Identification of Bacterial Wilt (Erwinia tracheiphila) Resistances in USDA Melon Collection

Bacterial wilt (BW) caused by the Gram-negative bacterium, Erwinia tracheiphila (Et.), is an important disease in melon (Cucumis melo L.). BW-resistant commercial melon varieties are not widely available. There are also no effective pathogen-based disease management strategies as BW-infected plants ultimately die. The purpose of this study is to identify BW-resistant melon accessions in the United States Department of Agriculture (USDA) collection. We tested 118 melon accessions in two inoculation trials under controlled environments. Four-week-old seedlings of test materials were mechanically inoculated with the fluorescently (GFP) labeled or unlabeled E. tracheiphila strain, Hca1-5N. We recorded the number of days to wilting of inoculated leaf (DWIL), days to wilting of whole plant (DWWP) and days to death of the plant (DDP). We identified four melon lines with high resistance to BW inoculation based on all three parameters. Fluorescent microscopy was used to visualize the host colonization dynamics of labeled bacteria from the point of inoculation into petioles, stem and roots in resistant and susceptible melon accessions, which provides an insight into possible mechanisms of BW resistance in melon. The resistant melon lines identified from this study could be valuable resistance sources for breeding of BW resistance as well as the study of cucurbit—E. tracheiphila interactions.

The contributions to virulence of the effectors Eop1 and DspE differ between two clades of Erwinia tracheiphila strains

Strains of Erwinia tracheiphila, causal agent of bacterial wilt of cucurbits, are divided into distinct clades. Et-melo clade strains wilt Cucumis spp. but not Cucurbita spp., thus exhibiting host specificity, whereas Et-C1 clade strains wilt Cucurbita spp. more rapidly than Cucumis melo, thus exhibiting a host preference. This study investigated the contribution of the effector proteins Eop1 and DspE to E. tracheiphila pathogenicity and host adaptation. Loss of eop1 did not enable Et-melo strains to infect squash (Cucurbita pepo) or an Et-C1 strain to induce a more rapid wilt of muskmelon (Cucumis melo), indicating that Eop1 did not function in host specificity or preference as in the related pathogen Erwinia amylovora. However, over-expression of eop1 from Et-melo strain MDCuke, but not from Et-C1 strain BHKY, increased the virulence of a BHKY eop1 deletion mutant on muskmelon, demonstrating that the Eop1 variants in the two clades are distinct in their virulence functions. Loss of dspE from Et-melo strains reduced but did not eliminate virulence on hosts muskmelon and cucumber, whereas loss of dspE from an Et-C1 strain eliminated pathogenicity on hosts squash, muskmelon and cucumber. Thus, the centrality of DspE to virulence differs in the two clades. Et-melo mutants lacking the chaperone DspF exhibited similar virulence to mutants lacking DspE, indicating that DspF is the sole chaperone for DspE in E. tracheiphila, unlike in E. amylovora. Collectively, these results provide the first functional evaluation of effectors in E. tracheiphila and demonstrate clade-specific differences in the roles of Eop1 and DspE.

Biomolecular characterization and efficacy evaluation of pathogen Erwinia tracheiphila against plant oils

Aim: To investigate patho-dyanamics of Erwinia tracheiphila on Cucurbita pepo with biochemical, molecular characterization and eco-friendly management approaches. Methodology: Variability study of Erwinia tracheiphila was done by collecting the isolates from different places in Odisha. Disease severity was calculated and analyzed from 2016-18 to predict trend of emergence and disease progress. Molecular detection of pathogen was done through 16S rRNA sequencing. Biochemical characterization and identification of pathogenic nature of Erwinia tracheiphila was performed through Ortho-Nitrophenyl β-Galactoside, Esculin hydrolysis, Citrate and Malonate utilization in the presence of different carbohydrate molecules. Efficacy of eight different plant oils were tested by in-vitro inhibition zone (IVIZ) assay against Erwinia tracheiphila as eco-friendly management approaches. Results: Analysis of disease severity data from 2016-18 ranged between 29% to 33%. Molecular detection of causal pathogen was further confirmed by 16S rRNA region sequencing with 99% identity of NCBI-GenBank published Erwinia tracheiphila (MK356446 and MK356441) database and phylogenetic analysis. The results of IVIZT revealed that the clove oil treatment showed maximum inhibitory effect on the growth of Erwinia tracheiphila followed by mustard oil and neem oil. Sesamum oil showed the lowest efficacy against Erwinia tracheiphila. Optimum temperature range between 25-28°C, 80-85% relative humidity and 150-180 mm average rainfall triggered the disease progress and turned to be a severe one under experimental agro-climatic situation. Interpretation: Biochemical characterization and molecular detection of causal isolate confirmed the pathogen as Erwinia tracheiphila, causing wilt disease of Cucurbita pepo. This study also revealed the potential efficacy of clove oil and mustard oil against Erwinia tracheiphila as compared to other selected plant oils.

