Bacterial gall caused by Pseudomonas amygdali pv. loropetali (PAL) is a prevalent problem on Loropetalum chinense shrubs in commercial plant nurseries. A method was developed to reliably detect PAL on the surface of loropetalum twigs. A whole-genome analysis resulted in the identification of a locus encoding an AraC regulator that is specific to PAL. A pair of primers and a TaqMan probe were designed based on a 71-base-pair sequence in this locus. Positive results of PCR amplification were obtained with genomic DNA samples from all PAL strains but not from those of other Pseudomonas species, Agrobacterium tumefaciens, or Burkholderia contaminans. Melting curve analysis demonstrated that all PAL PCR products shared the same melting temperature of 79°C. TaqMan-based quantitative PCR (qPCR) analysis of the serially diluted genomic DNA from PAL strain AAC exhibited a strong linear response for regressed cycle threshold and logarithm copy values (adjusted R2 = 0.9944) with a high amplification efficiency (E = 1.96), whereas the linear response (adjusted R2 = 0.8885) for PAL genomic DNA extracted from serially diluted bacterial cell suspension had a reduction in detection sensitivity. The limits of detection and quantification of PAL from the spiked plant twigs (diameter × length = ∼0.45 × 2.45 cm) were 873 and 14,724 cells, respectively, using a modified Promega Wizard extraction protocol. These limits of the qPCR method, although restrictive, still allow a practical detection of PAL strains associated with plant tissue that can be used in epidemiological studies to develop disease management options.

Bacterial gall of Loropetalum chinense, caused by Pseudomonas amygdali pv. loropetali (PAL), was reported to have become an invasive problem in nurseries due to a lack of proven control methods. We surveyed six nurseries to assess the frequency of loropetalum plants with and without galls and to assess PAL density on and in woody twigs in three vertical strata (base, mid-height, and new growth sections) of plants. Quantitative counts were determined using a quantitative real-time polymerase chain reaction method involving primers and probe specific for PAL. The whole-plant frequency of galled plants within a single cultivar block of loropetalum varied from 8 to 92%, with three nurseries having moderate levels of 27 to 39%. No consistent relationship was observed between gall frequency and PAL abundance across canopy strata. The pattern of the mean DNA copy number of PAL in twigs of the three vertical plant strata varied between nurseries, with the highest copy number occurring more frequently in the base stratum and least frequently in the new stratum. The presence of PAL in the phyllosphere of loropetalum plants indicates a risk for spreading the pathogen on vegetatively propagated stem cuttings.The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2026.

Biofilms are an important colonization mechanism employed by several microbial species to better establish themselves and monopolize the acquisition of resources across different environs. Some bacteria have evolved specialized metabolites that, when secreted, disrupt the formation and stability of biofilms generated by competing heterospecies, providing the producing organism with an ecological advantage. Soil-derived species are probable candidates for the identification of such compounds, given the intense level of competition that occurs within the terrestrial ecosystem. The MS14 strain of Burkholderia contaminans isolated from soil in Mississippi has previously been shown to produce antimicrobial compounds like occidiofungin and ornibactin. In this report, we demonstrate that this strain also produces 4-hydroxy-3-methyl-2-alkenylquinoline (HMAQ-7), an alkaloid-based metabolite structurally similar to others produced by Burkholderia. HMAQ-7 was isolated and purified in sufficient quantities to enable the elucidation of its covalent structure and the evaluation of its biological effects. The compound was found to possess a unique ability to inhibit biofilm biosynthesis in several species, including opportunistic pathogens like Staphylococcus haemolyticus and within saliva-derived multispecies biofilms. HMAQ-7 also demonstrated an ability to modulate additional cellular behaviors in Bacillus subtilis, including motility and sporulation, suggesting that this molecule is important to the interspecies dynamics present across many diverse microenvironments.IMPORTANCEThe present study furthers our understanding of the structural complexity and the biological functions of the 2-alkyl-4(1H)-quinolone metabolites produced by Burkholderia spp. Low micromolar concentrations of HMAQ-7' induced observable bacterial growth morphology differences. The antibiofilm properties of the HMAQ-7' characterized in this study will promote future investigations into possible biological and applied roles. The ability to alter biofilm formation using HMAQ-7' may facilitate Burkholderia spp. colonization in a multitude of environments, that is, aquatic, soil, and possibly during infection. HMAQ may subvert competition by potential competitor species in natural environments of Burkholderia spp. and possibly lung infections of cystic fibrosis patients.

