Phenazine-1-Carboxylic Acid Production by Pseudomonas fluorescens LBUM636 Alters Phytophthora infestans Growth and Late Blight Development

Phytophthora infestans causes late blight of potato, one of the most devastating diseases affecting potato production. Alternative approaches for controlling late blight are being increasingly sought due to increasing environmental concerns over the use of chemical pesticides and the increasing resistance of P. infestans to fungicides. Our research group has isolated a new strain of Pseudomonas fluorescens (LBUM636) of biocontrol interest producing the antibiotic phenazine-1-carboxylic acid (PCA). Wild-type LBUM636 was shown to significantly inhibit the growth of Phytophthora infestans in in vitro confrontational assays whereas its isogenic mutant (phzC−; not producing PCA) only slightly altered the pathogen’s growth. Wild-type LBUM636 but not the phzC− mutant also completely repressed disease symptom development on tubers. A pot experiment revealed that wild-type LBUM636 can significantly reduce P. infestans populations in the rhizosphere and in the roots of potato plants, as well as reduce in planta disease symptoms due to PCA production. The expression of eight common plant defense-related genes (ChtA, PR-1b, PR-2, PR-5, LOX, PIN2, PAL-2, and ERF3) was quantified in tubers, roots, and leaves by reverse-transcription quantitative polymerase chain reaction and revealed that the biocontrol observed was not associated with the induction of a plant defense response by LBUM636. Instead, a direct interaction between P. infestans and LBUM636 is required and PCA production appears to be a key factor for LBUM636’s biocontrol ability.

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Respuesta de biosurfactantes producidos por Pseudomonas fluorescens para el control de la gota de la papa Phytophthora infestans (Mont) de Bary, bajo condiciones controlada.

Ciencia y Tecnología Agropecuaria ◽

10.21930/rcta.vol11_num1_art:191 ◽

2010 ◽

Vol 11 (1) ◽

pp. 21

Author(s):

Hugo F. Rivera ◽

Erika P. Martínez ◽

Jairo A. Osorio ◽

Edgar Martínez

Keyword(s):

Late Blight ◽

Phytophthora Infestans ◽

Pseudomonas Fluorescens ◽

Negative Impact ◽

Potato Late Blight ◽

Pathogen Resistance ◽

Production Costs ◽

In Vitro Assays

Phytophthora infestans (Mont.) de Bary, agente causal de la gota de la papa, es considerado la principal limitante de la producción de este cultivo en Colombia. El control habitual del patógeno se realiza con fungicidas de tipo sistémico, que incrementan los costos de producción, pueden inducir la resistencia del patógeno y tiene un impacto negativo en el ambiente. Por tanto, se llevó a cabo este estudio con el propósito de buscar alternativas amigables con el ambiente, que hagan parte de un paquete tecnológico eficaz de control. Dos cepas nativas de Psedomonas fluorescens (039T y 021V), provenientes de cultivos de papa, fueron evaluadas contra P. infestans. Las suspensiones bacterianas y los biosurfactantes parcialmente purificados (BPP), producidos por éstas (obtenidos en medio mínimo de sales con querosén), fueron aplicados sobre foliolos desprendidos en ensayos in vitro y experimentos in vivo en plantas de papa, en condiciones controladas en casa de malla. Los resultados demostraron la capacidad que tienen los biosurfactantes y las suspensiones bacterianas para controlar al patógeno, ya que el BPP 039T logró reducir el nivel de severidad de la enfermedad en 79,9% in vitro y 38,5% in vivo, mientras que el BPP 021V redujo en 78,7% in vitro y 30,2% in vivo. Las suspensiones bacterianas redujeron el nivel de severidad en 72,4% (039T) y 66,1% (021V) en las evaluaciones in vitro y 35% en los experimentos in vivo. Los resultados de esta investigación muestran el potencial que tienen los biosurfactantes para el control de la gota en Colombia. Evaluation of Biosurfactants Produced by Pseudomonas fluorescens for Potato Late Blight Control (Phytophthora infestans (Mont) de Bary) Under Controlled ConditionsPhytophthora infestans (Mont.) de Bary, causal agent of potato late blight is considered the main limiting pathogen for the production of this crop in Colombia. The usual control of the disease has been performed with systemic fungicides which increase production costs, can induce pathogen resistance and have a negative impact on the environment. Therefore, this study was carried out in order to find effective and environmentally friendly control alternatives for potato late blight. Two Pseudomonas fluorescens native strains (039T and 021V) isolated from potato crops were evaluated against P. infestans. Bacterial suspensions (obtained from minimal salts medium added with kerosene) and partially purified biosurfactants (BPP) were applied on detached leaflets for in vitro assays and on potato plants in greenhouse, for in vivo assays and the measure of inhibitory effect of the disease was assessed. The results showed the ability of P. fluorescens biosurfactants and bacterial suspensions to control the pathogen. BPP 039T was able to reduce the level of severity disease by 79.9% in vitro and 38.5% in vivo, whereas BPP 021V decreased 78.7% in vitro and 30.2% in vivo. Bacterial suspensions reduced the severity level in 72.4% (039T) and 66.1% (021V) in vitro assessments and 35% in the in vivo experiment. These results show the potential of P. fluorescens biosurfactants to control the potato late blight in Colombia.

