Phylogenomic analysis of 343 Xanthomonas citri pv. citri strains unravels introduction history and dispersal paths.

Xanthomonas citri pv. citri (Xcc) causes the devastating citrus canker disease. Xcc is known to have been introduced into Florida, USA in at least three different events in 1915, 1986 and 1995 with the first two claimed to be eradicated. It was questioned whether the Xcc introduction in 1986 has been successfully eradicated. Furthermore, it is unknown how Xcc has spread throughout the citrus groves in Florida. In this study, we investigated the population structure of Xcc to address these questions. We sequenced the whole genome of 343 Xcc strains collected from Florida groves between 1997 and 2016. Our analysis revealed two distinct clusters of Xcc. Our data strongly indicate that the claimed eradication of the 1986 Xcc introduction was not successful and Xcc strains from 1986 introduction were present in samples from at least 8 counties collected after 1994. Importantly, our data revealed that the Cluster 2 strains, which are present in all 20 citrus-producing counties sampled in Florida, originated from the Xcc introduction event in the Miami area in 1995. Our data suggest that Polk County is the epicenter of the dispersal of Cluster 2 Xcc strains, which is consistent with the fact that three major hurricanes passed through Polk County in 2004. As copper-based products have been extensively used to control citrus canker, we also investigated whether Xcc strains have developed resistance to copper. Notably, none of the 343 strains contained known copper resistance genes. Twenty randomly selected Xcc strains displayed sensitivity to copper. Overall, this study provides valuable insights into the introduction, eradication, spread, and copper resistance of Xcc in Florida.

Bacterial Mutation During Seasonal Epidemics.

Rapidly evolving bacterial pathogens pose a unique challenge for long-term plant disease management. In this study, we investigated the types and rate of mutations in bacterial populations during seasonal disease epidemics. Two phylogenetically distinct strains of the bacterial spot pathogen, Xanthomonas perforans, were marked, released in tomato fields, and recaptured at several time points during the growing season. Genomic variations in recaptured isolates were identified by comparative analysis of their whole-genome sequences. In total, 180 unique variations (116 substitutions, 57 insertions/deletions, and 7 structural variations) were identified from 300 genomes, resulting in the overall host-associated mutation rate of ∼0.3 to 0.9/genome/week. This result serves as a benchmark for bacterial mutation during epidemics in similar pathosystems. [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.

Phylodynamic Insights into Global Emergence and Diversification of the Tomato Pathogen Xanthomonas hortorum pv. gardneri.

The emergence of plant pathogens is often associated with waves of unique evolutionary and epidemiological events. Xanthomonas hortorum pv. gardneri is one of the major pathogens causing bacterial spot disease of tomatoes. After its first report in the 1950s, there were no formal reports on this pathogen until the 1990s, despite active global research on the pathogens that cause tomato and pepper bacterial spot disease. Given the recently documented global distribution of X. hortorum pv. gardneri, our objective was to examine genomic diversification associated with its emergence. We sequenced the genomes of X. hortorum pv. gardneri strains collected in eight countries to examine global population structure and pathways of emergence using phylodynamic analysis. We found that strains isolated post-1990 group by region of collection and show minimal impact of recombination on genetic variation. A period of rapid geographic expansion in X. hortorum pv. gardneri is associated with acquisition of a large plasmid conferring copper tolerance by horizontal transfer and coincides with the burgeoning hybrid tomato seed industry through the 1980s. The ancestry of X. hortorum pv. gardneri is consistent with introduction to hybrid tomato seed production and dissemination during the rapid increase in trade of hybrid seeds.

Elucidating the Mode of Action of Hybrid Nanoparticles of Cu/Zn Against Copper-Tolerant Xanthomonas euvesicatoria.