Bacterial strain WP18 was isolated in the Mississippi Delta from the rhizosphere of a healthy soybean plant growing in a charcoal rot disease patch, caused by the fungal pathogen Macrophomina phaseolina. The 16S rDNA sequence analysis demonstrated that the strain belongs to the genus Pseudomonas. Preliminary studies show that strain WP18 possesses antimicrobial activity against known plant pathogens. Pseudomonas sp. strain WP18 was found to have a single, circular chromosome that is 6,336,986 base pairs in size. No plasmids were detected. There are a total of 5,669 coding genes in the WP18 genome, including 5,533 putative protein-coding genes, 50 putative pseudogenes, 82 genes encoding RNAs (16 rRNAs and 66 tRNAs), and a non-coding RNA. The WP18 genome has two predicted gene clusters that may encode the antimicrobial compounds hydrogen cyanide and 2,4-diacetylphloroglucinol. However, the essential genes for the production of phenazines, the Pseudomonas type III secretion system, and phytotoxins were not found. The findings of this study provide a genome resource for the bacterium.Copyright (c) 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Charcoal rot disease of soybean is a major threat to soybean harvests in the United States. This disease, caused by the fungal pathogen Macrophomina phaseolina (MP), cannot be economically treated in agronomic field conditions, and breeding of resistant cultivars has been largely unsuccessful due to multiple gene resistance. For difficult-to-treat phytopathogens, nascent research has emphasized the potential utility of endophyte modulation of disease symptoms. Most research into biocontrol of MP in soybeans has focused on in vitro or limited greenhouse/pot experiments, which may not emulate field conditions. Here, using a paired framework of diseased and asymptomatic soybeans exposed to MP in a production field setting, coupled with model-based community analyses, we identify several fungal endophytic taxa that apparently modulate disease pathogenicity. We use a multilevel analytical framework to provide evidence of taxa that respond to the disease state by querying plants across years and plant compartments. Additional work is needed to test the mechanisms by which these identified taxa may modulate MP, but this work provides the strongest evidence to date that endophytes may modulate charcoal rot disease levels in production field conditions. We posit that breeding for improved colonization of disease-modulating endophytes is a promising option to manage charcoal rot in soybean. [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 .

Non-genetic variation limits the identification of novel maize germplasm with genetic markers for reduced Aspergillus flavus infection and aflatoxin contamination. Aflatoxin measurements can vary substantially within fields containing the same germplasm following inoculation with A. flavus. While some variation is expected due to microenvironmental differences, components of field screening methodologies may also contribute to variability in collected data. Therefore, the objective of this study is to test the effects of three different shelling methods (whole ear (WE), ear end removal (EER), and inoculation site-surrounding (ISS)) to obtain bulk samples from maize on aflatoxin measurements. Five ears per row of three inbred lines and two hybrids were inoculated with A. flavus, then shelled using the three different methods, and aflatoxin was quantified. Overall, EER and ISS resulted in reduced coefficients of variance (CVs) in comparison to WE for both inbred and hybrid maize lines, with two exceptions. Susceptible B73 showed increased CVs with both EER and ISS compared to WE, and resistant Mp719’s EER CVs marginally increased compared to WE. While WE is the standard practice for most breeding programs due to its technical simplicity, EER and ISS may allow for finely phenotyping parental lines for further breeding applications.

Endophytic bacteria are endosymbionts that colonize a portion of plants without harming the plant for at least a part of its life cycle. Bacterial endophytes play an essential role in promoting plant growth using multiple mechanisms. The genus Burkholderia is an important member among endophytes and encompasses bacterial species with high genetic versatility and adaptability. In this study, the endophytic characteristics of Burkholderia species are investigated via comparative genomic analyses of several endophytic Burkholderia strains with pathogenic Burkholderia strains. A group of bacterial genes was identified and predicted as the putative endophytic behavior genes of Burkholderia. Multiple antimicrobial biosynthesis genes were observed in these endophytic bacteria; however, certain important pathogenic and virulence genes were absent. The majority of resistome genes were distributed relatively evenly among the endophytic and pathogenic bacteria. All known types of secretion systems were found in the studied bacteria. This includes T3SS and T4SS, which were previously thought to be disproportionately represented in endophytes. Additionally, questionable CRISPR-Cas systems with an orphan CRISPR array were prevalent, suggesting that intact CRISPR-Cas systems may not exist in symbiotes of Burkholderia. This research not only sheds light on the antimicrobial activities that contribute to biocontrol but also expands our understanding of genomic variations in Burkholderia's endophytic and pathogenic bacteria.

