First Report of Neofabraea actinidiae causing a Cranberry Fruit Rot in Oregon.

Cranberry (Vaccinium macrocarpon, L.) is a commercial small fruit that is native to North America. Oregon ranks fourth in cranberry production in the U.S.A. with 1052 Ha of cranberry beds and annual production of 23,590 metric tonnes (USDA NASS 2021). Cranberry fruit rot diseases are caused by a complex of 15 fungal pathogens belonging to 10 genera (Polashock et al. 2017). In fruit rot surveys of 'Stevens' cranberry beds in Coos and Curry Counties, Oregon, berries were collected before harvest and sorted by symptoms (e.g. softening, shriveling, or discoloration). Cranberries with field rot symptoms were surface-disinfested and lesion margin tissue placed on V8 agar. Cultures were incubated at room temperature and outgrowing fungi were transferred twice onto fresh V8 agar to obtain single isolates. Storage rots that developed on asymptomatic cranberries held at 4°C for 8 weeks were processed similarly. Since 2017, we periodically isolated Neofabraea actinidiae, which is not a member of the cranberry fruit rot complex, from rotted cranberries (Polashock et al. 2017). In 2022, N. actinidiae was isolated from 22% of 45 cranberries collected from an organically managed farm and developed storage rot and from 6% of 50 storage-rot cranberries from three conventionally managed farms. Symptoms associated with N. actinidiae on 'Stevens' cranberries often include softening and brown tissue surrounded by a yellow-colored ring. On V8 agar, N. actinidiae grew as compact white circular colonies with dense aerial hyphae near the center and accompanied by a red pigment in the agar. Pink-colored mucoidal irregular conidiomata often developed on the colony after 3 weeks. Conidia were hyaline, aseptate, and ellipsoidal to fusiform ranging from 7.5 to 12.6 µm long X 3.5 to 5.6 µm wide (n=100). Genomic DNA was extracted from N. actinidiae isolates from cranberries in 2017 and 2022. ß-tubulin and the ITS 5/4 region were amplified and sequenced with primers Bt-T2m-Up/Bt-LEV-Lo1 and ITS5/ITS4 using conditions of de Jong et al. (2001) and White et al. (1990), respectively. Sequences were deposited in NCBI Genbank (Accessions: OR606303, OR606305, OR606306, & OR606309 for ITS; OR610314, OR610316, OR610317, & OR610320 for ß-tubulin). BlastN analysis of ß-tubulin (695 bp) and ITS (489-490 bp) had a 99.6 to 99.8% and 99.8 to 100% identity, respectively, to Neofabraea actinidiae (CBS 121403) (Accession numbers: KR859285 ß-tubulin and KR859079 ITS). Phylogenetic analysis of concatenated sequences using Tamura-Nei neighbor-joining (Tamura et al. 2004) confirmed identity of cranberry isolates as N. actinidiae. Koch's Postulates were confirmed with four N. actinidiae isolates from cranberry. Agar or hyphal plugs were placed in a 3 mm wound on the side of six surface-disinfested, asymptomatic berries and incubated in a moist chamber at 20°C for 15 days or 4°C for 55 days. Similar symptoms developed on each berry inoculated with N. actinidiae, but not agar alone. The fungus was recovered from symptomatic tissues and identity confirmed by colony morphology. N. actinidiae causes a ripe rot and storage rot in kiwifruit and is one of the species causing Bull's Eye Rot of pome fruits (Tyson et al. 2019). Cryptosporiopsis actinidiae (anamorph) was isolated from cranberries roots in British Columbia, CA, but considered unlikely to be the causal agent of dieback disease of cranberry vines (Sabaratnam et al. 2009). We have demonstrated that N. actinidiae causes a cranberry fruit rot in beds and in storage. Its prevalence, associated fruit rot symptoms, and disease incidence will continue to be monitored.

Untargeted Identification of Alkyne-Containing Natural Products Using Ruthenium-Catalyzed Azide Alkyne Cycloaddition Reactions Coupled to LC-MS/MS.