The widespread presence of tolerance to copper in Xanthomonas species has resulted in the need to develop alternative approaches to control plant diseases caused by xanthomonads. In recent years, nanotechnological approaches have resulted in the identification of novel materials to control plant pathogens. With many metal-based nanomaterials having shown promise for disease control, an important question relates to the mode of action of these new materials. In this study, we used several approaches, such as scanning electron microscopy, propidium monoazide quantitative polymerase chain reaction, epifluorescence microscopy, and RNA sequencing to elucidate the mode of action of a Cu/Zn hybrid nanoparticle against copper-tolerant strains of Xanthomonas euvesicatoria. We demonstrate that Cu/Zn did not activate copper resistance genes (i.e., copA and copB) in the copper-tolerant bacterium but functioned by disrupting the bacterial cell structure and perturbing important biological processes such as cell respiration and chemical homeostasis.

Whole-Genome-Sequence-Based Classification of Xanthomonas euvesicatoria pv. eucalypti and Computational Analysis of the Type III Secretion System.

Xanthomonas spp. infect a wide range of annual and perennial plants. Bacterial blight in young seedlings of Eucalyptus spp. in Indonesia was originally identified as X. perforans. However, these strains failed to elicit a hypersensitive response (HR) on either tomatoes or peppers. Two of the strains, EPK43 and BCC 972, when infiltrated into tomato and pepper leaves, failed to grow to significant levels in comparison with well-characterized X. euvesicatoria pv. perforans (Xp) strains. Furthermore, spray inoculation of 'Bonny Best' tomato plants with a bacterial suspension of the Eucalyptus strains resulted in no obvious symptoms. We sequenced the whole genomes of eight strains isolated from two Eucalyptus species between 2007 and 2015. The strains had average nucleotide identities (ANIs) of at least 97.8 with Xp and X. euvesicatoria pv. euvesicatoria (Xeu) strains, both of which are causal agents of bacterial spot of tomatoes and peppers. A comparison of the Eucalyptus strains revealed that the ANI values were >99.99% with each other. Core genome phylogeny clustered all Eucalyptus strains with X. euvesicatoria pv. rosa. They formed separate clades, which included X. euvesicatoria pv. alangii, X. euvesicatoria pv. citrumelonis, and X. euvesicatoria pv. alfalfae. Based on ANI, phylogenetic relationships, and pathogenicity, we designated these Eucalyptus strains as X. euvesicatoria pv. eucalypti (Xee). Comparative analysis of sequenced strains provided unique profiles of type III secretion effectors. Core effector XopD, present in all pathogenic Xp and Xeu strains, was absent in the Xee strains. Comparison of the hrp clusters of Xee, Xp, and Xeu genomes revealed that HrpE in Xee strains was very different from that in Xp and Xeu. To determine if it was functional, we deleted the gene and complemented with the Xee hrpE, confirming it was essential for secretion of type III effectors. HrpE has a hypervariable N-terminus in Xanthomonas spp., in which the N-terminus of Xee strains differs significantly from those of Xeu and Xp strains.

Xanthomonas as a Model System for Studying Pathogen Emergence and Evolution.

In this review, we highlight studies in which whole-genome sequencing, comparative genomics, and population genomics have provided unprecedented insights into past and ongoing pathogen evolution. These include new understandings of the adaptive evolution of secretion systems and their effectors. We focus on Xanthomonas pathosystems that have seen intensive study and improved our understanding of pathogen emergence and evolution, particularly in the context of host specialization: citrus canker, bacterial blight of rice, and bacterial spot of tomato and pepper. Across pathosystems, pathogens appear to follow a pattern of bursts of evolution and diversification that impact host adaptation. There remains a need for studies on the mechanisms of host range evolution and genetic exchange among closely related but differentially host-specialized species and to start moving beyond the study of specific strain and host cultivar pairwise interactions to thinking about these pathosystems in a community context.

Phenotypic and genetic diversity of xanthomonads isolated from pepper (Capsicum spp.) in Taiwan from 1989 to 2019.