Natural environment serves as a reservoir for Burkholderia cepacia complex organisms, including the highly transmissible opportunistic human pathogen B. cenocepacia. Currently, there is a lack of an effective and quantitative method for B. cenocepacia detection in fresh food and other environmental niches. A quantitative real-time PCR (qPCR) detection method for B. cenocepacia bacteria was established in this study and validated using artificially inoculated fresh vegetable samples. Genome-wide comparative methods were applied to identify target regions for the design of species-specific primers. Assay specificity was measured with 12 strains of closely related Burkholderia bacteria and demonstrated the primer pair BCF6/R6 were 100% specific for detection of B. cenocepacia. The described qPCR assay evaluated B. cenocepacia with a 2 pg μl-1 limit of detection and appropriate linearity (R2 = 0.999). In 50 samples of experimentally infected produce (lettuce, onion, and celery), the assay could detect B. cenocepacia as low as 2.6 × 102 cells in each sample equal to 1 g. The established qPCR method quantitatively detects B. cenocepacia with high sensitivity and specificity, making it a promising technique for B. cenocepacia detection and epidemiological research on B. cepacia complex organisms from fresh vegetables.

Cyclic diguanylate (c-di-GMP) affects bacterial physiological and biochemical functions like biofilm, motility, virulence, and bacterial secretion systems. GGDEF/EAL-domain proteins, participating in c-di-GMP synthesis and degradation, are widely present in Pseudomonas, with various structures and functions. Pseudomonas glycinae MS82 is a rhizosphere bacterium that protects mushroom against the pathogenic fungi. Although 14 genes encoding GGDEF/EAL-domain proteins have been identified in the genome of MS82, c-di-GMP regulation is poorly understood as a facilitator or repressor of physiological phenotypes. Here, PafQ and PafR, encoding the proteins with the tandem GGDEF/EAL domain, were functionally analyzed and found to regulate antifungal activity. Individual deletion mutants of PafQ and PafR were constructed in P. glycinae MS82 through biparental conjugation and homologous recombination. Subsequently, antifungal activity, biofilm formation, motility, and expression of the genes related to antifungal substance synthesis were examined and contrasted with those of wild-type P. glycinae MS82. Most phenotypes of physiological activities were significantly reduced after knocking out PafQ or PafR. In other members of the genus Pseudomonas, homologous genes of PafQ and PafR possess different functions in c-di-GMP regulation. In P. glycinae, the positive regulation of PafQ and PafR on fungistatic substance synthesis, biofilm formation, and motility is crucial in the biocontrol of mushroom diseases.

Aflatoxin is a secondary metabolite produced by Aspergillus fungi and presents a major food safety concern globally. Among the available methods for prevention and control of aflatoxin, the application of antifungal bacteria has gained favor in recent years. An endophytic bacterium MS455, isolated from soybean, exhibited broad-spectrum antifungal activity against economically important pathogens, including Aspergillus flavus. MS455 was identified as a strain of Burkholderia based on genomic analysis. Random and site-specific mutations were used in discovery of the genes that share high homology to the ocf gene cluster of Burkholderia contaminans strain MS14, which is responsible for production of the antifungal compound occidiofungin. RNA sequencing analysis demonstrated that ORF1, a homolog to the ambR1 LuxR-type regulatory gene, regulates occidiofungin biosynthesis in MS455. Additionally, 284 differentially expressed genes, including 138 upregulated and 146 downregulated genes, suggesting that, in addition to its role in occidiofungin production, ORF1 is involved in expression of multiple genes, especially those involved in ornibactin biosynthesis. Plate bioassays showed the growth of A. flavus was significantly inhibited by the wild-type strain MS455 as compared with the ORF1 mutant. Similarly, corn kernel assays showed that growth of A. flavus and aflatoxin production were reduced significantly by MS455 as compared with buffer control and the ORF1 mutant. Collectively, the results demonstrated that production of occidiofungin is essential for antifungal activity of the endophytic bacterium MS455. This research has provided insights about antifungal mechanisms of MS455 and development of biological approaches to prevent aflatoxin contamination in plant production.