Characterization of Erwinia tracheiphila bacteriophage FBB1 isolated from spotted cucumber beetles that vector E. tracheiphila

Erwinia tracheiphila, the causal pathogen of bacterial wilt of cucurbit crops, is disseminated by cucumber beetles. A bacteriophage, designated FBB1, was isolated from spotted cucumber beetles (Diabrotica undecimpunctata) that were collected from a field where E. tracheiphila is endemic. FBB1 was classified into the Myoviridae family based on its morphology, which includes an elongated icosahedral head (106 × 82 nm) and a putatively contractile tail (120 nm). FBB1 infected all 62 E. tracheiphila strains examined and also three Pantoea spp. strains. FBB1 virions were stable at 55°C for 1 h and tolerated a pH range from 3 to 12. FBB1 has a genome of 175,994 bp with 316 predicted coding sequences and a GC content of 36.5%. The genome contains genes for a major bacterial outer-membrane protein, a putative exopolysaccharide depolymerase, and 22 predicted tRNAs. The morphology and genome indicate that FBB1 is a T4-like virus and thus in the Tevenvirinae subfamily. FBB1 is the first virulent phage of E. tracheiphila to be reported, and to date, is one of only two bacteriophages to be isolated from insect vectors of phytopathogens. Collectively, the results support FBB1 as a promising candidate for biocontrol of E. tracheiphila based on its virulent (lytic) rather than lysogenic lifestyle, its infection of all E. tracheiphila strains examined to date, and its infection of a few non-pathogenic bacteria that could be used to support phage populations when pathogen numbers are low.

A horizontally acquired expansin gene increases virulence of the emerging plant pathogen Erwinia tracheiphila

Erwinia tracheiphila is a bacterial plant pathogen that causes a fatal wilt infection in some cucurbit crop plants. Wilt symptoms are thought to be caused by systemic bacterial colonization through xylem that impedes sap flow. However, the genetic determinants of within-plant movement are unknown for this pathogen species. Here, we find that E. tracheiphila has horizontally acquired an operon with a microbial expansin (exlx) gene adjacent to a glycoside hydrolase family 5 (gh5) gene. Plant inoculation experiments with deletion mutants in the individual genes (Δexlx and Δgh5) and the full operon (Δexlx–gh5) resulted in decreased severity of wilt symptoms, decreased mortality rate, and impaired systemic colonization compared to the Wt strain. Co-inoculation experiments with Wt and Δexlx–gh5 rescued the movement defect of the mutant strain, suggesting that expansin and GH5 function extracellularly. Together, these results show that expansin–GH5 contributes to systemic movement through xylem, leading to rapid wilt symptom development and higher rates of plant death. The presence of expansin genes in diverse species of bacterial and fungal wilt-inducing pathogens suggests that microbial expansin proteins may be an under-appreciated virulence factor for many pathogen species.

Draft Genome Sequence Data of Glutamicibacter sp. FBE-19, a Bacterium Antagonistic to the Plant Pathogen Erwinia tracheiphila

Glutamicibacter sp.FBE-19 was isolated based on its strong antagonism to the cucurbit bacterial blight pathogen Erwinia tracheiphila on plates. Members of the Glutamicibacter genus can promote plant growth under saline conditions and antagonize fungi on plates via chitinolytic activity, but their production of antibacterial compounds has not been examined. Here we report the genome sequence of strain FBE-19. The genome is 3.85 Mbp with a G+C content of 60.1% and comprised of 3,791 genes. Genes that may contribute to its antagonistic activity include genes for the secondary metabolites stenothricin, salinosporamide A, a second beta-lactone compound, and a carotenoid. The Glutamicibacter sp. FBE-19 genome data may be a useful resource if this strain proves an effective biocontrol agent against E. tracheiphila.

Discovery and Characterization of Low-Molecular Weight Inhibitors of Erwinia tracheiphila

