Transcriptome analysis unravels the biocontrol mechanism of Serratia plymuthica A30 against potato soft rot caused by Dickeya solani.

Endophytic bacterium Serratia plymuthica A30 was identified as a superior biocontrol agent due to its effective colonization of potato tuber, tolerance to cold conditions, and strong inhibitory action against various soft rot pathogens, including Dickeya solani. We characterized transcriptome changes in potato tubers inoculated with S. plymuthica A30, D. solani, or both at the early and the late phases of interaction. At the early phase and in the absence of the pathogen, A30 influenced the microbial recognition system to initiate plant priming. In the presence of the pathogen alongside biocontrol strain, defense signaling was highly stimulated, characterized by the induction of genes involved in the detoxification system, reinforcement of cell wall structure, and production of antimicrobial metabolites, highlighting A30's role in enhancing the host resistance against pathogen attack. This A30-induced resistance relied on the early activation of jasmonic acid signaling and its production in tubers, while defense signaling mediated by salicylic acid was suppressed. In the late phase, A30 actively interferes with plant immunity by inhibiting stress- and defense-related genes expression. Simultaneously, the genes involved in cell wall remodeling and indole-3-acetic acid signaling were activated, thereby enhancing cell wall remodeling to establish symbiotic relationship with the host. The endophytic colonization of A30 coincided with the induction of genes involved in the biosynthesis and signaling of ethylene and abscisic acid, while downregulating those related to gibberellic acid and cytokinin. This combination suggested fitness benefits for potato tubers by preserving dormancy, and delaying sprouting, which affects durability of tubers during storage. This study contributes valuable insights into the tripartite interaction among S. plymuthica A30, D. solani, and potato tubers, facilitating the development of biocontrol system for soft rot pathogens under storage conditions.

Differences in the constituents of bacterial microbiota of soils collected from two fields of diverse potato blackleg and soft rot diseases incidences, a case study.

The presence of bacteria from the Dickeya spp. and Pectobacterium spp. in farmlands leads to global crop losses of over $420 million annually. Since 1982, the scientists have started to suspect that the development of disease symptoms in crops might be inhibited by bacteria present in the soil. Here, we characterized in terms of physicochemical properties and the composition of bacterial soil microbiota two fields differing, on the basis of long-term studies, in the occurrence of Dickeya spp.- and Pectobacterium spp.-triggered infections. Majority, i.e. 17 of the investigated physicochemical features of the soils collected from two fields of either low or high potato blackleg and soft rot diseases incidences turned out to be similar, in contrast to the observed 4 deviations in relation to Mg, Mn, organic C and organic substance contents. By performing microbial cultures and molecular diagnostics-based identification, 20 Pectobacterium spp. strains were acquired from the field showing high blackleg and soft rot incidences. In addition, 16S rRNA gene amplicon sequencing followed by bioinformatic analysis revealed differences at various taxonomic levels in the soil bacterial microbiota of the studied fields. We observed that bacteria from the genera Bacillus, Rumeliibacillus, Acidobacterium and Gaiella turned out to be more abundant in the soil samples originating from the field of low comparing to high frequency of pectinolytic bacterial infections. In the herein presented case study, it is shown for the first time that the composition of bacterial soil microbiota varies between two fields differing in the incidences of soft rot and blackleg infections.

Genetic Loci of Plant Pathogenic Dickeya solani IPO 2222 Expressed in Contact with Weed-Host Bittersweet Nightshade (Solanum dulcamara L.) Plants.

Dickeya solani, belonging to the Soft Rot Pectobacteriaceae, are aggressive necrotrophs, exhibiting both a wide geographic distribution and a wide host range that includes many angiosperm orders, both dicot and monocot plants, cultivated under all climatic conditions. Little is known about the infection strategies D. solani employs to infect hosts other than potato (Solanum tuberosum L.). Our earlier study identified D. solani Tn5 mutants induced exclusively by the presence of the weed host S. dulcamara. The current study assessed the identity and virulence contribution of the selected genes mutated by the Tn5 insertions and induced by the presence of S. dulcamara. These genes encode proteins with functions linked to polyketide antibiotics and polysaccharide synthesis, membrane transport, stress response, and sugar and amino acid metabolism. Eight of these genes, encoding UvrY (GacA), tRNA guanosine transglycosylase Tgt, LPS-related WbeA, capsular biosynthesis protein VpsM, DltB alanine export protein, glycosyltransferase, putative transcription regulator YheO/PAS domain-containing protein, and a hypothetical protein, were required for virulence on S. dulcamara plants. The implications of D. solani interaction with a weed host, S. dulcamara, are discussed.