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Differential Activity of Carboxylic Acid Amide Fungicides Against Various Developmental Stages of Phytophthora infestans

Phytopathology ◽

10.1094/phyto-97-10-1274 ◽

2007 ◽

Vol 97 (10) ◽

pp. 1274-1283 ◽

Cited By ~ 29

Author(s):

Yigal Cohen ◽

Ulrich Gisi

Keyword(s):

Carboxylic Acid ◽

Late Blight ◽

Phytophthora Infestans ◽

Developmental Stages ◽

Acid Amide ◽

Intrinsic Activity ◽

Dependent Manner ◽

Carboxylic Acid Amide ◽

In Planta ◽

Mycelium Growth

Three carboxylic acid amide (CAA) fungicides, mandipropamid (MPD), dimethomorph (DMM) and iprovalicarb (IPRO) were examined for their effects on various asexual developmental stages of Phytophthora infestans in vitro and in planta. Germination of cystospores and direct germination of sporangia were inhibited with nanomole concentrations of MPD (0.005 μg/ml) and micromole concentrations of DMM (0.05 μg/ml) or IPRO (0.5 μg/ml). A temporary exposure of 1 h to CAAs was not detrimental to germination and infectivity of sporangia or cystospores. CAAs applied to cystospores at 1 h after the onset of germination did not prevent the emergence of germ tubes, but inhibited their further growth and deformed their shape. None of the fungicides affected discharge of zoospores from sporangia or the encystment (cell wall formation/assembly) of the zoospores. Mycelium growth in solid or liquid media was inhibited with micromole concentrations. CAAs mixed with sporangia and drop inoculated onto detached leaves strongly suppressed infection. Curative application at 1 day postinoculation (dpi) required higher concentrations of CAAs than preventive application to inhibit infection and lost its effectiveness at 2 dpi. When sprayed on established late blight lesions 4 days after inoculation, CAAs reduced sporangial production in a dose-dependent manner. Trans-laminar protection of potato or tomato leaves, although achieved with higher doses, was more effective with MPD than with DMM or IPRO. Shade house studies demonstrated superior control of late blight epidemics by MPD compared with the other molecules. The data suggest that germ tube formation by cystospores or sporangia is the most sensitive stage in the life cycle of P. infestans to CAAs. Of the three CAAs, MPD had the highest intrinsic activity against spore germination. This property, together with its better trans-laminar activity, makes MPD more effective than DMM or IPRO in controlling epidemics caused by P. infestans.