Occidiofungin is a broad-spectrum antifungal compound produced by Burkholderia contaminans MS14. It is a cyclic glycol-lipopeptide with a novel beta-amino acid (NAA2) containing a hydroxylated C18 fatty acid chain with a xylose sugar. This study reports a strategy to produce semisynthetic analogs of occidiofungin to further explore the structure activity relationships of this class of compounds. Oxidative cleavage of the diol present on carbons five C(5) and six C(6) removes the xylose and twelve carbons of the fatty acid chain. The resulting cyclic peptide product, occidiofungin aldehyde, is devoid of antifungal activity. However, the free aldehyde group on this product can be subjected to reductive amination reactions to provide interesting semisynthetic analogs. This chemistry allows the quick generation of analogs to study the structure activity relationships of this class of compounds. Despite restoring the length of the aliphatic side chain by reductive amination addition with undecylamine or dodecylamine to the free aldehyde group, the obtained analogs did not demonstrate any antifungal activity. The antifungal activity was partially restored by the addition of a DL-dihydrosphingosine. The dodecylamine analog was demonstrated to still bind to the cellular target actin, suggesting that the diol on the side chain of native occidiofungin is important for entry into the cell enabling access to cellular target F-actin. These results show that the alkyl side chain on NAA2 along with the diol present on this side chain is important for occidiofungin's antifungal activity.