Bacterial spot caused by Xanthomonas spp. is an economically important disease of pepper causing significant yield losses in Taiwan. Monitoring the pathogen population on a continuous basis is necessary for developing disease management strategies. We analyzed a collection of xanthomonad strains isolated from pepper in Taiwan between 1989 and 2019. Among the sequenced genomes, sixty-five were identified as Xanthomonas euvesicatoria and ten were X. perforans. Thirty-five X. euvesicatoria and ten X. perforans strains were copper tolerant, whereas only five X. euvesicatoria and none of the X. perforans strains were tolerant to streptomycin. Nine X. euvesicatoria strains were amylolytic, which is considered an unusual characteristic for X. euvesicatoria. Bayesian analysis of the population structure based on core gene SNPs clustered the strains into five clusters for X. euvesicatoria and three clusters for X. perforans. One X. perforans cluster, designated as TP-2019, appears to be a novel genetic cluster based on core genes, accessory gene content, and effector profile. This knowledge of pathogen diversity with whole genomic information will be useful in future comparative studies and in improving breeding programs to develop disease-resistant cultivars and other disease management options.

Population Genomics Reveals an Emerging Lineage of Xanthomonas perforans on Pepper.

Xanthomonas perforans-the dominant causal agent of bacterial leaf spot of tomato-is an emerging pathogen of pepper, indicative of a potential host expansion across the southeastern United States. However, studies of the genetic diversity and evolution of X. perforans from pepper remain limited. In this study, the whole-genome sequences of 35 X. perforans strains isolated from pepper from four fields and two transplant facilities across southwest Florida between 2019 and 2021 were used to compare genomic divergence, evolution, and variation in type III secreted effectors. Phylogenetic analysis based on core genes revealed that all 35 X. perforans strains formed one genetic cluster with pepper and tomato strains from Alabama and Turkey and were closely related to strains isolated from tomato in Indiana, Mexico, and Louisiana. The in planta population growth of tomato strains isolated from Indiana, Mexico, Louisiana, and Turkey in pepper leaf mesophyll was on par with pepper X. perforans and X. euvesicatoria strains. Molecular clock analysis of the 35 Florida strains dated their emergence to approximately 2017. While strains varied in copper tolerance, all sequenced strains harbored the avrHah1 transcription activation-like effector located on a conjugative plasmid, not previously reported in Florida. Our findings suggest that there is a geographically distributed lineage of X. perforans strains on tomato that has the genetic background to cause disease on pepper. Moreover, this study clarifies potential adaptive variants of X. perforans on pepper that could help forecast the emergence of such strains and enable immediate or preemptive intervention.

Unexpected Diversity of Pseudomonads Associated with Bacterial Leaf Spot of Cucurbits in the Southeastern United States.

Bacterial leaf spot of cucurbits (BLS) is an emerging disease in the southeastern United States that is capable of causing widespread outbreaks under conducive conditions. Historically attributed solely to the bacterium Pseudomonas syringae pv. lachrymans, recent studies have identified additional P. syringae pathovars as causal agents of the disease. To further investigate the identity and diversity of P. syringae strains associated with BLS in the southeastern United States, 47 bacterial isolates were recovered from symptomatic cucurbits from Florida, Alabama, and Georgia. Strains were characterized using the LOPAT testing scheme, fluorescence, and pathogenicity to watermelon and squash seedlings. Thirty-eight fluorescent isolates underwent whole-genome sequencing and were further characterized with 16S rRNA, four gene multilocus sequence analysis (MLSA) phylogeny, and average nucleotide identity analysis. Thirty-four isolates were identified as members of the P. syringae species complex, including P. syringae sensu stricto (12), P. alliivorans (12), P. capsici (nine), and P. viridiflava (one). An additional four isolates were found to belong to the Pseudomonas genus outside of the syringae species complex, though they did not share 95% or greater average nucleotide identity to any validly published species and are believed to belong to three novel Pseudomonas species. These results reveal an unpredicted level of diversity of Pseudomonas strains associated with BLS in the region and show the benefits of whole-genome sequencing for strain identification. Identification of P. capsici, which is capable of causing disease at higher temperatures than P. syringae, as a causal agent of BLS may also affect management strategies in the future.