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Mutagenesis of Phytophthora infestans for Resistance Against Carboxylic Acid Amide and Phenylamide Fungicides

Plant Disease ◽

10.1094/pdis-92-5-0675 ◽

2008 ◽

Vol 92 (5) ◽

pp. 675-683 ◽

Cited By ~ 20

Author(s):

Avia (Evgenia) Rubin ◽

Dror Gotlieb ◽

Ulrich Gisi ◽

Yigal Cohen

Keyword(s):

Carboxylic Acid ◽

Phytophthora Infestans ◽

Mating Type ◽

Uv Light ◽

Acid Amide ◽

Late Blight Resistance ◽

Plasmopara Viticola ◽

Carboxylic Acid Amide ◽

In Planta

The carboxylic acid amide (CAA) fungicides mandipropamid, dimethomorph, iprovalicarb, and the phenylamide fungicide mefenoxam (MFX, the active enantiomer of metalaxyl) are anti-oomycete fungicides effective against downy mildews and late blight. Resistance against MFX was reported in nature in several oomycetes including Phytophthora infestans and Plasmopara viticola, whereas resistance against CAAs was reported in P. viticola but not in P. infestans. In this study the mutability of P. infestans for resistance against CAAs and MFX (as a control) was explored under laboratory conditions. UV light or chemical mutagens (e.g., ethyl methan sulfonate [EMS]) were applied to sporangia, and the emergence of mutants resistant to CAAs or MFX, or with altered mating type, was followed. Many mutants resistant to CAAs developed at generation 0 after mutagenesis, but all showed erratic, instable resistance in planta, diminishing after 1 to 8 asexual infection cycles, and failed to grow on CAA-amended medium. In contrast, 19 mutants resistant to MFX were obtained: 6 with UV irradiation (in isolates 28 or 96) and 13 with EMS (in isolates 408, 409, and 410). In three experiments, a shift in mating type, from A1 to A2, was detected. To elucidate whether or not resistance to CAAs is recessive and therefore might emerge only after sexual recombination, A1 and A2 mutants were crossed and the F1 and F2 progeny isolates were tested for resistance. Offspring isolates segregated for resistance to MFX, with resistant isolates maintaining stable resistance in vitro and in planta, whereas all progeny isolates failed to show stable resistance to CAAs in planta or in vitro. The data suggest that P. infestans could be artificially mutated for resistance against MFX, but not against CAAs.

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The Role of a P1-Type ATPase from Pseudomonas fluorescens SBW25 in Copper Homeostasis and Plant Colonization

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-5-0581 ◽

2007 ◽

Vol 20 (5) ◽

pp. 581-588 ◽

Cited By ~ 21

Author(s):

Xue-Xian Zhang ◽

Paul B. Rainey

Keyword(s):

Pseudomonas Fluorescens ◽

Mutant Strain ◽

Copper Ions ◽

Biological Significance ◽

Copper Homeostasis ◽

Plant Colonization ◽

Wild Type ◽

Pseudomonas Fluorescens Sbw25 ◽

Plant Surfaces

The genome of the plant-colonizing bacterium Pseudomonas fluorescens SBW25 possesses a putative copper-transporting P1-type ATPase (CueA) that is induced on the plant surfaces. Using a chromosomally-integrated cueA-'lacZ fusion, we show that transcription of cueA can be induced (in vitro) by ions of copper, silver, gold, and mercury. To investigate the biological significance of cueA, a nonpolar cueA deletion mutant (SBW25ΔcueA) was constructed. This mutant strain displayed a twofold reduction in its tolerance to copper compared with the wild-type strain; however, no change was observed in the sensitivity of the mutant strain to silver, gold, or mercury ions. To obtain insight into the ecological significance of cueA, the competitive ability of SBW25ΔcueA was determined relative to wild-type SBW25 in three environments (none contained added copper): minimal M9 medium, the root of sugar beet (Beta vulgaris), and the root of pea (Pisum sativum). Results showed that the fitness of SBW25ΔcueA was not different from the wild type in laboratory medium but was compromised in the two plant environments. Taken together, these data demonstrate a functional role for CueA in copper homeostasis and reveal an ecologically significant contribution to bacterial fitness in the plant rhizosphere. They also suggest that copper ions accumulate on plant surfaces.

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Triple Gene Block Protein Interactions Involved in Movement of Barley Stripe Mosaic Virus

Journal of Virology ◽

10.1128/jvi.02586-07 ◽

2008 ◽

Vol 82 (10) ◽

pp. 4991-5006 ◽

Cited By ~ 42

Author(s):

Hyoun-Sub Lim ◽

Jennifer N. Bragg ◽

Uma Ganesan ◽

Diane M. Lawrence ◽

Jialin Yu ◽

...