Plant pathogenic bacteria in the genus Erwinia cause economically important diseases, including bacterial wilt of cucurbits caused by Erwinia tracheiphila. Conventional bactericides are insufficient to control this disease. Using high-throughput screening, 464 small molecules (SMs) with either cidal or static activity at 100 µM against a cucumber strain of E. tracheiphila were identified. Among them, 20 SMs (SM1 to SM20), composed of nine distinct chemical moiety structures, were cidal to multiple E. tracheiphila strains at 100 µM. These lead SMs had low toxicity to human cells and honey bees at 100 µM. No phytotoxicity was observed on melon plants at 100 µM, except when SM12 was either mixed with Silwet L-77 and foliar sprayed or when delivered through the roots. Lead SMs did not inhibit the growth of beneficial Pseudomonas and Enterobacter species but inhibited the growth of Bacillus species. Nineteen SMs were cidal to Xanthomonas cucurbitae and showed >50% growth inhibition against Pseudomonas syringae pv. lachrymans. In addition, 19 SMs were cidal or static against Erwinia amylovora in vitro. Five SMs demonstrated potential to suppress E. tracheiphila when foliar sprayed on melon plants at 2× the minimum bactericidal concentration. Thirteen SMs reduced Et load in melon plants when delivered via roots. Temperature and light did not affect the activity of SMs. In vitro cidal activity was observed after 3 to 10 h of exposure to these five SMs. Here, we report 19 SMs that provide chemical scaffolds for future development of bactericides against plant pathogenic bacterial species.

Bacterial communities of herbivores and pollinators that have co-evolved Cucurbita spp

Insects, like all animals, are exposed to diverse environmental microbes throughout their life cycle. Yet, we know little about variation in the microbial communities associated with the majority of wild, unmanaged insect species. Here, we use a 16S rRNA gene metabarcoding approach to characterize temporal and geographic variation in the gut bacterial communities of herbivores (Acalymma vittatum and A. trivittatum) and pollinators (Eucera (Peponapis) pruinosa) that have co-evolved with the plant genus Cucurbita (pumpkin, squash, zucchini and gourds). Overall, we find high variability in the composition of bacterial communities in squash bees and beetles collected from different geographic locations and different time points throughout a growing season. Still, many of the most common OTUs are shared in E. (P.) pruinosa, A. vittatum and A. trivittatum. This suggests these insects may be exposed to similar environmental microbial sources while foraging on the same genus of host plants, and that similar microbial taxa may aid in digestion of Cucurbita plant material. The striped cucumber beetle A. vittatum can also transmit Erwinia tracheiphila, the causal agent of bacterial wilt of cucurbits. We find that few field-collected A. vittatum individuals have detectable E. tracheiphila, and when this plant pathogen is detected, it comprises less than 1% of the gut bacterial community. Together, these results are consistent with previous studies showing that plant feeding insects have highly variable gut bacterial communities, and provides a first step towards understanding the spatiotemporal variation in the microbial communities associated with herbivores and pollinators that depend on Cucurbita host plants.

A horizontally acquired expansin gene increases virulence of the emerging plant pathogen Erwinia tracheiphila

All land plants depend on proteins called ‘expansins’ that non-enzymatically loosen structural cellulose, enabling cell wall extension during normal growth. Surprisingly, expansin genes are also present – but functionally uncharacterized – in taxonomically diverse bacteria and fungi that do not produce cellulosic cell walls. Here, we find that Erwinia tracheiphila (Enterobacteriaceae), the causative agent of bacterial wilt of cucurbits, has horizontally acquired an operon with a microbial expansin (exlx) gene and a glycoside hydrolase family 5 (gh5) gene. E. tracheiphila is an unusually virulent plant pathogen that induces systemic wilt symptoms followed by plant death, and has only recently emerged into cultivated cucurbit populations in temperate Eastern North America. Plant inoculation experiments with deletion mutants show that EXLX-GH5 is a secreted virulence factor that confers efficient xylem movement and colonization ability to E. tracheiphila. Bacterial colonization of xylem blocks sap flow, inducing wilt symptoms and causing plant death. Together, these results suggest that the horizontal acquisition of the exlx-gh5 locus was likely a key step driving the recent emergence of E. tracheiphila. The increase in E. tracheiphila virulence conferred by microbial expansins, the presence of this gene in many other bacterial and fungal wilt-inducing plant pathogen species, and the amenability of microbial expansins to horizontal gene transfer suggest this gene may be an under-appreciated virulence factor in taxonomically diverse agricultural pathogens.ImportanceErwinia tracheiphila is a bacterial plant pathogen that causes a fatal wilt infection in cucurbit crop plants. Here, we report that E. tracheiphila has horizontally acquired a microbial expansin gene (exlx) adjacent to a glycoside hydrolase family 5 (gh5) gene. Expansins are predominantly associated with plants due to their essential role in loosening structural cell wall cellulose during normal growth. We find that the EXLX and GH5 proteins in E. tracheiphila function as a single complex to facilitate xylem colonization, possibly by manipulating the size of xylem structures that normally exclude the passage of bacteria. This suggests that horizontal acquisition of the exlx-gh5 locus was likely a key step in the recent emergence of E. tracheiphila as an unusually virulent plant pathogen. The presence of microbial expansin genes in diverse species of bacterial and fungal wilt-inducing pathogens suggests it may be an under-appreciated virulence factor for other microbes.