Combining recombinase polymerase amplification with tyrosine modified 2'-deoxyuridine-5'-triphosphate for direct voltammetric detection of double-stranded DNA: Application to potato pathogen Dickeya solani.

The approach based on a combination of isothermal recombinase polymerase amplification (RPA), 2'-deoxyuridine-5'-triphosphate modified with tyrosine aromatic group (dUTP-Y1), and direct voltammetric detection of RPA product carrying electroactive labels was successfully applied to the potato pathogen Dickeya solani. The artificial nucleotide dUTP-Y1 demonstrated a good compatibility with RPA, enabling by targeting a section of D. solani genome with a unique sequence to produce the full-size modified products at high levels of substitution of dTTP by dUTP-Y1 (up to 80-90 %) in the reaction mixture. The optimized procedure of square wave voltammetry allowed to reliably detect the product generated by RPA at 80 % substitution of dTTP by dUTP-Y1 (dsDNA-Y1) in microliter sample volumes on the surface of disposable carbon screen printed electrodes at the potential of about 0.6 V. The calibration curve for the amplicon detection was linear in coordinates 'Ip, A vs. Log (c, M)' within the 0.05-1 µM concentration range. The limit of detection for dsDNA-Y1 was estimated as 8 nM. The sensitivity of the established electrochemical approach allowed to detect amplicons generated in a single standard 50 µL RPA reaction after their purification with silica-coated magnetic beads. The overall detectability of D. solani with the suggested combination of RPA and voltammetric registration of dsDNA-Y1 can be as low as a few copies of bacterial genome per standard reaction. In total, amplification, purification, and electrochemical detection take about 120-150 min. Considering the potential of direct electrochemical analysis for miniaturization, as well as compliance with low-cost and low-power requirements, the findings provide grounds for future development of microfluidic devices integrating isothermal amplification, amplicon purification and detection based on the tyrosine modified nucleotide for the purpose of 'on-site' detection of various pathogens.

Surviving the Potato Stems: Differences in Genes Required for Fitness by Dickeya dadantii and Dickeya dianthicola.

Bacteria belonging to the genus Dickeya cause blackleg and soft rot symptoms on many plant hosts, including potato. Although there is considerable knowledge about the genetic determinants that allow Dickeya to colonize host plants, as well as the genes that contribute to virulence, much is still unknown. To identify the genes important for fitness in potato stems, we constructed and evaluated randomly barcoded transposon mutant (RB-TnSeq) libraries of Dickeya dadantii and Dickeya dianthicola. We identified 169 and 157 genes important for growth in D. dadantii and D. dianthicola in stems, respectively. This included genes related to metabolic pathways, chemotaxis and motility, transcriptional regulation, transport across membranes, membrane biogenesis, detoxification mechanisms, and virulence-related genes, including a potential virulence cluster srfABC, c-di-GMP modulating genes, and pectin degradation genes. When we compared the results of the stem assay with other datasets, we identified genes important for growth in stems versus tubers and in vitro conditions. Additionally, our data showed differences in fitness determinants for D. dadantii and D. dianthicola. These data provide important insights into the mechanisms used by Dickeya when interacting with and colonizing plants and thus might provide targets for management.

Impact of climate change on potential distribution of Dickeya zeae causal agent of stalk rot of maize in Sialkot district Pakistan.

Maize (Zea mays) is an influential crop in its production across the world. However, the invasion of many phytopathogens greatly affects the maize crop yield at various hotspot areas. Of many diseases, bacterial stalk rot of maize caused by Dickeya zeae results in severe yield reduction, thus the need for efficient management is important. Further, to produce epidemiological information for control of disease outbreaks in the hot spot regions of Sialkot District, Punjab Pakistan, extensive field surveys during 2021 showed that out of 266 visited areas, the highest disease incidence ranging from 66.5 to 78.5% while the lowest incidence was ranging from 9 to 20%. The Maxent modeling revealed that among 19 environmental variables, four variables including temperature seasonality (bio-4), mean temperature of the wettest quarter (bio-8), annual precipitation (bio-12), and precipitation of driest month (bio-14) were significantly contributing to disease distribution in current and coming years. The study outcomes revealed that disease spread will likely increase across four tehsils of Sialkot over the years 2050 and 2070. Our findings will be helpful to policymakers and researchers in devising effective disease management strategies against bacterial stalk rot of maize outbreaks in Sialkot, Pakistan.