A promoter trap in transgenic citrus mediates recognition of a broad spectrum of Xanthomonas citri pv. citri TALEs, including in planta-evolved derivatives.

Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), causes dramatic losses to the citrus industry worldwide. Transcription activator-like effectors (TALEs), which bind to effector binding elements (EBEs) in host promoters and activate transcription of downstream host genes, contribute significantly to Xcc virulence. The discovery of the biochemical context for the binding of TALEs to matching EBE motifs, an interaction commonly referred to as the TALE code, enabled the in silico prediction of EBEs for each TALE protein. Using the TALE code, we engineered a synthetic resistance (R) gene, called the Xcc-TALE-trap, in which 14 tandemly arranged EBEs, each capable of autonomously recognizing a particular Xcc TALE, drive the expression of Xanthomonas avrGf2, which encodes a bacterial effector that induces plant cell death. Analysis of a corresponding transgenic Duncan grapefruit showed that transcription of the cell death-inducing executor gene, avrGf2, was strictly TALE-dependent and could be activated by several different Xcc TALE proteins. Evaluation of Xcc strains from different continents showed that the Xcc-TALE-trap mediates resistance to this global panel of Xcc isolates. We also studied in planta-evolved TALEs (eTALEs) with novel DNA-binding domains and found that these eTALEs also activate the Xcc-TALE-trap, suggesting that the Xcc-TALE-trap is likely to confer durable resistance to Xcc. Finally, we show that the Xcc-TALE-trap confers resistance not only in laboratory infection assays but also in more agriculturally relevant field studies. In conclusion, transgenic plants containing the Xcc-TALE-trap offer a promising sustainable approach to control CBC.

Genome-Wide Association to Study the Host-Specificity Determinants of Xanthomonas perforans.

Xanthomonas perforans and X. euvesicatoria are the causal agents of bacterial spot disease of tomato and pepper, endemic to the Southeastern United States. Although very closely related, the two bacterial species differ in host specificity, where X. perforans is the dominant pathogen of tomato and X. euvesicatoria that of pepper. This is in part due to the activity of avirulence proteins that are secreted by X. perforans strains and elicit effector-triggered immunity in pepper leaves, thereby restricting pathogen growth. In recent years, the emergence of several pepper-pathogenic X. perforans lineages has revealed variability within the bacterial species to multiply and cause disease in pepper, even in the absence of avirulence gene activity. Here, we investigated the basal evolutionary processes underlying the host range of this species using multiple genome-wide association analyses. Surprisingly, we identified two novel gene candidates that were significantly associated with pepper-pathogenic X. perforans and X. euvesicatoria. Both candidates were predicted to be involved in the transport/acquisition of nutrients common to the plant cell wall or apoplast and included a TonB-dependent receptor, which was disrupted through independent mutations within the X. perforans lineage. The other included a symporter of protons/glutamate, gltP, enriched with pepper-associated mutations near the promoter and start codon of the gene. Functional analysis of these candidates revealed that only the TonB-dependent receptor had a minor effect on the symptom development and growth of X. perforans in pepper leaves, indicating that pathogenicity to this host might have evolved independently within the bacterial species and is likely a complex, multigenic trait.

A Brief Evaluation of a Copper Resistance Mobile Genetic Island in the Bacterial Leaf Spot Pathogen Xanthomonas euvesicatoria pv. perforans.

Due to the continuous use of copper containing bactericides without effective alternative bactericides, copper resistance has become more prevalent in plant pathogens, including Xanthomonas euvesicatoria pv. perforans (formerly Xanthomonas perforans), a predominant cause of bacterial leaf spot disease of tomato and pepper in the Southeastern United States. Previously, reports of copper resistance have been associated with a large conjugative plasmid. However, we have characterized a copper resistance genomic island located within the chromosome of multiple X. euvesicatoria pv. perforans strains. The island is distinct from a previously described chromosomally encoded copper resistance island in X. vesicatoria strain XVP26. Computational analysis revealed the genomic island to contain multiple genes associated with genetic mobility, including both phage-related genes and transposase. Among copper-tolerant strains of X. euvesicatoria pv. perforans isolated from Florida, the majority of strains were found to have the copper resistance chromosomally encoded rather than plasmid borne. Our results suggest that this copper resistance island may have two modes of horizontal gene transfer and that chromosomally encoded copper resistance genes may provide a fitness advantage over plasmid-borne resistance.

