scholarly journals A Powerful LAMP Weapon against the Threat of the Quarantine Plant Pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens

2020 ◽  
Vol 8 (11) ◽  
pp. 1705
Author(s):  
Stefania Tegli ◽  
Carola Biancalani ◽  
Aleksandr N. Ignatov ◽  
Ebrahim Osdaghi
Keyword(s):  
Plant Pathogen ◽  
Genomic Dna ◽  
Economic Losses ◽  
Early 20Th Century ◽  
Visual Examination ◽  
Phytopathogenic Bacterium ◽  
Eu Legislation ◽  
Leguminous Crops ◽  
Specific Diagnostic Test ◽  
Union Territory

Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) is a Gram-positive phytopathogenic bacterium attacking leguminous crops and causing systemic diseases such as the bacterial wilt of beans and bacterial spot of soybeans. Since the early 20th century, Cff is reported to be present in North America, where it still causes high economic losses. Currently, Cff is an emerging plant pathogen, rapidly spreading worldwide and occurring in many bean-producing countries. Infected seeds are the main dissemination pathway for Cff, both over short and long distances. Cff remains viable in the seeds for long times, even in field conditions. According to the most recent EU legislation, Cff is included among the quarantine pests not known to occur in the Union territory, and for which the phytosanitary inspection consists mainly of the visual examination of imported bean seeds. The seedborne nature of Cff combined with the globalization of trades urgently call for the implementation of a highly specific diagnostic test for Cff, to be routinely and easily used at the official ports of entry and into the fields. This paper reports the development of a LAMP (Loop-Mediated Isothermal Amplification) specific for Cff, that allows the detection of Cff in infected seeds, both by fluorescence and visual monitoring, after 30 min of reaction and with a detection limit at around 4 fg/μL of pure Cff genomic DNA.

scholarly journals Detection of fish pathogen Saprolegnia parasitica in environmental DNA samples by droplet digital PCR

2021 ◽  
Vol 4 ◽  
Author(s):  
Dora Pavić ◽  
Anđela Miljanović ◽  
Uršula Prosenc-Zmrzljak ◽  
Rok Košir ◽  
Dorotea Grbin ◽  
...  
Keyword(s):  
Water Samples ◽  
Genomic Dna ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Economic Losses ◽  
Internal Transcribed Spacer Region ◽  
Saprolegnia Parasitica ◽  
Oomycete Pathogen ◽  
Total Dna