Alkyne-containing natural products have been identified from plants, insects, algae, fungi, and bacteria. This class of natural products has been characterized as having a variety of biological activities. Polyynes are a subclass of acetylenic natural products that contain conjugated alkynes and are underrepresented in natural product databases due to the fact that they decompose during purification. Here we report a workflow that utilizes alkyne azide cycloaddition (AAC) reactions followed by LC-MS/MS analysis to identify acetylenic natural products. In this report, we demonstrate that alkyne azide cycloaddition reactions with p-bromobenzyl azide result in p-bromobenzyl-substituted triazole products that fragment to a common brominated tropylium ion. We were able to identify a synthetic alkyne spiked into the extract of Anabaena sp. PCC 7120 at a concentration of 10 µg/mL after optimization of MS/MS conditions. We then successfully identified the known natural product fischerellin A in the extract of Fischerella muscicola PCC 9339. Lastly, we identified the recently identified natural products protegenins A and C from Pseudomonas protegens Pf-5 through a combination of genome mining and RuAAC reactions. This is the first report of RuAAC reactions to detect acetylenic natural products. We also compare CuAAC and RuAAC reactions and find that CuAAC reactions produce fewer byproducts compared to RuAAC but is limited to terminal-alkyne-containing compounds. In contrast, RuAAC is capable of identification of both terminal and internal acetylenic natural products, but byproducts need to be eliminated from analysis by creation of an exclusion list. We believe that both CuAAC and RuAAC reactions coupled to LC-MS/MS represent a method for the untargeted identification of acetylenic natural products, but each method has strengths and weaknesses.

Genome-based evolutionary history of Pseudomonas spp.

Pseudomonas is a large and diverse genus of Gammaproteobacteria. To provide a framework for discovery of evolutionary and taxonomic relationships of these bacteria, we compared the genomes of type strains of 163 species and 3 additional subspecies of Pseudomonas, including 118 genomes sequenced herein. A maximum likelihood phylogeny of the 166 type strains based on protein sequences of 100 single-copy orthologous genes revealed thirteen groups of Pseudomonas, composed of two to sixty three species each. Pairwise average nucleotide identities and alignment fractions were calculated for the data set of the 166 type strains and 1224 genomes of Pseudomonas available in public databases. Results revealed that 394 of the 1224 genomes were distinct from any type strain, suggesting that the type strains represent only a fraction of the genomic diversity of the genus. The core genome of Pseudomonas was determined to contain 794 genes conferring primarily housekeeping functions. The results of this study provide a phylogenetic framework for future studies aiming to resolve the classification and phylogenetic relationships, identify new gene functions and phenotypes, and explore the ecological and metabolic potential of the Pseudomonas spp.

Investigations into the Biosynthesis, Regulation, and Self-Resistance of Toxoflavin in Pseudomonas protegens Pf-5.

Pseudomonas spp. are prolific producers of natural products from many structural classes. Here we show that the soil bacterium Pseudomonas protegens Pf-5 is capable of producing trace levels of the triazine natural product toxoflavin (1) under microaerobic conditions. We evaluated toxoflavin production by derivatives of Pf-5 with deletions in specific biosynthesis genes, which led us to propose a revised biosynthetic pathway for toxoflavin that shares the first two steps with riboflavin biosynthesis. We also report that toxM, which is not present in the well-characterized cluster of Burkholderia glumae, encodes a monooxygenase that degrades toxoflavin. The toxoflavin degradation product of ToxM is identical to that of TflA, the toxoflavin lyase from Paenibacillus polymyxa. Toxoflavin production by P. protegens causes inhibition of several plant-pathogenic bacteria, and introduction of toxM into the toxoflavin-sensitive strain Pseudomonas syringae DC3000 results in resistance to toxoflavin.

An Interspecies Signaling System Mediated by Fusaric Acid Has Parallel Effects on Antifungal Metabolite Production by Pseudomonas protegens Strain Pf-5 and Antibiosis of Fusarium spp.