Strength in Numbers: Density-Dependent Volatile-Induced Antimicrobial Activity by Xanthomonas perforans.

For most of the 20th century, Xanthomonas euvesicatoria was the only known bacterium associated with bacterial spot of tomato in Florida. X. perforans quickly replaced X. euvesicatoria, mainly because of production of three bacteriocins (BCNs) against X. euvesicatoria; however, X. perforans outcompeted X. euvesicatoria even when the three known BCNs were deleted. Surprisingly, we observed antimicrobial activity against X. euvesicatoria in the BCN triple mutant when the triple mutant was grown in Petri plates containing multiple spots but not in Petri plates containing only one spot. We determined that changes in the headspace composition (i.e., volatiles) rather than a diffusible signal in the agar were required for induction of the antimicrobial activity. Other Xanthomonas species also produced volatile-induced antimicrobial compounds against X. euvesicatoria and elicited antimicrobial activity by X. perforans. A wide range of plant pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis, Pantoea stewartii, and Pseudomonas cichorii, also elicited antimicrobial activity by X. perforans when multiple spots of the species were present. To identify potential antimicrobial compounds, we performed liquid chromatography with high-resolution mass spectrometry of the agar surrounding the spot in the high cell density Petri plates where the antimicrobial activity was present compared with agar surrounding the spot in Petri plates with one spot where antimicrobial activity was not observed. Among the compounds identified in the zone of inhibition were N-butanoyl-L-homoserine lactone and N-(3-hydroxy-butanoyl)-homoserine lactone, which are known quorum-sensing metabolites in other bacteria.

First report of a Herbaspirillum sp. causing leaf spots on Boston Fern (Nephrolepis exaltata) in Florida.

Boston fern (Nephrolepis exaltata) samples were submitted by a nursery operation in Florida separately to the University of Florida Plant Diagnostic Center (UFPDC, Gainesville, FL) and to the North Carolina State University Plant and Pest Diagnostic Lab (NCSU PPDL, Raleigh, NC) in October 2021. Symptoms included tan spots on pinnules, some of which progressed into pinnule blight (Fig. S1). Bacterial streaming was noted from samples in both labs. Leaf spot margins were excised, macerated in sterile tap water, then streaked onto nutrient agar (NA) plates and incubated for 48 h at 27°C. Individual representative colonies that were opaque, creamy white, mucoid, and round with smooth margins were transferred and streaked onto additional NA plates. One strain from each lab (G21-1742, UFPDC and NC40101, NCSU PPDL) was selected for subsequent characterization. A suspension of each strain was adjusted to 108 CFU/mL and infiltrated into tobacco and tomato leaves, and confluent necrosis was observed 72 h after infiltration. The isolates were Gram-negative, oxidase-positive, HR-positive on tomato and tobacco, aerobic, not pectolytic, and nonfluorescent on King's Medium B. DNA was extracted from G21-1742 using Qiagen Stool kit (Qiagen cat#51604) and the 16S rRNA gene from strain G21-1742 was amplified using 16SrRNA universal primers UP1 (5'-TACGTGCCAGCAGCCGCGGTAATA-3') and UP2 (5'-AGTAAGGAGGGTATCCAACCGCA-3') (Kuppusamy et al. 2014). The amplicon was sequenced and submitted to NCBI (Genbank Accession No. OR004801). BLASTn analysis of 16S rRNA of G21-1742 resulted in 99.7% sequence identity to the type strain of Herbaspirillum huttiense subsp. huttiense ATCC 14670T (Genbank Accession NR_024698). The 16S rRNA sequence of NC40101 was identical to that of G21-1742. To determine if the G21-1742 strain was pathogenic, Boston fern plants were inoculated by suspending bacterial cells in tap water from a 24h culture grown on NA, adjusting the suspension to 108 CFU/mL and spraying the suspension on one three-week old frond from each of three healthy Boston fern plants. A second frond from each plant was sprayed with sterile tap water. Each treated frond was individually sealed in a clear plastic bag for 24h at approximately 25°C. Inoculated plants remained on the greenhouse bench after the plastic bags were removed. The inoculation experiment was repeated once. After 4 days, tan spots were observed on pinnules of inoculated plants that were identical to the original submitted samples, while no symptoms developed on water-inoculated plants. Bacterial strains were reisolated from symptomatic plants and were morphologically identical to G21-1742. The 16S rRNA sequence of the reisolated strain was identical to G21-1742. Additionally, we conducted MLSA analysis using 12 housekeeping genes (See Table S2 for housekeeping genes and accession numbers) from the fern strains and the corresponding housekeeping genes for the type strains of 13 Herbaspirillum species, which placed the fern strains most closely with H. huttiense (see Fig. S2). This is the first known report of a Herbaspirillum sp. on Boston fern, an important ornamental crop, that renders the plants aesthetically unsaleable. Previously, a Herbaspirillum sp. was reported in Florida to cause a leaf spot and blight on greenhouse grown tomato seedlings (Obradovic et al. 2007).