Oomycetes are fungal-like microorganisms parasitic towards a large number of plant and animal species. Genera from order Saprolegniales, such as Saprolegnia and Aphanomyces, cause devastating infections of freshwater animals. Saprolegnia parasitica is a widely distributed oomycete pathogen that causes saprolegniosis, a disease responsible for significant economic losses in aquaculture, as well as declines of natural populations of fish and other freshwater organisms. Despite its negative impact, no monitoring protocol for S. parasitica has been established to date. Thus, we aimed to develop a droplet digital PCR (ddPCR) assay for the detection and quantification of S. parasitica in environmental DNA samples. Saprolegnia parasitica-specific primers were designed to target internal transcribed spacer region 2 (ITS 2), based on the alignment of ITS sequences of S. parasitica and a range of Saprolegnia spp., as well as other oomycetes. The specificity of primers was tested using genomic DNA of S. parasitica (as positive control) and DNA of non–S. parasitica oomycete isolates, as well as trout/crayfish DNA (as negative control). The primers specifically amplified a segment of the ITS region of oomycete pathogen S. parasitica, while no amplification (i.e. no positive droplets) was obtained for closely related Saprolegnia spp. (e.g. Saprolegnia sp. 1 and S. ferax) and other more distantly related oomycetes. Next, the limit of detection (LOD) of the assay was established by using serial dilutions of the S. parasitica genomic DNA. The determined sensitivity of the assay was high: LOD was 15 fg of pathogen’s genomic DNA per µL of the reaction mixture. Assay performance was further assessed with environmental DNA samples isolated from water from the trout farms and natural environments, as well as (ii) biofilm from the host surface (swab samples). Water samples were collected from 21 different locations in Croatia, while swab samples were collected from S. parasitica host/carrier species: (i) skin and eggs of the rainbow trout (Oncorhynchus mykiss Walbaum, 1792) and brown trout (Salmo trutta Linnaeus, 1758), and (ii) cuticle of signal crayfish (Pacifastacus leniusculus Dana, 1852) and narrow clawed crayfish (Pontastacus leptodactylus Eschscholtz, 1823). Samples were classified into agent levels A0 to A6, depending of the number of S. parasitica ITS copies per ng of total DNA. Saprolegnia parasitica was detected in 76 % of water samples (16/21) and the range of pathogen’s ITS copies in positive samples was between 0.02 and 14 copies/ng of total DNA (agent levels A1 to A3). Regarding the swab samples, S. parasitica load was significantly higher in diseased trout than in those with healthy appearance: 9375 vs 3.28 S. parasitica copies/ng of total swab DNA (average agent level A6 vs. A2, respectively). Despite the fact that none of the sampled crayfish had signs of infection, the pathogen was detected in all tested cuticle swabs. Swabs of P. leniusculus, a known S. parasitica host, had significantly higher S. parasitica load than swabs of P. leptodactylus, S. parasitica carrier: 390 vs 83 S. parasitica copies/ng (agent level A5 vs. A4, respectively). In conclusion, our results demonstrate the applicability of the newly developed ddPCR assay in monitoring and early detection of S. parasitica in aquaculture facilities and natural freshwater environments. This would help in a better understanding of S. parasitica ecology and its effects on the host populations.

scholarly journals Potential Distribution and the Risks of Bactericera cockerelli and Its Associated Plant Pathogen Candidatus Liberibacter Solanacearum for Global Potato Production

Insects ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 298
Author(s):  
Jing Wan ◽  
Rui Wang ◽  
Yonglin Ren ◽  
Simon McKirdy
Keyword(s):  
At Risk ◽  
South America ◽  
Plant Pathogen ◽  
Potato Production ◽  
Economic Losses ◽  
Bactericera Cockerelli ◽  
Ecological Niche Models ◽  
Potato Acreage ◽  
Candidatus Liberibacter Solanacearum ◽  
Candidatus Liberibacter

The tomato potato psyllid (TPP), Bactericera cockerelli, is a psyllid native to North America that has recently invaded New Zealand and Australia. The potential for economic losses accompanying invasions of TPP and its associated bacterial plant pathogen Candidatus Liberibacter solanacearum (CLso), has caused much concern. Here, we employed ecological niche models to predict environments suitable for TPP/CLso on a global scale and then evaluated the extent to which global potato cultivation is at risk. In addition, at a finer scale the risk to the Australian potato acreage was evaluated. A total of 86 MaxEnt models were built using various combinations of settings and climatic predictors, and the best model based on model evaluation metrics was selected. Climatically suitable habitats were identified in Eurasia, Africa, South America, and Australasia. Intersecting the predicted suitability map with land use data showed that 79.06% of the global potato cultivation acreage, 96.14% of the potato production acreage in South America and Eurasia, and all the Australian potato cropping areas are at risk. The information generated by this study increases knowledge of the ecology of TPP/CLso and can be used by government agencies to make decisions about preventing the spread of TPP and CLso across the globe.

scholarly journals Generation of PCV2 in PK15 cells transfected with recombinant baculovirus containing a 1.1 copy of the PCV2 genome

2017 ◽  
Vol 65 (2) ◽  
pp. 278-290 ◽  
Author(s):  
Jie Cai ◽  
Xiaohong Xie ◽  
Yi Hu ◽  
Yang Zhan ◽  
Wanting Yu ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Recombinant Baculovirus ◽  
Porcine Circovirus ◽  
Economic Losses ◽  
Loop Structure ◽  
Swine Industry ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Pcv2 Replication ◽  
Pcv2 Genome