Svx Peptidases of Phytopathogenic Pectolytic Bacteria: Structural, Catalytic and Phytoimmune Properties.

Svx proteins are virulence factors secreted by phytopathogenic bacteria of the Pectobacterium genus into the host plant cell wall. Svx-encoding genes are present in almost all species of the soft rot Pectobacteriaceae (Pectobacterium and Dickeya genera). The Svx of P. atrosepticum (Pba) has been shown to be a gluzincin metallopeptidase that presumably targets plant extensins, proteins that contribute to plant cell wall rigidity and participate in cell signaling. However, the particular "output" of the Pba Svx action in terms of plant-pathogen interactions and plant immune responses remained unknown. The Svx proteins are largely unexplored in Dickeya species, even though some of them have genes encoding two Svx homologs. Therefore, our study aims to compare the structural and catalytic properties of the Svx proteins of Pba and D. solani (Dso) and to test the phytoimmune properties of these proteins. Two assayed Dso Svx proteins, similar to Pba Svx, were gluzincin metallopeptidases with conservative tertiary structures. The two domains of the Svx proteins form electronegative clefts where the active centers of the peptidase domains are located. All three assayed Svx proteins possessed phytoimmunosuppressory properties and induced ethylene-mediated plant susceptible responses that play a decisive role in Pba-caused disease.

Reproducible Quantitative Trait Loci for Resistance to Soft Rot Caused by Dickeya dianthicola Derived from the Wild Potato Solanum microdontum (PI 458355) Are Located on Chromosomes 1, 3, and 5.

The potato wild relative Solanum microdontum is a breeder-friendly source of genetic resistance to soft rot. Our objectives were to (i) identify loci associated with soft rot resistance in S. microdontum germplasm and (ii) develop bi-parental populations in a self-compatible S. tuberosum genetic background to recover segregating F2 progenies, construct a linkage map, and identify quantitative trait loci (QTLs). Under objective (i), tubers from 103 S. microdontum genotypes from the United States Potato Genebank were inoculated with a high virulence strain of Dickeya dianthicola, and lesion size was measured after a 24-h incubation period at 30°C. Association analysis using 3,490 polymorphic Infinium array SNP markers identified soft rot resistance loci on chromosomes 1, 2, 3, 5, 7, 8, 11, and 12. Under objective (ii), a resistant S. microdontum accession PI 458355 was crossed with a highly fertile, self-compatible, diploid S. tuberosum pollen parent (PI 654351) to generate segregating F2 populations. Composite interval mapping was conducted using a genetic linkage map with 970 GBS-based SNP markers. Reproducible QTLs were detected on chromosomes 1, 3, and 5, explaining 11, 13, and 23% of the phenotypic variation, respectively. Homozygous S. microdontum alleles at the QTL on chromosome 5 and heterozygous or homozygous S. microdontum alleles at QTLs on chromosomes 1 and 3 significantly decrease lesion size compared with the homozygous S. tuberosum parent. The germplasm created in these studies provides a resource for studying traits from S. microdontum, and we can use the advanced F2 selections for future potato improvement. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Multilocus sequence and phenotypic analysis of Pectobacterium and Dickeya type strains for identification of soft rot Pectobacteriaceae from symptomatic potato stems and tubers in Pennsylvania.