Keyword(s):

Mosaic Virus ◽

Triple Gene Block ◽

Cell Movement ◽

Wild Type Virus ◽

Barley Stripe Mosaic Virus ◽

Wild Type ◽

In Planta ◽

Gene Block ◽

Physical Interactions

ABSTRACT Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.

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A Polyamine Oxidase from Selaginella lepidophylla (SelPAO5) can Replace AtPAO5 in Arabidopsis through Converting Thermospermine to Norspermidine instead to Spermidine

Plants ◽

10.3390/plants8040099 ◽

2019 ◽

Vol 8 (4) ◽

pp. 99 ◽

Cited By ~ 1

Author(s):

G. H. M. Sagor ◽

Tomonobu Kusano ◽

Thomas Berberich

Keyword(s):

Normal Values ◽

Normal Growth ◽

Polyamine Oxidase ◽

Loss Of Function ◽

Wild Type ◽

In Planta ◽

Selaginella Lepidophylla ◽

Growth Phenotype ◽

Polyamine Oxidases

Of the five polyamine oxidases in Arabidopsis thaliana, AtPAO5 has a substrate preference for the tetraamine thermospermine (T-Spm) which is converted to triamine spermidine (Spd) in a back-conversion reaction in vitro. A homologue of AtPAO5 from the lycophyte Selaginella lepidophylla (SelPAO5) back-converts T-Spm to the uncommon polyamine norspermidine (NorSpd) instead of Spd. An Atpao5 loss-of-function mutant shows a strong reduced growth phenotype when growing on a T-Spm containing medium. When SelPAO5 was expressed in the Atpao5 mutant, T-Spm level decreased to almost normal values of wild type plants, and NorSpd was produced. Furthermore the reduced growth phenotype was cured by the expression of SelPAO5. Thus, a NorSpd synthesis pathway by PAO reaction and T-Spm as substrate was demonstrated in planta and the assumption that a balanced T-Spm homeostasis is needed for normal growth was strengthened.

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Direct evidence for a new mode of plant defense against insects via a novel polygalacturonase-inhibiting protein expression strategy

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014027 ◽

2020 ◽

Vol 295 (33) ◽

pp. 11833-11844

Author(s):

Wiebke Haeger ◽

Jana Henning ◽

David G. Heckel ◽

Yannick Pauchet ◽

Roy Kirsch

Keyword(s):

Cell Wall ◽

Plant Defense ◽

Plant Cell Wall ◽

Direct Evidence ◽

Larval Growth ◽

Gene Families ◽

Preliminary Evidence ◽

Cell Wall Polysaccharide ◽

In Planta

Plant cell wall–associated polygalacturonase-inhibiting proteins (PGIPs) are widely distributed in the plant kingdom. They play a crucial role in plant defense against phytopathogens by inhibiting microbial polygalacturonases (PGs). PGs hydrolyze the cell wall polysaccharide pectin and are among the first enzymes to be secreted during plant infection. Recent studies demonstrated that herbivorous insects express their own PG multi-gene families, raising the question whether PGIPs also inhibit insect PGs and protect plants from herbivores. Preliminary evidence suggested that PGIPs may negatively influence larval growth of the leaf beetle Phaedon cochleariae (Coleoptera: Chrysomelidae) and identified BrPGIP3 from Chinese cabbage (Brassica rapa ssp. pekinensis) as a candidate. PGIPs are predominantly studied in planta because their heterologous expression in microbial systems is problematic and instability and aggregation of recombinant PGIPs has complicated in vitro inhibition assays. To minimize aggregate formation, we heterologously expressed BrPGIP3 fused to a glycosylphosphatidylinositol (GPI) membrane anchor, immobilizing it on the extracellular surface of insect cells. We demonstrated that BrPGIP3_GPI inhibited several P. cochleariae PGs in vitro, providing the first direct evidence of an interaction between a plant PGIP and an animal PG. Thus, plant PGIPs not only confer resistance against phytopathogens, but may also aid in defense against herbivorous beetles.

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