Porcine circovirus associated diseases (PCVAD) caused by PCV2 are responsible for severe economic losses in the swine industry. The mechanism of PCV2 replication has not been fully elucidated yet. PCV2 may be successfully rescued by means of either an infectious DNA clone containing the full length of the viral genomic DNA, or from PCV2-infected clinical tissues in PK15 cell culture. However, viruses harvested by both methods have low titres. In this study, PCV2 was prepared with a higher titre from PK15 cells infected by recombinant baculoviruses containing 1PCV2 (one stem-loop structure) or 1.1PCV2 (two stem-loop structure) genomic DNA copy. In addition, infectious DNA clones containing two stem-loop structures in either plasmid or baculovirus backbones are capable of generating a higher virus titre than the DNA clones with only one copy of stem-loop structure.

scholarly journals Two DNA Methyltransferases for Site-Specific 6mA and 5mC DNA Modification in Xanthomonas euvesicatoria

2021 ◽  
Vol 12 ◽  
Author(s):  
Hye-Jee Park ◽  
Hoon Je Seong ◽  
Jongchan Lee ◽  
Lynn Heo ◽  
Woo Jun Sul ◽  
...  
Keyword(s):  
Single Molecule ◽  
Restriction Enzymes ◽  
Polymyxin B ◽  
Dna Methyltransferases ◽  
Economic Losses ◽  
Biological Processes ◽  
Label Free ◽  
Phytopathogenic Bacterium ◽  
Bacterial Spot Disease ◽  
Xanthomonas Euvesicatoria

Xanthomonas euvesicatoria (Xe) is a gram-negative phytopathogenic bacterium that causes bacterial spot disease in tomato/pepper leading to economic losses in plantations. DNA methyltransferases (MTases) are critical for the survival of prokaryotes; however, their functions in phytopathogenic bacteria remain unclear. In this study, we characterized the functions of two putative DNA MTases, XvDMT1 and XvDMT2, in Xe by generating XvDMT1- and XvDMT2-overexpressing strains, Xe(XvDMT1) and Xe(XvDMT2), respectively. Virulence of Xe(XvDMT2), but not Xe(XvDMT1), on tomato was dramatically reduced. To postulate the biological processes involving XvDMTs, we performed a label-free shotgun comparative proteomic analysis, and results suggest that XvDMT1 and XvDMT2 have distinct roles in Xe. We further characterized the functions of XvDMTs using diverse phenotypic assays. Notably, both Xe(XvDMT1) and Xe(XvDMT2) showed growth retardation in the presence of sucrose and fructose as the sole carbon source, with Xe(XvDMT2) being the most severely affected. In addition, biofilm formation and production of exopolysaccharides were declined in Xe(XvDMT2), but not Xe(XvDMT1). Xe(XvDMT2) was more tolerant to EtOH than Xe(XvDMT1), which had enhanced tolerance to sorbitol but decreased tolerance to polymyxin B. Using single-molecule real-time sequencing and methylation-sensitive restriction enzymes, we successfully predicted putative motifs methylated by XvDMT1 and XvDMT2, which are previously uncharacterized 6mA and 5mC DNA MTases, respectively. This study provided new insights into the biological functions of DNA MTases in prokaryotic organisms.

scholarly journals Genome sequence of Dichelobacter nodosus JKS-07B isolate from J&K, India associated with virulent footrot of sheep

2021 ◽  
Vol 104 (4) ◽  
pp. 003685042110576
Author(s):  
Sabia Qureshi ◽  
Shakil A Wani ◽  
Shaheen Farooq ◽  
Zahid Kashoo ◽  
Basharat Bhat ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Gc Content ◽  
Economic Losses ◽  
Illumina Hiseq ◽  
Protein Coding ◽  
Growth And Survival ◽  
Dichelobacter Nodosus ◽  
New Genes ◽  
Potential Vaccine ◽  
Efficacious Vaccine