Outbreaks of potato blackleg and soft rot caused by Pectobacterium species and more recently Dickeya species across the U.S. mid-Atlantic region have caused yield loss due to poor emergence as well as losses from stem and tuber rot. To develop management strategies for soft rot diseases, we must first identify which members of the soft rot Pectobacteriaceae are present in regional potato plantings. However, the rapidly expanding number of soft rot Pectobacteriaceae species and the lack of readily available comparative data for type strains of Pectobacterium and Dickeya hinder quick identification. This manuscript provides a comparative analysis of soft rot Pectobacteriaceae and a comprehensive comparison of type strains from this group using rep-PCR, MLSA and 16S sequence analysis, as well as phenotypic and physiological analyses using Biolog GEN III plates. These data were used to identify isolates cultured from symptomatic potato stems collected between 2016 and 2018. The isolates were characterized for phenotypic traits and by sequence analysis to identify the bacteria from potatoes with blackleg and soft rot symptoms in Pennsylvania potato fields. In this survey, P. actinidiae, P. brasiliense, P. polonicum, P. polaris, P. punjabense, P. parmentieri, and P. versatile were identified from Pennsylvania for the first time. Importantly, the presence of P. actinidiae in Pennsylvania represents the first report of this organism in the U.S. As expected, P. carotorvorum and D. dianthicola were also isolated. In addition to a resource for future work studying the Dickeya and Pectobacterium associated with potato blackleg and soft rot, we provide recommendations for future surveys to monitor for quarantine or emerging soft rot Pectobacteriace regionally.

Clonality and Diversity in the Soft Rot Dickeya solani Phytopathogen.

Bacterial diversity analyses often suffer from a bias due to sampling only from a limited number of hosts or narrow geographic locations. This was the case for the phytopathogenic species Dickeya solani, whose members were mainly isolated from a few hosts-potato and ornamentals-and from the same geographical area-Europe and Israel, which are connected by seed trade. Most D. solani members were clonal with the notable exception of the potato isolate RNS05.1.2A and two related strains that are clearly distinct from other D. solani genomes. To investigate if D. solani genomic diversity might be broadened by analysis of strains isolated from other environments, we analysed new strains isolated from ornamentals and from river water as well as strain CFBP 5647 isolated from tomato in the Caribbean island Guadeloupe. While water strains were clonal to RNS05.1.2A, the Caribbean tomato strain formed a third clade. The genomes of the three clades are highly syntenic; they shared almost 3900 protein families, and clade-specific genes were mainly included in genomic islands of extrachromosomal origin. Our study thus revealed both broader D. solani diversity with the characterisation of a third clade isolated in Latin America and a very high genomic conservation between clade members.

Phyloproteomic study by MALDI-TOF MS in view of intraspecies variation in a significant homogenous phytopathogen Dickeya solani.

Dickeya solani is an economically significant pectinolytic phytopathogen belonging to the Pectobacteriaceae family, which causes soft rot and blackleg diseases. Despite its notable impact on global potato production, there are no effective methods to control this pest. Here, we undertook a phyloproteomic study on 20 D. solani strains, of various origin and year of isolation, with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) supported by an in-depth characterization of the strains in terms of the virulence-associated phenotype. In spite of high homogeneity in this species, we herein revealed for the first time intraspecies variation in the MALDI-TOF MS protein profiles among the studied D. solani isolates. Finally, representative mass spectra for the four delineated clades are presented. A majority of the analysed D. solani strains showed high virulence potential, while two strains stood out in their growth dynamics, virulence factors production and ability to macerate plant tissue. Nonetheless, the metabolic profiles of D. solani strains turned out to be uniform, except for gelatinase activity. Given that all D. solani isolates distinctly grouped from the other Dickeya species in the MALDI-TOF MS analysis, there is strong evidence supporting the potential routine use of this method for fast and reliable to-species identification of D. solani isolates of environmental origin.

Structure-Function Characterisation of Eop1 Effectors from the Erwinia-Pantoea Clade Reveals They May Acetylate Their Defence Target through a Catalytic Dyad.

The YopJ group of acetylating effectors from phytopathogens of the genera Pseudomonas and Ralstonia have been widely studied to understand how they modify and suppress their host defence targets. In contrast, studies on a related group of effectors, the Eop1 group, lag far behind. Members of the Eop1 group are widely present in the Erwinia-Pantoea clade of Gram-negative bacteria, which contains phytopathogens, non-pathogens and potential biocontrol agents, implying that they may play an important role in agroecological or pathological adaptations. The lack of research in this group of YopJ effectors has left a significant knowledge gap in their functioning and role. For the first time, we perform a comparative analysis combining AlphaFold modelling, in planta transient expressions and targeted mutational analyses of the Eop1 group effectors from the Erwinia-Pantoea clade, to help elucidate their likely activity and mechanism(s). This integrated study revealed several new findings, including putative binding sites for inositol hexakisphosphate and acetyl coenzyme A and newly postulated target-binding domains, and raises questions about whether these effectors function through a catalytic triad mechanism. The results imply that some Eop1s may use a catalytic dyad acetylation mechanism that we found could be promoted by the electronegative environment around the active site.

