line compared to those of untransformed 'Gala Galaxy'. Despite the fact that the pathogen can overcome this resistance by a single nucleotide mutation, this is, to our knowledge, the first prototype of a cisgenic apple with increased resistance to fire blight.
| S-EPMC4666654 | biostudies-literature
Project description:Key messagepPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes. Intragenesis is an important alternative to transgenesis to produce modified plants containing native DNA only. A key point to develop such a strategy is the availability of regulatory sequences controlling the expression of the gene of interest. With the aim of finding apple gene promoters either inducible by the fire blight pathogen Erwinia amylovora (Ea) or moderately constitutive, we focused on polyphenoloxidase genes (PPO). These genes encode oxidative enzymes involved in many physiological processes and have been previously shown to be upregulated during the Ea infection process. We found ten PPO and two PPO-like sequences in the apple genome and characterized the promoters of MdPPO16 (pPPO16) and MdKFDV02 PPO-like (pKFDV02) for their potential as Ea-inducible and low-constitutive regulatory sequences, respectively. Expression levels of reporter genes fused to these promoters and transiently or stably expressed in apple were quantified after various treatments. Unlike pKFDV02 which displayed a variable activity, pPPO16 allowed a fast and strong expression of transgenes in apple following Ea infection in a Type 3 Secretion System dependent manner. Altogether our results does not confirmed pKFDV02 as a constitutive and weak promoter whereas pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes.
| S-EPMC9270298 | biostudies-literature
Project description:miRNAs are key players in multiple biological processes, therefore analysis and characterization of these small regulatory RNAs is a critical step towards better understanding of animal and plant biology. In apple (Malus domestica) two hundred microRNAs are known, which most probably represents only a fraction of miRNAome diversity. As a result, more effort is required to better annotate miRNAs and their functions in this economically important species. We performed deep sequencing of twelve small RNA libraries obtained for fire blight resistant and fire blight sensitive trees. In the sequencing results we identified 116 novel microRNAs and confirmed a majority of previously reported apple miRNAs. We then experimentally verified selected candidates with RT-PCR and stem-loop qPCR and performed differential expression analysis. Finally, we identified and characterized putative targets of all known apple miRNAs. In this study we considerably expand the apple miRNAome by identifying and characterizing dozens of novel microRNAs. Moreover, our data suggests that apple microRNAs might be considered as regulators and markers of fire blight resistance. Actively-growing shoot tip tissue samples were collected from twelve apple trees, which includes three biological replicates of each following scion-rootstock combinations: B.9, G.30, M.111 and M.27.
2015-01-09 | E-GEOD-59482 | biostudies-arrayexpress
Project description:Fast and accurate diagnosis is needed to eradicate and manage economically important and invasive diseases like fire blight. Loop-mediated isothermal amplification (LAMP) is known as the best on-site diagnostic, because it is fast, highly specific to a target, and less sensitive to inhibitors in samples. In this study, LAMP assay that gives more consistent results for on-site diagnosis of fire blight than the previous developed LAMP assays was developed. Primers for new LAMP assay (named as DS-LAMP) were designed from a histidine-tRNA ligase gene (EAMY_RS32025) of E. amylovora CFBP1430 genome. The DS-LAMP amplified DNA (positive detection) only from genomic DNA of E. amylovora strains, not from either E. pyrifoliae (causing black shoot blight) or from Pseudomonas syringae pv. syringae (causing shoot blight on apple trees). The detection limit of DS-LAMP was 10 cells per LAMP reaction, equivalent to 104 cells per ml of the sample extract. DS-LAMP successfully diagnosed the pathogens on four fire-blight infected apple and pear orchards. In addition, it could distinguish black shoot blight from fire blight. The Bühlmann-LAMP, developed previously for on-site diagnosis of fire blight, did not give consistent results for specificity to E. amylovora and on-site diagnosis; it gave positive reactions to three strains of E. pyrifoliae and two strains of P. syringae pv. syringae. It also, gave positive reactions to some healthy sample extracts. DS-LAMP, developed in this study, would give more accurate on-site diagnosis of fire blight, especially in the Republic of Korea, where fire blight and black shoot blight coexist.
| S-EPMC5985645 | biostudies-literature
Project description: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.
| S-EPMC7033628 | biostudies-literature
Project description:Fire blight, caused by the enterobacterium Erwinia amylovora, is a destructive disease, which can affect most members of the Rosaceae family. Since no significant genomic differences have been found by others to explain differences in virulence, we used here a gel-based proteomic approach to elucidate mechanisms and key players that allow the pathogen to survive, grow and multiply inside its host. Therefore, two strains with proven difference in virulence were grown under controlled conditions in vitro as well as in planta (infected apple rootstocks). Proteomic analysis including 2DE and mass spectrometry revealed that proteins involved in transcription regulation were more abundant in the in planta condition for both strains. In addition, genes involved in RNA processing were upregulated in planta for the highly virulent strain PFB5. Moreover, the upregulation of structural components of the F0F1-ATP synthase are major findings, giving important information on the infection strategy of this devastating pathogen. Overall, this research provides the first proteomic profile of E. amylovora during infection of apple rootstocks and insights into the response of the pathogen in interaction with its host.
| S-EPMC6076297 | biostudies-literature