Introduction: Virulent footrot of sheep caused by Dichelobacter nodosus is associated with tremendous economic losses due to recurrent treatment costs and increased culling rates. This organism being a fastidious anaerobe is difficult to isolate on ordinary media that does not support its growth. The D. nodosus serogroup B isolate described in the present study has been used in the preparation of the whole-cell killed vaccine against footrot in India. D. nodosus serogroup B is the predominant serogroup involved in virulent footrot (lesion score 4) in India as well as in many sheep-rearing countries of the globe. Methods: Genomic DNA was extracted using wizard Genomic DNA purification kit. The whole genome of the D. nodosus strain B was sequenced using an Illumina HiSeq 2500 platform and annotated according to functional gene categories. Annotations were performed using in-house developed Perl scripts using Nr/Nt database, uniprot, Pfam, KEGG, Panther DB, and GO database. Result: The assembled genome size is 1.311,533 Mb and GC content is 44.38. A total of 1215 protein-coding genes, 44tRNA and 7 rRNA were identified. The genome shows 98.63% sequence homology with the reference genome. However, 21 new genes have been identified in this genome. The information will provide insights into the various genes and regulators necessary for D. nodosus growth and survival. Discussion: The genome information of this serogroup B of D. nodosus isolate involved in 85–90% cases of virulent footrot of sheep in India provides further insights for improvement of the killed vaccine (B serogroup) developed recently in India. For the development of an efficacious vaccine against virulent footrot, it is essential to know the serological diversity as well as the virulent status of the strains of the D. nodosus. This serogroup isolate is a potential vaccine candidate to mitigate ovine footrot in India as the majority of virulent footrot cases belong to serogroup B of D. nodosus.

scholarly journals Linear Plasmid in the Genome of Clavibacter michiganensis subsp. sepedonicus

2002 ◽  
Vol 184 (10) ◽  
pp. 2841-2844 ◽  
Author(s):  
Susan E. Brown ◽  
Dennis L. Knudson ◽  
Carol A. Ishimaru
Keyword(s):  
Electric Field ◽  
Plant Pathogen ◽  
Genomic Dna ◽  
Linear Plasmid ◽  
Homogeneous Electric Field ◽  
Clavibacter Michiganensis ◽  
Clavibacter Michiganensis Subsp ◽  
Extrachromosomal Element

ABSTRACT Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen Clavibacter michiganensis subsp. sepedonicus revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid.

scholarly journals Genome Analysis of Phytophthora nicotianae JM01 Provides Insights into Its Pathogenicity Mechanisms

Plants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1620
Author(s):  
Xiao-Long Yuan ◽  
Cheng-Sheng Zhang ◽  
Fan-Yu Kong ◽  
Zhong-Feng Zhang ◽  
Feng-Long Wang
Keyword(s):  
Genome Analysis ◽  
Plant Pathogen ◽  
Gc Content ◽  
Gene Families ◽  
Phytophthora Nicotianae ◽  
Glycoside Hydrolases ◽  
Economic Losses ◽  
Whole Genome ◽  
Pathogenic Mechanisms ◽  
A Genome