Pseudomonas chlororaphis L5 and Enterobacter asburiae L95 biocontrol Dickeya soft rot diseases by quenching virulence factor modulating quorum sensing signal.

Virulence factor modulating (VFM) is a quorum sensing (QS) signal shared by and specific to Dickeya bacteria, regulating the production of plant cell wall degrading enzymes (PCWDEs) and virulence of Dickeya. High polarity and trace of VFM signal increase the difficulty of signal separation and structure identification, and thus limit the development of quorum quenching strategy to biocontrol bacterial soft rot diseases caused by Dickeya. In order to high-throughput screen VFM quenching bacteria, a vfmE-gfp biosensor VR2 (VFM Reporter) sensitive to VFM signal was first constructed. Subsequently, two bacterial strains with high quenching efficiency were screened out by fluorescence intensity measurement and identified as Pseudomonas chlororaphis L5 and Enterobacter asburiae L95 using multilocus sequence analysis (MLSA). L5 and L95 supernatants reduced the expression of vfm genes, and both strains also decreased the production of PCWDEs of D. zeae MS2 and significantly reduced the virulence of D. oryzae EC1 on rice seedlings, D. zeae MS2 on banana seedlings, D. dadantii 3937 on potato and D. fangzhongdai CL3 on taro. Findings in this study provide a method to high-throughput screen VFM quenching bacteria and characterize novel functions of P. chlororaphis and E. asburiae in biocontrolling plant diseases through quenching VFM QS signal.

Pseudomonas PA14H7: Identification and Quantification of the 7-Hydroxytropolone Iron Complex as an Active Metabolite against Dickeya, the Causal Agent of Blackleg on the Potato Plant.

Soft rot Pectobacteriaceae (SRP), such as Pectobacterium and Dickeya, are phytopathogenic agents responsible for blackleg disease on several crops, such as potatoes, affecting the yield and depressing the seed production quality. However, neither conventional nor biocontrol products are available on the market to control this disease. In this study Pseudomonas PA14H7, a bacteria isolated from potato rhizosphere, was selected as a potential antagonist agent against Dickeya solani. In order to understand the mechanism involved in this antagonism, we managed to identify the main active molecule(s) produced by PA14H7. Cell-free supernatant (CFS) of PA14H7 cultures were extracted and analyzed using LC-MS, GC-MS, and NMR. We further correlated the biological activity against Dickeya solani of extracted CFS-PA14H7 to the presence of 7-hydroxytropolone (7-HT) complexed with iron. In a second time, we have synthesized this molecule and determined accurately using LC-UV, LC-MS, and GC-MS that, after 48 h incubation, PA14H7 released, in its CFS, around 9 mg/L of 7-HT. The biological activities of CFS-PA14H7 vs. synthetic 7-HT, at this concentration, were evaluated to have a similar bacteriostatic effect on the growth of Dickeya solani. Even if 7-HT is produced by other Pseudomonas species and is mostly known for its antibacterial and antifungal activities, this is the first description of its involvement as an effective molecule against pectinolytic bacteria. Our work opens the way for the comprehension of the mode of action of PA14H7 as a biocontrol agent against potato blackleg.

Fast genome-based delimitation of Enterobacterales species.