Pseudomonas protegens strain Pf-5 is a rhizosphere bacterium that suppresses soilborne plant diseases and produces at least seven different secondary metabolites with antifungal properties. We derived mutants of Pf-5 with single and multiple mutations in biosynthesis genes for seven antifungal metabolites: 2,4-diacetylphoroglucinol (DAPG), pyrrolnitrin, pyoluteorin, hydrogen cyanide, rhizoxin, orfamide A, and toxoflavin. These mutants were tested for inhibition of the pathogens Fusarium verticillioides and Fusarium oxysporum f. sp. pisi. Rhizoxin, pyrrolnitrin, and DAPG were found to be primarily responsible for fungal antagonism by Pf-5. Previously, other workers showed that the mycotoxin fusaric acid, which is produced by many Fusarium species, including F. verticillioides, inhibited the production of DAPG by Pseudomonas spp. In this study, amendment of culture media with fusaric acid decreased DAPG production, increased pyoluteorin production, and had no consistent influence on pyrrolnitrin or orfamide A production by Pf-5. Fusaric acid also altered the transcription of biosynthetic genes, indicating that the mycotoxin influenced antibiotic production by Pf-5 at the transcriptional level. Addition of fusaric acid to the culture medium reduced antibiosis of F. verticillioides by Pf-5 and derivative strains that produce DAPG but had no effect on antibiosis by Pf-5 derivatives that suppressed F. verticillioides due to pyrrolnitrin or rhizoxin production. Our results demonstrated the importance of three compounds, rhizoxin, pyrrolnitrin, and DAPG, in suppression of Fusarium spp. by Pf-5 and confirmed that an interspecies signaling system mediated by fusaric acid had parallel effects on antifungal metabolite production and antibiosis by the bacterial biological control organism.

Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions.

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45-52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire.

Pseudomonas protegens Pf-5 causes discoloration and pitting of mushroom caps due to the production of antifungal metabolites.

Bacteria in the diverse Pseudomonas fluorescens group include rhizosphere inhabitants known for their antifungal metabolite production and biological control of plant disease, such as Pseudomonas protegens Pf-5, and mushroom pathogens, such as Pseudomonas tolaasii. Here, we report that strain Pf-5 causes brown, sunken lesions on peeled caps of the button mushroom (Agaricus bisporus) that resemble brown blotch symptoms caused by P. tolaasii. Strain Pf-5 produces six known antifungal metabolites under the control of the GacS/GacA signal transduction system. A gacA mutant produces none of these metabolites and did not cause lesions on mushroom caps. Mutants deficient in the biosynthesis of the antifungal metabolites 2,4-diacetylphloroglucinol and pyoluteorin caused less-severe symptoms than wild-type Pf-5 on peeled mushroom caps, whereas mutants deficient in the production of lipopeptide orfamide A caused similar symptoms to wild-type Pf-5. Purified pyoluteorin and 2,4-diacetylphloroglucinol mimicked the symptoms caused by Pf-5. Both compounds were isolated from mushroom tissue inoculated with Pf-5, providing direct evidence for their in situ production by the bacterium. Although the lipopeptide tolaasin is responsible for brown blotch of mushroom caused by P. tolaasii, P. protegens Pf-5 caused brown blotch-like symptoms on peeled mushroom caps through a lipopeptide-independent mechanism involving the production of 2,4-diacetylphloroglucinol and pyoluteorin.

Genes expressed by the biological control bacterium Pseudomonas protegens Pf-5 on seed surfaces under the control of the global regulators GacA and RpoS.

Gene expression profiles of the biological control strain Pseudomonas protegens Pf-5 inhabiting pea seed surfaces were revealed using a whole-genome oligonucleotide microarray. We identified genes expressed by Pf-5 under the control of two global regulators (GacA and RpoS) known to influence biological control and secondary metabolism. Transcript levels of 897 genes, including many with unknown functions as well as those for biofilm formation, cyclic diguanylate (c-di-GMP) signalling, iron homeostasis and secondary metabolism, were influenced by one or both regulators, providing evidence for expression of these genes by Pf-5 on seed surfaces. Comparison of the GacA and RpoS transcriptomes defined for Pf-5 grown on seed versus in broth culture overlapped, but most genes were regulated by GacA or RpoS under only one condition, likely due to differing levels of expression in the two conditions. We quantified secondary metabolites produced by Pf-5 and gacA and rpoS mutants on seed and in culture, and found that production profiles corresponded generally with biosynthetic gene expression profiles. Future studies evaluating biological control mechanisms can now focus on genes expressed by Pf-5 on seed surfaces, the habitat where the bacterium interacts with seed-infecting pathogens to suppress seedling diseases.

pA506, a conjugative plasmid of the plant epiphyte Pseudomonas fluorescens A506.