Phytophthora nicotianae is a widely distributed plant pathogen that can cause serious disease and cause significant economic losses to various crops, including tomatoes, tobacco, onions, and strawberries. To understand its pathogenic mechanisms and explore strategies for controlling diseases caused by this pathogen, we sequenced and analyzed the whole genome of Ph. nicotianae JM01. The Ph. nicotianae JM01 genome was assembled using a combination of approaches including shotgun sequencing, single-molecule sequencing, and the Hi-C technique. The assembled Ph. nicotianae JM01 genome is about 95.32 Mb, with contig and scaffold N50 54.23 kb and 113.15 kb, respectively. The average GC content of the whole-genome is about 49.02%, encoding 23,275 genes. In addition, we identified 19.15% of interspersed elements and 0.95% of tandem elements in the whole genome. A genome-wide phylogenetic tree indicated that Phytophthora diverged from Pythium approximately 156.32 Ma. Meanwhile, we found that 252 and 285 gene families showed expansion and contraction in Phytophthora when compared to gene families in Pythium. To determine the pathogenic mechanisms Ph. nicotianae JM01, we analyzed a suite of proteins involved in plant–pathogen interactions. The results revealed that gene duplication contributed to the expansion of Cell Wall Degrading Enzymes (CWDEs) such as glycoside hydrolases, and effectors such as Arg-Xaa-Leu-Arg (RXLR) effectors. In addition, transient expression was performed on Nicotiana benthamiana by infiltrating with Agrobacterium tumefaciens cells containing a cysteine-rich (SCR) protein. The results indicated that SCR can cause symptoms of hypersensitive response. Moreover, we also conducted comparative genome analysis among four Ph. nicotianae genomes. The completion of the Ph. nicotianae JM01 genome can not only help us understand its genomic characteristics, but also help us discover genes involved in infection and then help us understand its pathogenic mechanisms.

scholarly journals CRISPR-Cas systems in the plant pathogen Xanthomonas spp. and their impact on genome plasticity

2019 ◽  
Author(s):  
Paula Maria Moreira Martins ◽  
Andre da Silva Xavier ◽  
Marco Aurelio Takita ◽  
Poliane Alfemas-Zerbini ◽  
Alessandra Alves de Souza
Keyword(s):  
Phylogenetic Analysis ◽  
Plant Pathogen ◽  
Significant Variation ◽  
Crop Production ◽  
Plant Pathogens ◽  
Economic Losses ◽  
Genome Plasticity ◽  
Plant Pathogen Interactions ◽  
Cas Proteins

AbstractXanthomonas is one of the most important bacterial genera of plant pathogens causing economic losses in crop production worldwide. Despite its importance, many aspects of basic Xanthomonas biology remain unknown or understudied. Here, we present the first genus-wide analysis of CRISPR-Cas in Xanthomonas and describe specific aspects of its occurrence. Our results show that Xanthomonas genomes harbour subtype I-C and I-F CRISPR-Cas systems and that species belonging to distantly Xanthomonas-related genera in Xanthomonadaceae exhibit the same configuration of coexistence of the I-C and I-F CRISPR subtypes. Additionally, phylogenetic analysis using Cas proteins indicated that the CRISPR systems present in Xanthomonas spp. are the result of an ancient acquisition. Despite the close phylogeny of these systems, they present significant variation in both the number and targets of spacers. An interesting characteristic observed in this study was that the identified plasmid-targeting spacers were always driven toward plasmids found in other Xanthomonas strains, indicating that CRISPR-Cas systems could be very effective in coping with plasmidial infections. Since many effectors are plasmid encoded, CRISPR-Cas might be driving specific characteristics of plant-pathogen interactions.

scholarly journals Comparative Proteomic Analysis of Plant–Pathogen Interactions in Resistant and Susceptible Poplar Ecotypes Infected with Botryosphaeria dothidea

2019 ◽  
Vol 109 (12) ◽  
pp. 2009-2021 ◽  
Author(s):  
Yongxia Li ◽  
Yuqian Feng ◽  
Quan Lü ◽  
Donghui Yan ◽  
Zhenyu Liu ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Plant Pathogen ◽  
Molecular Mechanisms ◽  
Triosephosphate Isomerase ◽  
Hydrolytic Enzymes ◽  
Economic Losses ◽  
Botryosphaeria Dothidea ◽  
Bisphosphate Carboxylase ◽  
Populus Tomentosa Carr ◽  
Plant Pathogen Interactions