HomePlant DiseaseVol. 106, No. 9Complete Genome Sequence Resource for Pseudomonas amygdali pv. loropetali Strain AAC Causing Bacterial Gall of Loropetalum chinense PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseComplete Genome Sequence Resource for Pseudomonas amygdali pv. loropetali Strain AAC Causing Bacterial Gall of Loropetalum chinenseJiayuan Jia, Warren E. Copes, Kate Phillips, and Shi-En LuJiayuan JiaDepartment of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mail Stop 9655, Mississippi State, MS 39762Search for more papers by this author, Warren E. CopesUnited States Department of Agriculture–Agricultural Research Service, Thad Cochran Southern Horticultural Research Laboratory, Poplarville, MS 39740Search for more papers by this author, Kate PhillipsDepartment of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mail Stop 9655, Mississippi State, MS 39762Search for more papers by this author, and Shi-En Lu†Corresponding author: S.-E. Lu; E-mail Address: sl332@msstate.eduhttps://orcid.org/0000-0003-2255-1404Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mail Stop 9655, Mississippi State, MS 39762Search for more papers by this authorAffiliationsAuthors and Affiliations Jiayuan Jia1 Warren E. Copes2 Kate Phillips1 Shi-En Lu1 † 1Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mail Stop 9655, Mississippi State, MS 39762 2United States Department of Agriculture–Agricultural Research Service, Thad Cochran Southern Horticultural Research Laboratory, Poplarville, MS 39740 Published Online:26 Jul 2022https://doi.org/10.1094/PDIS-04-22-0919-AAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleGenome AnnouncementThe plant-pathogenic Pseudomonas syringae species complex (Pssc) represents a group of closely related bacteria that cause diseases on hundreds of plant species that comprise monocots, herbaceous dicots, and woody dicots (Young 2010). A reclassification revealed that the Pssc group contains 10 species with 60 pathovars, which includes P. amygdali (Gardan et al. 1999; Young 2010). Specifically, several species were classified into P. amygdali, including P. savastanoi, P. ficuserectae, P. meliae, and P. tremae, based on DNA relatedness ribotyping studies, repetitive element sequence-based PCR, and rpoD analyses (Gardan et al. 1999; Marques et al. 2008; Parkinson et al. 2011). Currently, the genomes of the majority of P. amygdali strains were drafted as contigs and scaffolds, and only a few complete genomes are available in GenBank, which include the pathovars tabaci, lachrymans, and morsprunorum. However, no complete genome sequence of the pathovar loropetali is available.Loropetalum chinense, also known as the Chinese fringe flower or loropetalum, is a well-adapted woody ornamental in regions of the southeastern United States that is propagated by cuttings. The bacterial knot disease caused by P. amygdali pv. loropetali on loropetalum cultivars was first reported in 2013 (Conner et al. 2013; Harmon et al. 2018). Disease symptoms include inconspicuous bacterial galls of 0.2 to 1.0 cm in diameter and irregular dark callus formation on limbs or girdled stems, with progressive death of twigs and branches and foliar nutrient deficiency symptoms distal of stem symptoms. This disease has led to a significant problem in commercial plant nurseries and has been given accelerative prevalence in the landscape of the same region. Thus, to further expand the genome information of P. amygdali and understand the genomic basis underlying host–pathogen interaction of pathovar loropetali, we report the complete genome sequence of P. amygdali pv. loropetali strain AAC, which was originally isolated from loropetalum shrubs purchased at a local retail outlet in Forest County, Mississippi, United States (Copes et al. 2019).Bacterial strain AAC from a stock at the Thad Cochran Southern Horticultural Research Laboratory, United States Department of Agriculture–Agricultural Research Service, was streaked onto nutrient broth yeast extract agar medium, then incubated at 28°C (Vidaver 1967). Genomic DNA was extracted from overnight bacterial cultures using Promega Wizard Genomic DNA Purification Kit (Promega Corp.), following the manufacturer’s protocols, with RNase A treatment. DNA quantity and quality were assessed using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). The library preparation and genome sequencing were conducted at Novogene (Beijing Novogene Corporation, China) by PacBio single-molecule real-time (SMRT) DNA sequencing. To prepare SMRTbell libraries, DNA (5 µg) was fragmented, damage repaired, end repaired, adapter ligated, primers annealed, and polymerase bound. The library was checked with Qubit for quantification and bioanalyzer for size distribution detection (Mardis and McCombie 2017). Quantified libraries were pooled and sequenced on the PacBio Sequel II system. Default parameters were used for all software. In total, 194,745 subreads with an N50 value of 15,678 bp and average read length of 12,468 bp were obtained, which provided approximately 391-fold coverage. Genome assembly was carried out using FALCON (FALCON-kit = 1.8.1) with hierarchical genome assembly process algorithm version 4. Merged contigs were circularized with the software Circlator (1.5.5) (Hunt et al. 2015). All reads and contigs after circularization were polished with Arrow (2.3.3). Based on evolutionarily informed expectations of gene content from near-universal single-copy orthologs, the benchmarking universal single-copy orthologous (BUSCO 4.0.2) analysis was used to verify the completeness of the genome assembly with database Pseudomonadales_odb10 (Simão et al. 2015). The BUSCO analysis showed that 124 complete and single-copy BUSCOs (100.00%) were detected. Clusters of orthologous groups of proteins (COGs) (Galperin et al. 2015) and gene ontology (GO) (Ashburner et al. 2000) were used for functional analysis. The genome was automatically annotated using the NCBI’s Prokaryotic Genome Annotation Pipeline (Tatusova