Joyce E. Loper

Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5

Pseudomonas fluorescens Pf-5 is a plant commensal bacterium that inhabits the rhizosphere and produces secondary metabolites that suppress soilborne plant pathogens. The complete sequence of the 7.1-Mb Pf-5 genome was determined. We analyzed repeat sequences to identify genomic islands that, together with other approaches, suggested P. fluorescens Pf-5s recent lateral acquisitions include six secondary metabolite gene clusters, seven phage regions and a mobile genomic island. We identified various features that contribute to its commensal lifestyle on plants, including broad catabolic and transport capabilities for utilizing plant-derived compounds, the apparent ability to use a diversity of iron siderophores, detoxification systems to protect from oxidative stress, and the lack of a type III secretion system and toxins found in related pathogens. In addition to six known secondary metabolites produced by P. fluorescens Pf-5, three novel secondary metabolite biosynthesis gene clusters were also identified that may contribute to the biocontrol properties of P. fluorescens Pf-5.

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.

Soilborne Plant Diseases Caused by Pythium spp.: Ecology, Epidemiology, and Prospects for Biological Control

Soilborne root diseases caused by plant pathogenic Pythium species cause serious losses in a number of agricultural production systems, which has led to a considerable effort devoted to the development of biological agents for disease control. In this article we review information on the ecology and biological control of these pathogens with the premise that a clear understanding of the ecology of the pathogen will assist in the development of efficacious biocontrol agents. The lifecycles of the pathogens and etiology of host infection also are reviewed, as are epidemiological concepts of inoculum-disease relationships and the influence of environmental factors on pathogen aggressiveness and host susceptibility. A number of fungal and bacterial biocontrol agents are discussed and parallels between their ecology and that of the target pathogens highlighted. The mechanisms by which these microbial agents suppress diseases caused by Pythium spp., such as interference with pathogen survival, disruption of the...

Evaluation of streptomycin, oxytetracycline, and copper resistance of Erwinia amylovora isolated from pear orchards in Washington State

One hundred and thirty-eight pathogenic strains of E. amylovora were isolated from fire blight cankers of pear trees from 44 orchards in the major pear-growing regions of Washington State. Ninety-eight strains, isolated from 38 of the orchards sampled, were resistant to streptomycin (1 mg/ml). Streptomycin-resistant strains of E. amylovora were ubiquitous in all of the major pear-growing regions of Washington. None of the strains tested were resistant to oxytetracycline (25 μg/ml) or CuSO 4 (0.16 mM). Nevertheless, spontaneous mutants with tolerance to 0.16 mM CuSO 4 were observed at a frequency of 10 −6 -10 −7 mutant colonies per wild type colony in most strains

Microbial Antagonism at the Root Level Is Involved in the Suppression of Fusarium Wilt by the Combination of Nonpathogenic Fusarium oxysporum Fo47 and Pseudomonas putida WCS358

ABSTRACT Two biological control agents, nonpathogenic Fusarium oxysporum Fo47 and Pseudomonas putida WCS358, were evaluated for suppression of Fusarium wilt of flax grown in nutrient solution and for suppression of the population density and metabolic activity of the causal organism F. oxysporum f. sp. lini strain Foln3GUS on root surfaces. Due to the presence of an introduced gusA reporter gene construct in Foln3GUS, the pathogen expressed beta-glucuronidase activity that was related to its carbon metabolism. At a Fo47 to Foln3GUS inoculum ratio of 100:1, both the population density of the pathogen and the beta-glucuronidase activity on and in flax roots were reduced by the nonpathogenic strain, and Fusarium wilt was suppressed. At a Fo47 to Foln3GUS inoculum ratio of 10:1, Fo47 decreased the severity of Fusarium wilt to a smaller extent and it also reduced beta-glucuronidase activity without reducing the density of Foln3GUS on flax roots. At a nonpathogenic to pathogenic Fusarium strains ratio of 10:1, the addition of P. putida WCS358 further suppressed Fusarium wilt and the density of the pathogen at the root level, whereas a mutant of WCS358 deficient in pseudobactin production had no significant effect. Iron availability to WCS358 on flax roots, assessed by ice-nucleation activity conferred from a transcriptional fusion (pvd-inaZ) of an ice-nucleation reporter gene to an iron-regulated promoter, was sufficiently low to allow pseudobactin production. P. putida WCS358 did not reduce the severity of Fusarium wilt of flax when inoculated without Fo47, and it did not improve disease suppression achieved by high inoculum doses of Fo47 (a Fo47 to Foln3GUS ratio of 100:1). Together, these data provide evidence that (i) suppression of Fusarium wilt of flax by Fo47 is related to reductions in the population density and metabolic activity of the pathogen on the root surface; (ii) WCS358 can enhance the biological control activity of Fo47, but this enhancement depends on the population of Fo47 relative to the pathogen; and (iii) pseudobactin contributes to suppression of Fusarium wilt by the combination of Fo47 and WCS358 on roots in which conditions are conducive to pseudobactin production by the bacterium.

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.

Involvement of Phenazines and Anthranilate in the Antagonism of Pseudomonas aeruginosa PNA1 and Tn5 Derivatives Toward Fusarium spp. and Pythium spp.