Exploring Diversity of Bacterial Spot Associated Xanthomonas Population of Pepper in Southwest Florida.

Bacterial spot caused by Xanthomonas spp. is a significant disease that challenges pepper growers worldwide and is particularly severe in a hot and humid environment. Understanding the pathogen's population biology is critical for sustainable disease management. The goal of this study was to characterize the species, race, and bactericide sensitivity of bacterial spot-associated Xanthomonas collected from pepper in Florida. A survey of pepper production fields in southwest Florida between 2019 and 2021-covering two counties, eight farms, and two transplant facilities-resulted in the isolation of 542 Xanthomonas euvesicatoria and 35 Xanthomonas perforans strains. Four races were identified on pepper, of which most strains were race P1 (42%), race P6 (26%), race P3 (24%), and less common was race P4 (8%). All X. perforans strains were characterized as race P1 and showed a compatible reaction on tomato. Sixty-two and 96% of strains were sensitive to copper sulfate and streptomycin, respectively. One farm that did not use copper to manage the disease contained only copper-sensitive strains and was the only farm with race P3 strains. Strains were assayed for starch hydrolysis activity of which a third of X. euvesicatoria strains were strongly amylolytic, a characteristic not typically observed in X. euvesicatoria. All X. perforans strains produced bacteriocins against X. euvesicatoria in vitro. The Xanthomonas population causing bacterial spot on pepper in southwest Florida is diverse and dynamic; thus, regular monitoring provides pertinent information to plant breeders and growers for designing disease management strategies.

Improved Persistence of Bacteriophage Formulation with Nano N-Acetylcysteine-Zinc Sulfide and Tomato Bacterial Spot Disease Control.