et al. 2016). The Microbial Genomes Atlas (MiGA) webserver was used to establish the taxonomic classification of the strain AAC (Rodriguez-R et al. 2018). To evaluate genetic relatedness between closely related genomes, average nucleotide identity (ANI) was calculated by using the OrthoANIu v0.93 with OrthoANIu algorithm (Yoon et al. 2017). BLAST comparison of genomes was conducted by BRIG (Alikhan et al. 2011).The genome of P. amygdali pv. loropetali AAC comprises one circular chromosome (Fig. 1). The genome features of strain AAC are summarized in Table 1. The chromosome of AAC is 6,241,584 bp in length, with 5,279 protein-encoding genes and 58.20% G+C content, 16 ribosomal RNAs, and 66 transfer RNAs. According to functional protein alignments, 4,318 proteins were assigned to the COGs database, and GO terms were assigned to 3,927 genes. Strain AAC was preliminarily identified as P. amygdali based on the multilocus sequence analysis in a previous study (Copes et al. 2019). In this study, the MiGA analysis revealed that the closest related type strain was P. amygdali CFBP 3205T. In addition, comparisons of the complete genome assemblies of nine close type strains of the genus Pseudomonas demonstrated that strain AAC shares the highest ANI value of 98.78% to the type strain CFBP 3205T of P. amygdali, which is above the benchmark for species demarcation (95 to 96%) as described by Richter and Rosselló-Móra (2009) (Supplementary Table S1). Therefore, strain AAC was further identified as P. amygdali based on the MiGA and ANI results.Fig. 1. Circular representation of the complete genome of Pseudomonas amygdali pv. loropetali AAC compared with eight sequenced P. amygdali complete genomes. Rings from inside to the outside represent (1) Scale (black kilobase-pair-scaled ring), (2) GC content (black ring of segmented domains), (3) GC skew(−) (purple ring of segmented domain); GC skew(+) (green ring of segmented domains), (4) BLAST comparison with P. amygdali 35-1 (lavender ring), (5) BLAST comparison with P. amygdali HS1 (blizzard blue ring), (6) BLAST comparison with P. amygdali pv. morsprunorum 15244 (blue ring), (7) BLAST comparison with P. amygdali pv. tabaci 6605 (cornflower blue ring), (8) BLAST comparison with P. amygdali pv. tabaci ATCC 11528 (yellow ring), (9) BLAST comparison with P. amygdali pv. lachrymans 8 (Caribbean green ring), (10) BLAST comparison with P. amygdali pv. lachrymans M301315 (bud green ring), and (11) BLAST comparison with P. amygdali pv. lachrymans NM002 (red ring).Download as PowerPointTable 1. Genome features of Pseudomonas amygdali pv. loropetali strain AACFeatureAACSequence stateCompleteGenomic typeChromosomeAccession numberCP089282Size (bp)6,241,584Genes5,647Coding sequences5,279Pseudogenes282Ribosomal RNAs16Transfer RNAs66Noncoding RNAs4G+C content (%)58.20Table 1. Genome features of Pseudomonas amygdali pv. loropetali strain AACView as image HTML Blast research of the AAC genome against all available complete genomes of P. amygdali, including P. amygdali 35-1 (CP084212), P. amygdali HS1 (CP079716), P. amygdali pv. morsprunorum 15244 (CP026558), P. amygdali pv. tabaci 6605 (AP024464), P. amygdali pv. tabaci ATCC 11528 (CP042804), P. amygdali pv. lachrymans 8 (CP075686), P. amygdali pv. lachrymans M301315 (CP031225), and P. amygdali pv. lachrymans NM002 (CP020351), revealed noticeable genome diversity and multiple unique gene regions which were only found in the AAC genome (Fig. 1). In addition, the typical virulence factors of Pseudomonas spp. were analyzed in the AAC genome. The type III secretion system (T3SS), which delivers type III secretion effectors (T3SEs) into the host cell, where they act by suppressing the plant immune defenses and promoting virulence by different mechanisms, was found (Green and Mecsas 2016). As expected, two sets of T3SSs and 25 T3SEs were found in the AAC genome. The auxin phytohormone indole-3-acetic acid (IAA) has been described as a pathogenicity or virulence factor in P. savastanoi and P. syringae pathovars (Glickmann et al. 1998). The iaaM, iaaH, iaaL, aldA, and aldB genes that are involved in production of IAA (Aragón et al. 2014; Glickmann et al. 1998; McClerklin et al. 2018) were searched in the AAC genome. According to Glickmann et al. (1998), most of the IAA production pathovars of P. syringae harbor the iaaL gene. The entire iaaMH operon is absent in the AAC genome; however, it harbors part of the iaaL gene and the entire aldA and aldB genes. Thus, the possibility of AAC production of IAA could be attributed to aldehyde dehydrogenase family proteins, which are encoded by the genes aldA and aldB.To the best of our knowledge, this is the first reported complete genome of P. amygdali pv. loropetali. This genome sequence of P. amygdali pv. loropetali AAC will provide a valuable resource for future studies on the Loropetalum bacterial gall pathogen and its interactions with loropetalum.Data AvailabilityThe genome sequence of P. amygdali pv. loropetali AAC has been deposited in GenBank in BioProject accession number PRJNA786935 and the BioSample ID SAMN23753993. The sequence of the chromosome is deposited in GenBank under accession number CP089282.The author(s) declare no conflict of interest.Literature CitedAlikhan, N.-F., Petty, N. K., Ben Zakour, N. L., and Beatson, S. A. 2011. 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Lu.The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 106, No. 9 September 2022SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Metrics Article History Issue Date: 30 Aug 2022Published: 26 Jul 2022Accepted: 23 May 2022 Pages: 2502-2505 Information© 2022 The American Phytopathological SocietyFundingNon-Assistance Cooperative Agreement of United States Department of AgricultureGrant/Award Number: 6062-21430-004-00DNational Institute of Food and AgricultureGrant/Award Number: MS-401200Keywordsbacterial gallcomplete genomeLoropetalum chinensePseudomonas amygdaliThe author(s) declare no conflict of interest.PDF download