Conjugative plasmids are known to facilitate the acquisition and dispersal of genes contributing to the fitness of Pseudomonas spp. Here, we report the characterization of pA506, the 57-kb conjugative plasmid of Pseudomonas fluorescens A506, a plant epiphyte used in the United States for the biological control of fire blight disease of pear and apple. Twenty-nine of the 67 open reading frames (ORFs) of pA506 have putative functions in conjugation, including a type IV secretion system related to that of MOBP6 family plasmids and a gene cluster for type IV pili. We demonstrate that pA506 is self-transmissible via conjugation between A506 and strains of Pseudomonas spp. or the Enterobacteriaceae. The origin of vegetative replication (oriV) of pA506 is typical of those in pPT23A family plasmids, which are present in many pathovars of Pseudomonas syringae, but pA506 lacks repA, a defining locus for pPT23A plasmids, and has a novel partitioning region. We selected a plasmid-cured derivative of A506 and compared it to the wild type to identify plasmid-encoded phenotypes. pA506 conferred UV resistance, presumably due to the plasmid-borne rulAB genes, but did not influence epiphytic fitness of A506 on pear or apple blossoms in the field. pA506 does not appear to confer resistance to antibiotics or other toxic elements. Based on the conjugative nature of pA506 and the large number of its genes that are shared with plasmids from diverse groups of environmental bacteria, the plasmid is likely to serve as a vehicle for genetic exchange between A506 and its coinhabitants on plant surfaces.

Inactivation of the GacA response regulator in Pseudomonas fluorescens Pf-5 has far-reaching transcriptomic consequences.

The GacS/GacA signal transduction system is a central regulator in Pseudomonas spp., including the biological control strain P. fluorescens Pf-5, in which GacS/GacA controls the production of secondary metabolites and exoenzymes that suppress plant pathogens. A whole genome oligonucleotide microarray was developed for Pf-5 and used to assess the global transcriptomic consequences of a gacA mutation in P. fluorescens Pf-5. In cultures at the transition from exponential to stationary growth phase, GacA significantly influenced transcript levels of 635 genes, representing more than 10% of the 6147 annotated genes in the Pf-5 genome. Transcripts of genes involved in the production of hydrogen cyanide, the antibiotic pyoluteorin and the extracellular protease AprA were at a low level in the gacA mutant, whereas those functioning in siderophore production and other aspects of iron homeostasis were significantly higher in the gacA mutant than in wild-type Pf-5. Notable effects of gacA inactivation were also observed in the transcription of genes encoding components of a type VI secretion system and cytochrome c oxidase subunits. Two novel gene clusters expressed under the control of gacA were identified from transcriptome analysis, and we propose global-regulator-based genome mining as an approach to decipher the secondary metabolome of Pseudomonas spp.

Isolation and identification of rhizoxin analogs from Pseudomonas fluorescens Pf-5 by using a genomic mining strategy.

The products synthesized from a hybrid polyketide synthase/nonribosomal peptide synthetase gene cluster in the genome of Pseudomonas fluorescens Pf-5 were identified using a genomics-guided strategy involving insertional mutagenesis and subsequent metabolite profiling. Five analogs of rhizoxin, a 16-member macrolide with antifungal, phytotoxic, and antitumor activities, were produced by Pf-5, but not by a mutant with an insertion in the gene cluster. The five rhizoxin analogs, one of which had not been described previously, were differentially toxic to two agriculturally important plant pathogens, Botrytis cinerea and Phytophthora ramorum. The rhizoxin analogs also caused swelling of rice roots, a symptom characteristic of rhizoxin itself, but were less toxic to pea and cucumber roots. Of the rhizoxin analogs produced by Pf-5, the predominant compound, WF-1360 F, and the newly described compound 22Z-WF-1360 F were most toxic against the two plant pathogens and three plant species. These rhizoxin analogs were tested against a panel of human cancer lines, and they exhibited potent but nonselective cytotoxicity. This study highlights the value of the genomic sequence of the soil bacterium P. fluorescens Pf-5 in providing leads for the discovery of novel metabolites with significant biological properties.