Average Nucleotide Identity (ANI) is becoming a standard measure for bacterial species delimitation. However, its calculation can take orders of magnitude longer than similarity estimates based on sampling of short nucleotides, compiled into so-called sketches. These estimates are widely used. However, their variable correlation with ANI has suggested that they might not be as accurate. For a where-the-rubber-meets-the-road assessment, we compared two sketching programs, mash and dashing, against ANI, in delimiting species among Esterobacterales genomes. Receiver Operating Characteristic (ROC) analysis found Area Under the Curve (AUC) values of 0.99, almost perfect species discrimination for all three measures. Subsampling to avoid over-represented species reduced these AUC values to 0.92, still highly accurate. Focused tests with ten genera, each represented by more than three species, also showed almost identical results for all methods. Shigella showed the lowest AUC values (0.68), followed by Citrobacter (0.80). All other genera, Dickeya, Enterobacter, Escherichia, Klebsiella, Pectobacterium, Proteus, Providencia and Yersinia, produced AUC values above 0.90. The species delimitation thresholds varied, with species distance ranges in a few genera overlapping the genus ranges of other genera. Mash was able to separate the E. coli + Shigella complex into 25 apparent phylogroups, four of them corresponding, roughly, to the four Shigella species represented in the data. Our results suggest that fast estimates of genome similarity are as good as ANI for species delimitation. Therefore, these estimates might suffice for covering the role of genomic similarity in bacterial taxonomy, and should increase confidence in their use for efficient bacterial identification and clustering, from epidemiological to genome-based detection of potential contaminants in farming and industry settings.

Characterization of the Arn lipopolysaccharide modification system essential for zeamine resistance unveils its new roles in Dickeya oryzae physiology and virulence.

The zeamines produced by Dickeya oryzae are potent polyamine antibiotics and phytotoxins that are essential for bacterial virulence. We recently showed that the RND efflux pump DesABC in D. oryzae confers partial resistance to zeamines. To fully elucidate the bacterial self-protection mechanisms, in this study we used transposon mutagenesis to identify the genes encoding proteins involved in zeamine resistance in D. oryzae EC1. This led to the identification of a seven-gene operon, arnEC1 , that encodes enzyme homologues associated with lipopolysaccharide modification. Deletion of the arnEC1 genes in strain EC1 compromised its zeamine resistance 8- to 16-fold. Further deletion of the des gene in the arnEC1 mutant background reduced zeamine resistance to a level similar to that of the zeamine-sensitive Escherichia coli DH5α. Intriguingly, the arnEC1 mutants showed varied bacterial virulence on rice, potato, and Chinese cabbage. Further analyses demonstrated that ArnBCATEC1 are involved in maintenance of the bacterial nonmucoid morphotype by repressing the expression of capsular polysaccharide genes and that ArnBEC1 is a bacterial virulence determinant, influencing transcriptional expression of over 650 genes and playing a key role in modulating bacterial motility and virulence. Taken together, these findings decipher a novel zeamine resistance mechanism in D. oryzae and document new roles of the Arn enzymes in modulation of bacterial physiology and virulence.

A natural single nucleotide mutation in the small regulatory RNA ArcZ of Dickeya solani switches off the antimicrobial activities against yeast and bacteria.

The necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene clusters. Analogy with genes described in other bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster named sol was recently shown to produce an antifungal molecule. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters sol, ooc, and zms to compare first the phenotype of the D. solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The cluster sol, conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D. solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters sol and zms. A single-point mutation, conserved in some Dickeya wild-type strains, including the D. solani type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form.

Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region.

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18-20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Comparative Pathogenomic Analysis of Two Banana Pathogenic Dickeya Strains Isolated from China and the Philippines.

Dickeya is a major and typical member of soft rot Pectobacteriaceae (SRP) with a wide range of plant hosts worldwide. Previous studies have identified D. zeae as the causal agent of banana soft rot disease in China. In 2017, we obtained banana soft rot pathogen strain FZ06 from the Philippines. Genome sequencing and analysis indicated that FZ06 can be classified as D. dadantii and represents a novel subspecies of D. dadantii, which we propose to name as subsp. paradisiaca. Compared with Chinese banana soft rot pathogenic strain D. zeae MS2, strain FZ06 has a similar host range but different virulence; FZ06 is significantly less virulent to banana and potato but more virulent to Chinese cabbage and onion. Characterization of virulence factors revealed obviously less production of pectate lyases (Pels), polygalacturonases (Pehs), proteases (Prts), and extrapolysaccharides (EPSs), as well as lower swimming and swarming motility and biofilm formation in strain FZ06. Genomic comparison of the two strains revealed five extra gene clusters in FZ06, including one Stt-type T2SS, three T4SSs, and one T4P. Expression of cell wall degrading enzyme (CWDE)-encoding genes is significantly lower in FZ06 than in MS2.