Poplar are important forestry species in China, but the Botryosphaeria dothidea pathogen causes serious economic losses worldwide. To identify candidate B. dothidea resistance proteins and explore the molecular mechanisms involved in poplar–pathogen interactions, proteomic responses of stem samples from resistant and susceptible poplar ecotypes to B. dothidea were investigated using nanoflow liquid chromatography-tandem mass spectrometry with label-free quantitative analysis. We identified 588 proteins, divided into 21 biological process categories including 48 oxidoreductases, 72 hydrolytic enzymes, 80 metabolic enzymes, and 29 proteins of unknown function. Differential proteome analysis revealed large differences between resistant Populus tomentosa Carr and susceptible Populus beijingensis Hsu ecotypes before and after inoculation. Among 102 identified proteins, 22 were highly upregulated in the resistant genotype but downregulated in the susceptible genotype. Proteins induced in P. tomentosa Carr in response to B. dothidea are associated with plant defenses including oxidoreductase activity (catalase, isocitrate dehydrogenase, and superoxide dismutase), phenylpropanoid biosynthesis and phenylalanine metabolism (alcohol dehydrogenase), photosynthesis (ATP synthase subunit alpha, ATP synthase gamma chain, photosystem I P700 chlorophyll a apoprotein A2, photosystem II CP47 chlorophyll apoprotein), carbon fixation (pyruvate kinase, triosephosphate isomerase, malic enzyme, phosphoglycerate kinase, ribulose-1,5-bisphosphate carboxylase, and ribulose bisphosphate carboxylase small chain), and glycolysis/gluconeogenesis (fructose-bisphosphate aldolase). Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 168 proteins related to metabolic pathways, 41 proteins related to the biosynthesis of phenylpropanoids, and 36 proteins related to the biosynthesis of plant hormones, the biosynthesis of alkaloids derived from ornithine, lysine, and nicotinic acid, and photosynthesis in response to B. dothidea. Our findings provide insight into plant–pathogen interactions in resistant and susceptible poplar ecotypes infected with B. dothidea and could assist the development of novel strategies for fighting poplar canker disease.

scholarly journals 2-Amino-3-(Oxirane-2,3-Dicarboxamido)-Propanoyl-Valine, an Effective Peptide Antibiotic from the Epiphyte Pantoea agglomerans 48b/90

2009 ◽  
Vol 75 (24) ◽  
pp. 7710-7717 ◽  
Author(s):  
Ulrike F. Sammer ◽  
Beate Völksch ◽  
Ute Möllmann ◽  
Michaela Schmidtke ◽  
Peter Spiteller ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Pathogen ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Pantoea Agglomerans ◽  
Economic Losses ◽  
Peptide Antibiotic ◽  
Ionization Mass ◽  
Serratia Plymuthica ◽  
Phytopathogenic Bacteria

ABSTRACT The epiphyte Pantoea agglomerans 48b/90, which has been isolated from soybean leaves, belongs to the Enterobacteriaceae, as does the plant pathogen Erwinia amylovora, which causes fire blight on rosaceous plants such as apples and leads to severe economic losses. Since P. agglomerans efficiently antagonizes phytopathogenic bacteria, the P. agglomerans strain C9-1 is used as a biocontrol agent (BlightBan C9-1). Here we describe the bioassay-guided isolation of a peptide antibiotic that is highly active against the plant pathogen E. amylovora and pathovars of Pseudomonas syringae, and we elucidate its structure. Bioassay-guided fractionation using anion-exchange chromatography followed by hydrophobic interaction liquid chromatography yielded the bioactive, highly polar antibiotic. The compound was identified as 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine by using high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance techniques. This peptide was found to be produced by three of the nine P. agglomerans strains analyzed. Notably, the biocontrol strain P. agglomerans C9-1 also produces 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine. Previously, 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine has been characterized only from Serratia plymuthica. 2-Amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine has been shown to inhibit the growth of the human pathogen Candida albicans efficiently, but its involvement in the defense of epiphytes against phytopathogenic bacteria has not been investigated so far.

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