Pseudomonas aeruginosa PNA1, isolated from the rhizosphere of chickpea in India, suppressed Fusarium wilt of chickpea, caused by Fusarium oxysporum f. sp. ciceris, and Pythium damping-off of bean, caused by Pythium splendens. When grown in culture, PNA1 produced the phenazine antibiotics phenazine-1-carboxylic acid and oxychloraphine, and inhibited mycelial growth of F. oxysporum f. sp. ciceris, P. splendens, and certain other phytopathogenic fungi. Two mutants (FM29 and FM13) deficient in phenazine production were obtained following transposon mutagenesis of PNA1. The transposon in the genome of FM29 was localized to phnA, which is thought to encode a subunit of anthranilate synthase II involved in the phenazine biosynthesis. The FM13 mutation was complemented by trpC, which encodes indole glycerol phosphate synthase in the tryptophan biosynthesis pathway; consequently, FM13 could not grow on a minimal medium in the absence of tryptophan. Neither FM29 nor FM13 suppressed Fusarium wilt of chickpea to the ...

Molecular analysis of a novel gene cluster encoding an insect toxin in plant‐associated strains of Pseudomonas fluorescens

Pseudomonas fluorescens CHA0 and the related strain Pf-5 are well-characterized representatives of rhizosphere bacteria that have the capacity to protect crop plants from fungal root diseases, mainly by releasing a variety of exoproducts that are toxic to plant pathogenic fungi. Here, we report that the two plant-beneficial pseudomonads also exhibit potent insecticidal activity. Anti-insect activity is linked to a novel genomic locus encoding a large protein toxin termed Fit (for P. fluorescensinsecticidal toxin) that is related to the insect toxin Mcf (Makes caterpillars floppy) of the entomopathogen Photorhabdus luminescens, a mutualist of insect-invading nematodes. When injected into the haemocoel, even low doses of P. fluorescens CHA0 or Pf-5 killed larvae of the tobacco hornworm Manduca sexta and the greater wax moth Galleria mellonella. In contrast, mutants of CHA0 or Pf-5 with deletions in the Fit toxin gene were significantly less virulent to the larvae. When expressed from an inducible promoter in a non-toxic Escherichia coli host, the Fit toxin gene was sufficient to render the bacterium toxic to both insect hosts. Our findings establish the Fit gene products of P. fluorescens CHA0 and Pf-5 as potent insect toxins that define previously unappreciated anti-insect properties of these plant-colonizing bacteria.

The Genomic Sequence of Pseudomonas fluorescens Pf-5: Insights Into Biological Control

ABSTRACT The complete sequence of the 7.07 Mb genome of the biological control agent Pseudomonas fluorescens Pf-5 is now available, providing a new opportunity to advance knowledge of biological control through genomics. P. fluorescens Pf-5 is a rhizosphere bacterium that suppresses seedling emergence diseases and produces a spectrum of antibiotics toxic to plant-pathogenic fungi and oomycetes. In addition to six known secondary metabolites produced by Pf-5, three novel secondary metabolite biosynthesis gene clusters identified in the genome could also contribute to biological control. The genomic sequence provides numerous clues as to mechanisms used by the bacterium to survive in the spermosphere and rhizosphere. These features include broad catabolic and transport capabilities for utilizing seed and root exudates, an expanded collection of efflux systems for defense against environmental stress and microbial competition, and the presence of 45 outer membrane receptors that should allow for the uptake of iron from a wide array of siderophores produced by soil microorganisms. As expected for a bacterium with a large genome that lives in a rapidly changing environment, Pf-5 has an extensive collection of regulatory genes, only some of which have been characterized for their roles in regulation of secondary metabolite production or biological control. Consistent with its commensal lifestyle, Pf-5 appears to lack a number of virulence and pathogenicity factors found in plant pathogens.

Role of Iron in Rhizobacteria-Mediated Induced Systemic Resistance of Cucumber

ABSTRACT Seed treatment with the rhizosphere bacterium Serratia marcescens strain 90-166 suppressed anthracnose of cucumber, caused by Colleto-trichum orbiculare, through induced systemic resistance (ISR). When the iron concentration of a planting mix was decreased by addition of an iron chelator, suppression of cucumber anthracnose by strain 90-166 was significantly improved. Strain 90-166 produced 465 +/- 70 mg/liter of catechol siderophore, as determined by the Rioux assay in deferrated Kings medium B. The hypothesis that a catechol siderophore produced by strain 90-166 may be responsible for induction of systemic resistance by this strain was tested by evaluating disease suppression by a mini-Tn5-phoA mutant deficient in siderophore production. Sequence analysis of genomic DNA flanking the mini-Tn5-phoA insertion identified the target gene as entA, which encodes an enzyme in the catechol siderophore biosynthetic pathways of several bacteria. Severity of anthracnose of cucumbers treated with the entA mutant was not significantly different (P = 0.05) from the control, whereas plants treated with wild-type 90-166 had significantly less disease (P = 0.05) than the control. Total (internal and external) population sizes of 90-166 and the entA mutant on roots did not differ significantly (P = 0.05) at any sample time, whereas internal population sizes of the entA mutant were significantly lower (P = 0.05) than those of the wild-type strain at two sampling times. These data suggest that catechol siderophore biosynthesis genes in Serratia marcescens 90-166 are associated with ISR but that this role may be indirect via a reduction in internal root populations.