Cyclic dimeric guanosine monophosphate (c-di-GMP) is synthesized by diguanylate cyclase (DGC) with the GGDEF domain. As a ubiquitous bacterial second messenger, it regulates diverse life-activity phenotypes in some bacteria. Although 38 genes encoding GGDEF-domain-containing proteins have been identified in the genome of the Pseudomonas glycinae strain MS82, whether c-di-GMP functions as a facilitator or repressor of life-activity phenotypes is poorly understood. In this study, one of the 38 genes containing a GGDEF domain in MS82, PafS was investigated to explore its regulatory function in bacterial life activities. The PafS-deletion mutant ΔPafS and reversion mutant PafS-comp were constructed by the method of biparental conjugation and homologous recombination. The life activities of the mutants, such as antifungal activity, biofilm formation ability, polysaccharide content, and motor behavior, were explored. The results showed that all life-activity phenotypes were significantly reduced after knocking out PafS, whereas all were significantly restored to a similar level to that of MS82 after the complementation of PafS. These results suggested that PafS plays an important role in the regulation of a range of cellular activities by c-di-GMP in P. glycinae MS82.

The bacterial pathogen Acidovorax citrulli causes the destructive fruit blotch (BFB) on cucurbit plants. Pseudomonas chlororaphis YL-1 is a bacterial strain isolated from Mississippi soil and its genome harbors some antimicrobial-related gene clusters, such as phenazine, pyrrolnitrin, and pyoverdine. Here, we evaluated the antimicrobial activity of strain YL-1 as compared with its deficient mutants of antimicrobial-related genes, which were obtained using a sacB-based site-specific mutagenesis strategy. We found that only phenazine-deficient mutants ΔphzE and ΔphzF almost lost the inhibitory effects against A. citrulli in LB plates compared with the wild-type strain YL-1, and that the main antibacterial compound produced by strain YL-1 in LB medium was phenazine-1-carboxylic acid (PCA) based on the liquid chromatography-mass spectrometry (LC-MS) analysis. Gene expression analyses revealed that PCA enhanced the accumulation of reactive oxygen species (ROS) and increased the activity of catalase (CAT) in A. citrulli. The inhibition effect of PCA against A. citrulli was lowered by adding exogenous CAT. PCA significantly upregulated the transcript level of katB from 6 to 10 h, which encodes CAT that helps to protect the bacteria against oxidative stress. Collectively, the findings of this research suggest PCA is one of the key antimicrobial metabolites of bacterial strain YL-1, a promising biocontrol agent for disease management of BFB of cucurbit plants.

The results demonstrated that PVDs are essential for the broad-spectrum antibacterial activities of strain YL-1 against both Gram-positive and Gram-negative bacteria under low-iron conditions. Our findings also highlight the effect of exogenous iron on the production of PVD and the importance of this bacterial product in bacterial interactions. As a biocontrol agent, PVDs can directly inhibit the proliferation of the tested bacteria in addition to participating in iron competition.

Burkholderia sp. strain MS389, an endophytic bacterium, was isolated from a healthy soybean plant growing adjacent to a patch of plants affected by charcoal rot disease, caused by the fungal pathogen Macrophomina phaseolina. Preliminary studies demonstrated that strain MS389 possesses antimicrobial activities against multiple plant pathogens. Burkholderia sp. strain MS389 was found to have three circular chromosomes of 3,563,380 bp, 3,002,449 bp, and 1,180,421 bp in size, respectively. The 7,746,250-bp genome, with 66.73% G+C content, harbors 6,756 protein coding genes in the predicted 6,985 genes. In total, 18 rRNAs, 68 tRNAs, and four ncRNAs were identified and 139 pseudogenes were annotated as well. The findings of this study will provide valuable data to explore the antimicrobial mechanisms of the endophytic bacterial strain.

