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

Péchy-Tarr, Maria; Bruck, Denny J.; Maurhofer, M.; Fischer, Esther; Vogne, Christelle; Henkels, Marcella D.; Donahue, Kelly M.; Grunder, J.; Loper, Joyce E.; Keel, Christoph

doi:10.1111/j.1462-2920.2008.01662.x

Cited by 143 publications

(200 citation statements)

References 81 publications

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“…On top of plant-beneficial activity, genomics has revealed unexpected and broader ecological versatility for these bacteria (Paulsen et al, 2005;Loper et al, 2012). Three of the best-characterized biocontrol strains, Pseudomonas protegens strains CHA0 and Pf-5 and Pseudomonas chlororaphis PCL1391, were shown to have potent insecticidal activity (Péchy-Tarr et al, 2008;Ruffner et al, 2013). When injected into the hemocoel of Galleria mellonella or Manduca sexta larvae, they rapidly multiply and cause larval death within a few hours (Péchy-Tarr et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Insect pathogenicity in plant-beneficial pseudomonads: phylogenetic distribution and comparative genomics

et al. 2016

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Bacteria of the genus Pseudomonas occupy diverse environments. The Pseudomonas fluorescens group is particularly well-known for its plant-beneficial properties including pathogen suppression. Recent observations that some strains of this group also cause lethal infections in insect larvae, however, point to a more versatile ecology of these bacteria. We show that 26 P. fluorescens group strains, isolated from three continents and covering three phylogenetically distinct sub-clades, exhibited different activities toward lepidopteran larvae, ranging from lethal to avirulent. All strains of sub-clade 1, which includes Pseudomonas chlororaphis and Pseudomonas protegens, were highly insecticidal regardless of their origin (animals, plants). Comparative genomics revealed that strains in this sub-clade possess specific traits allowing a switch between plant-and insect-associated lifestyles. We identified 90 genes unique to all highly insecticidal strains (sub-clade 1) and 117 genes common to all strains of sub-clade 1 and present in some moderately insecticidal strains of sub-clade 3. Mutational analysis of selected genes revealed the importance of chitinase C and phospholipase C in insect pathogenicity. The study provides insight into the genetic basis and phylogenetic distribution of traits defining insecticidal activity in plant-beneficial pseudomonads. Strains with potent dual activity against plant pathogens and herbivorous insects have great potential for use in integrated pest management for crops.

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Section: Introductionmentioning

confidence: 99%

Insect pathogenicity in plant-beneficial pseudomonads: phylogenetic distribution and comparative genomics

et al. 2016

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“…The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species [1][2][3][4][5] , as well as the wealth of information available regarding the insect's immune system [6][7][8] , and the pending genome sequence 9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species.…”

Section: Introductionmentioning

confidence: 99%

Rearing and Injection of <em>Manduca sexta</em> Larvae to Assess Bacterial Virulence

Hussa

Goodrich-Blair

2012

JoVE

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Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species [1][2][3][4][5] , as well as the wealth of information available regarding the insect's immune system [6][7][8] , and the pending genome sequence 9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter 10 . The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking 11 and oral toxicity assays 12 . Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of wholecell inocula.The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides 7 ; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR 13 . The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes 6 . To analyze these responses, injected insects can be dissected and visualized by microscopy 13,14 . Video LinkThe video component of this article can be found at

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“…Currently, genomic analysis of the two isolates is in progress, which will demonstrate which biosynthetic gene clusters for antibiotic production and other antagonistic activities are present in the two well water isolates from Algeria. Like the reference strain P. protegens CHA0 T and related strains [41,47], the isolates S4LiBe and S5LiBe also harbor insecticidal activities, as could be demonstrated by the positive Galleria mellonella injection assay and the presence of the fitD gene (Figure 3). Since the infection and growth within insect larvae constitutes a second growth cycle of these bacteria -apart from plant roots , it may be hypothesized, that these types of bacteria were enriched in the well water also harboring insect larvae.…”

Section: Discussionmentioning

confidence: 99%

“…The injection assays for virulence determination used last-instar larvae of G. mellonella (Entomos AG, Grossdietwil, Switzerland) as described before by Péchy-Tarr et al, [41] with 18 larvae per tested bacterial strain.…”

Section: Insecticidal Activitymentioning

confidence: 99%

“…Plant growth promoting activities of Pseudomonas spp. include production of indole acetic acid (IAA) [16], phosphate solubilization [60], degradation of toxic compounds [38] and production of biocontrol agents against fungal phytopathogens like the production of siderophores [7,14] or antibiotics [21,24] as well as against insect pests by the production of insect toxins [41].…”

Section: Introductionmentioning

confidence: 99%

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Heavy metal tolerant Pseudomonas protegens isolates from agricultural well water in northeastern Algeria with plant growth promoting, insecticidal and antifungal activities

Bensidhoum

Nabti

Tabli

et al. 2016

European Journal of Soil Biology

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The application of plant growth-promoting bacteria (PGPB) with biocontrol activities as inoculants of crops plants against phytopathogenic fungi and insect pests provides a biological alternative to the use of agrochemicals. Two Pseudomonas sp. strains were isolated from agricultural well water in the area of Bejaia, northeastern Algeria, located rather closely to a lead mine deposit. The isolates S4LiBe and S5LiBe had 16S rRNA gene sequence similarities of 99.4% to 99.7% with Pseudomonas protegens CHAO T and other P. protegens strains. The phenotypic profiles tested with BIOLOG-GN2-microplates were very similar, but showed also some remarkable differences. The isolates S4LiBe and S5LiBe showed plant growthpromoting potential based on the production of the phytohormone indole acetic acid and siderophores and the solubilization of insoluble phosphate. In addition, they produced chitinase and other polymer degrading enzymes. Interestingly, while S4LiBe and S5LiBe were resistant against heavy metals (2.0 mM K2Cr2O7 and 3.0 mM CoSO4, HgSO4, CdSO4 8H2O and PbCl2), the reference strain P. protegens CHAO T was very sensitive to Hg 2+ and Cd 2+ and had lower tolerance towards Co 2+ and Pb 2+ . The isolates S4LiBe and S5LiBe were very active in mycelial growth inhibition assays against Botrytis cinerea, Verticillium dahlia, Fusarium graminearum, Aspergillus niger and A. flavus (growth inhibition between 88% and 48%). Furthermore, S4LiBe and S5LiBe showed effective insecticidal activities, when tested in the Galleria injection assay and they were tested positive for the insect toxin gene fitD alike the reference strain CHA0 T . Finally, inoculation of barley seeds with S5LiBe resulted in significantly stimulated germination rate and growth of seedlings, with increased shoot length, shoot and root fresh weight, shoot and root dry weight as compared to non-inoculated plants.Thus, the heavy metal tolerant isolates S4LiBe and S5LiBe harbor a diverse potential as beneficial bacteria for agricultural application. They may be very useful even in polluted soils for the stimulation of e.g. biomass crops. The demonstration of successful isolation from agricultural well water may open more ready access for a wide variety of this kind of beneficial bacteria for agricultural application.

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

Cited by 143 publications

References 81 publications

Insect pathogenicity in plant-beneficial pseudomonads: phylogenetic distribution and comparative genomics

Insect pathogenicity in plant-beneficial pseudomonads: phylogenetic distribution and comparative genomics

Rearing and Injection of <em>Manduca sexta</em> Larvae to Assess Bacterial Virulence

Heavy metal tolerant Pseudomonas protegens isolates from agricultural well water in northeastern Algeria with plant growth promoting, insecticidal and antifungal activities

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