Bacteriophages are biocontrol agents used to manage bacterial diseases. They have long been used against plant pathogenic bacteria; however, several factors impede their use as a reliable disease management strategy. Short-lived persistence on plant surfaces under field conditions results mainly from rapid degradation by exposure to ultraviolet (UV) light. Currently, there are no effective commercial formulations that protect phages from UV. The phage ΦXp06-02-1, which lyses strains of the tomato bacterial spot pathogen Xanthomonas perforans, was mixed with different concentrations of the nanomaterial N-acetylcysteine surface-coated manganese-doped zinc sulfide (NAC-ZnS; 3.5 nm). In vitro, NAC-ZnS at 10,000 µg/ml formulated phage, when exposed to UV for 1 min, provided statistically equivalent plaque-forming unit (PFU) recovery as phages that were not exposed to UV. NAC-ZnS had no negative effect on the phage's ability to lyse bacterial cells under in vitro conditions. NAC-ZnS reduced phage degradation over time in comparison with the nontreated control, whereas N-acetylcysteine-zinc oxide (NAC-ZnO) had no effect. In fluorescent light, without UV exposure, NAC-ZnO-formulated phages were more infective than NAC-ZnS-formulated phages. The nanomaterial-phage mixture did not cause any phytotoxicity when applied to tomato plants. Following exposure to sunlight, the NAC-ZnS formulation improved phage persistence in the phyllosphere by 15 times compared with nonformulated phages. NAC-ZnO-formulated phage populations were undetectable within 32 h, whereas NAC-ZnS-formulated phage populations were detected at 103 PFU/g. At 4 h of sunlight exposure, NAC-ZnS-formulated phages at 1,000 µg/ml significantly reduced tomato bacterial spot disease severity by 16.4% compared with nonformulated phages. These results suggest that NAC-ZnS can be used to improve the efficacy of phages for bacterial diseases.

Novel magnesium-copper hybrid nanomaterials for management of bacterial spot of tomato.

Bacterial spot of tomato (BST), predominantly caused by Xanthomonas perforans (Xp) in Florida, is one of the most devastating diseases in hot, humid environments. Bacterial resistance to copper-based bactericides and antibiotics makes disease management extremely challenging. This necessitates alternative solutions to manage the disease. In this study, we used two novel hybrid copper and magnesium nanomaterials noted as magnesium double-coated (Mg-Db) and magnesium-copper (Mg-Cu), to manage BST. In in vitro experiments, no viable cells were recovered following 4 h exposure to 500 µg/ml of both Mg-Db and Mg-Cu, while 100 and 200 µg/ml required 24 h of exposure for complete inhibition. In viability assay using live/dead cell straining method and epifluorescence microscopy, copper tolerant Xp cells were killed within 4 h by both Mg-Cu and Mg-Db nanomaterials at 500 µg/ml, but not by copper hydroxide (Kocide 3000). In the greenhouse, Mg-Db and Mg-Cu at 100-500 µg/ml significantly reduced BST severity compared to micron-sized commercial Cu bactericide Kocide 3000 and the growers' standard (copper hydroxide + mancozeb) (P < 0.05). In field studies, Mg-Db and Mg-Cu nanomaterials significantly reduced disease severity in two out for field trials. Mg-Db at 500 µg/ml reduced BST severity by 34% compared to the non-treated control without affecting yield in Fall, 2020. The use of hybrid nanomaterials at the highest concentrations (500 µg/ml) used in the field experiments can reduce copper use by 90% compared to the growers' standard. In addition, there was no phytotoxicity observed with the use of hybrid nanomaterials in the field. These results suggest the potential of novel magnesium-copper based hybrid nanomaterials to manage copper-tolerant bacterial pathogens.

GFP labeling of a Bradyrhizobium strain and an attempt to track the crack entry process during symbiosis with peanuts.

Compared to the well-studied model legumes, where symbiosis is established via root hair entry, the peanut is infected by Bradyrhizobium through the crack entry, which is less common and not fully understood. Crack entry is, however, considered a primitive symbiotic infection pathway, which could be potentially utilized for engineering non-legume species with nitrogen fixation ability. We utilized a fluorescence-labeled Bradyrhizobium strain to help in understanding the crack entry process at the cellular level. A modified plasmid pRJPaph-bjGFP, harboring the codon-optimized GFP gene and tetracycline resistance gene, was created and conjugated into Bradyrhizobium strain Lb8, an isolate from peanut nodules, through tri-parental mating. Microscopic observation and peanut inoculation assays confirmed the successful GFP tagging of Lb8, which is capable of generating root nodules. A marking system for peanut root potential infection sites and an optimized sample preparation protocol for cryostat sectioning was developed. The feasibility of using the GFP-tagged Lb8 for observing crack entry was examined. GFP signal was detected at the nodule primordial stage and the following nodule developmental stages with robust GFP signals observed in infected cells in the mature nodules. Spherical bacteroids in the root tissue were visualized at the nodules' inner cortex under higher magnification, reflecting the trace along the rhizobial infection path. The GFP labeled Lb8 can serve as an essential tool for plant-microbe studies between the cultivated peanut and Bradyrhizobium, which could facilitate further study of the crack entry process during the legume-rhizobia symbiosis.