Burkholderia contaminans MS14, isolated from a soil sample in Mississippi, is known for producing the novel antifungal compound occidiofungin. In addition, MS14 exhibits a broad range of antibacterial activities against common plant pathogens. Random mutagenesis and gene complementation indicate that four genes are required for antibacterial activity of strain MS14 against the fire blight pathogen Erwinia amylovora. With the aim of finding the biosynthetic gene cluster for the unknown antibacterial compound, we used RNA-seq to analyze the transcriptome of MS14 wild type and mutants lacking antibacterial activity. The twofold lower expressed genes in all mutants were studied, and a polyketide synthase (PKS) gene cluster was predicted to be directly involved in MS14 antibacterial activities. The nptII-resistance cassette and CRISPR-Cas9 systems were used to mutate the PKS gene cluster. Plate bioassays showed that either insertion or frame-shifting one of the PKS genes resulted in a loss of antibacterial activity. Considering that the antibacterial-defective mutants maintain the same antifungal activities as the wild-type strain, the results suggest that this PKS gene cluster is highly likely to be involved in or directly responsible for the production of MS14 antibacterial activity. Purification efforts revealed that the antibacterial activity of the compound synthesized by the gene cluster is sensitive to UV radiation. Nevertheless, these findings have provided more insights to understand the antibacterial activity of strain MS14.

Abstract Strains MS586T and MS82, which are aerobic, Gram‐negative, rod‐shaped, and polar‐flagellated bacteria, were isolated from the soybean rhizosphere in Mississippi. Taxonomic positions of MS586T and MS82 were determined using a polyphasic approach. 16S rRNA gene sequence analyses of the two strains showed high pairwise sequence similarities (>98%) to some Pseudomonas species. Analysis of the concatenated 16S rRNA, rpoB, rpoD, and gyrB gene sequences indicated that the strains belonging to the Pseudomonas koreensis subgroup (SG) shared the highest similarity with Pseudomonas kribbensis strain 46‐2T. Analyses of average nucleotide identity (ANI), genome‐to‐genome distance, delineated MS586T and MS82 from other species within the genus Pseudomonas. The predominant quinone system of the strain was ubiquinone 9 (Q‐9), and the DNA G+C content was 60.48 mol%. The major fatty acids were C16:0, C17:0 cyclo, and the summed features 3 and 8 consisting of C16:1ω7c/C16:1ω6c and C18:1ω7c/C18:1ω6c, respectively. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol. Based on these data, it is proposed that strains MS586T and MS82 represent a novel species within the genus Pseudomonas. The proposed name for the new species is Pseudomonas glycinae, and the type strain is MS586T (accession NRRL B‐65441 = accession LMG 30275).

Occidiofungin is a nonribosomally synthesized cyclic lipopeptide that possesses broad-spectrum antifungal properties at submicromolar concentrations. This report explores multiple routes of administration and formulations of occidiofungin, as well as its toxicity in mice. Further, infection studies were performed in mice to assess the application of occidiofungin for treating systemic and intravaginal yeast infections. Formulations for intravenous and intravaginal administration of occidiofungin were prepared. Pharmacokinetic analyses were performed in a murine model, and a liquid chromatography-mass spectrometry (LC-MS) method was developed and used to quantify occidiofungin in mouse plasma samples. Toxicological and histopathological analyses of two repeat-dose studies using occidiofungin were performed. In these animal models, following intravenous administration, a liposomal formulation of occidiofungin improved the half-life and peak plasma drug concentration over that with a liposome-free formulation. Two long-term repeat dosing toxicity studies of occidiofungin indicated the absence of toxicity in organ tissues. Murine models of a systemic yeast infection and a vulvovaginal yeast infection were performed. The findings of the systemic infection study revealed limitations in the use of occidiofungin that may be alleviated with the development of novel structural analogs or with further formulation studies. The gel formulation of occidiofungin demonstrated improved efficacy over that of the commercial product Monistat 3 in a vulvovaginal candidiasis study. This report outlines the optimal routes of administration of occidiofungin and demonstrates minimal toxicity following chronic exposure. Further, the results of these studies provide a clear indication for the use of occidiofungin for the treatment of recurrent vulvovaginal candidiasis (RVVC), which is a serious and clinically relevant issue.