Secondary Metabolism and Interspecific Competition Affect Accumulation of Spontaneous Mutants in the GacS-GacA Regulatory System in Pseudomonas protegens.

Secondary metabolites are synthesized by many microorganisms and provide a fitness benefit in the presence of competitors and predators. Secondary metabolism also can be costly, as it shunts energy and intermediates from primary metabolism. In Pseudomonas spp., secondary metabolism is controlled by the GacS-GacA global regulatory system. Intriguingly, spontaneous mutations in gacS or gacA (Gac- mutants) are commonly observed in laboratory cultures. Here we investigated the role of secondary metabolism in the accumulation of Gac- mutants in Pseudomonas protegens strain Pf-5. Our results showed that secondary metabolism, specifically biosynthesis of the antimicrobial compound pyoluteorin, contributes significantly to the accumulation of Gac- mutants. Pyoluteorin biosynthesis, which poses a metabolic burden on the producer cells, but not pyoluteorin itself, leads to the accumulation of the spontaneous mutants. Interspecific competition also influenced the accumulation of the Gac- mutants: a reduced proportion of Gac- mutants accumulated when P. protegens Pf-5 was cocultured with Bacillus subtilis than in pure cultures of strain Pf-5. Overall, our study associated a fitness trade-off with secondary metabolism, with metabolic costs versus competitive benefits of production influencing the evolution of P. protegens, assessed by the accumulation of Gac- mutants.IMPORTANCE Many microorganisms produce antibiotics, which contribute to ecologic fitness in natural environments where microbes constantly compete for resources with other organisms. However, biosynthesis of antibiotics is costly due to the metabolic burdens of the antibiotic-producing microorganism. Our results provide an example of the fitness trade-off associated with antibiotic production. Under noncompetitive conditions, antibiotic biosynthesis led to accumulation of spontaneous mutants lacking a master regulator of antibiotic production. However, relatively few of these spontaneous mutants accumulated when a competitor was present. Results from this work provide information on the evolution of antibiotic biosynthesis and provide a framework for their discovery and regulation.

Characterization of Toxin Complex Gene Clusters and Insect Toxicity of Bacteria Representing Four Subgroups of Pseudomonas fluorescens.

Ten strains representing four lineages of the Pseudomonas fluorescens group (P. chlororaphis, P. corrugata, P. koreensis, and P. fluorescens subgroups) were evaluated for toxicity to the tobacco hornworm Manduca sexta and the common fruit fly Drosophila melanogaster. The three strains within the P. chlororaphis subgroup exhibited both oral and injectable toxicity to the lepidopteran M. sexta. All three strains have the gene cluster encoding the FitD insect toxin and a ΔfitD mutant of P. protegens strain Pf-5 exhibited diminished oral toxicity compared to the wildtype strain. Only one of the three strains, P. protegens Pf-5, exhibited substantial levels of oral toxicity against the dipteran D. melanogaster. Three strains in the P. fluorescens subgroup, which lack fitD, consistently showed significant levels of injectable toxicity against M. sexta. In contrast, the oral toxicity of these strains against D. melanogaster was variable between experiments, with only one strain, Pseudomonas sp. BG33R, causing significant levels of mortality in repeated experiments. Toxin complex (Tc) gene clusters, which encode insecticidal properties in Photorhabdus luminescens, were identified in the genomes of seven of the ten strains evaluated in this study. Within those seven genomes, six types of Tc gene clusters were identified, distinguished by gene content, organization and genomic location, but no correlation was observed between the presence of Tc genes and insect toxicity of the evaluated strains. Our results demonstrate that members of the P. fluorescens group have the capacity to kill insects by both FitD-dependent and independent mechanisms.