Pseudomonas californiensis sp. nov. and Pseudomonas quasicaspiana sp. nov., isolated from ornamental crops in California.

Five bacterial strains were isolated from symptomatic leaves of Achillea millefolium, Delphinium sp. and Hydrangea sp. in California. Colonies isolated on King's medium B (KMB) appeared white, mucoid and round, similar to Pseudomonas species. Phylogenetic analyses based on 16S rRNA, rpoB, rpoD and gyrB genes placed the bacteria into three distinct groups within Pseudomonas that were most closely related to Pseudomonas viridiflava, Pseudomonas cichorii or Pseudomonas caspiana. To further characterize the strains, phenotypic analyses and the following tests were performed: fatty acid methyl ester composition, LOPAT, fluorescence on KMB, Biolog assay, and transmission electron microscopy. Finally, whole genome sequencing of the strains was conducted, and the sequences were compared with reference genomes of Pseudomonas species based on average nucleotide identity (ANI). The first group, which consists of three strains isolated from delphinium, hydrangea and achillea, had 95.6-96.9 % pairwise ANI between each other; the second group consists of two strains isolated from delphinium that had 100 % pairwise ANI. Although comparisons of the two groups with publicly available genomes revealed closest relationships with P. viridiflava (91.6 %), P. caspiana (88.3 %) and P. asturiensis (86.7 %), ANI values were less than 95 % compared to all validly published pseudomonads. Combining genomic and phenotypic data, we conclude that these strains represent two new species and the names proposed are Pseudomonas quasicaspiana sp. nov. (type strain DSMZ 11 30 42T=LMG 32 434T) for the strains isolated from delphinium, achillea and hydrangea and Pseudomonas californiensis sp. nov. (DSMZ 11 30 43T=LMG 32 432T) for the two strains isolated from delphinium. The specific epithets quasicaspiana and californiensis were selected based on the close phylogenetic relationship of strains with P. caspiana and on the geographic location of isolation, respectively.

Mutation of a Single Core Gene, tssM, of Type VI Secretion System of Xanthomonas perforans Influences Virulence, Epiphytic Survival, and Transmission During Pathogenesis on Tomato.

Xanthomonas perforans is a seedborne hemibiotrophic pathogen that successfully establishes infection in the phyllosphere of tomato. While most studies investigating mechanistic basis of pathogenesis have focused on successful apoplastic growth, factors important during asymptomatic colonization in the early stages of disease development are not well understood. In this study, we show that tssM gene of the type VI secretion system cluster i3* (T6SS-i3*) plays a significant role during initial asymptomatic epiphytic colonization at different stages during the life cycle of the pathogen. Mutation in a core gene, tssM of T6SS-i3*, imparted higher aggressiveness to the pathogen, as indicated by higher overall disease severity, higher in planta growth, and shorter latent infection period compared with the wild-type upon dip inoculation of 4- to 5-week-old tomato plants. Contribution of tssM toward aggressiveness was evident during vertical transmission from seed to seedling, with wild-type showing reduced disease severity as well as lower in planta populations on seedlings compared with the mutant. Presence of functional TssM offered higher epiphytic fitness as well as higher dissemination potential to the pathogen when tested in an experimental setup mimicking transplant house high-humidity conditions. We showed higher osmotolerance being one mechanism by which TssM offers higher epiphytic fitness. Taken together, these data reveal that functional TssM plays a larger role in offering ecological advantage to the pathogen. TssM prolongs the association of hemibiotrophic pathogen with the host, minimizing overall disease severity yet facilitating successful dissemination.