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Pathogenicity of Isolates of Serratia Marcescens towards Larvae of the Scarab Phyllophaga Blanchardi (Coleoptera).

Pineda-Castellanos ML, Rodríguez-Segura Z, Villalobos FJ, Hernández L, Lina L, Nuñez-Valdez ME - Pathogens (2015)

Bottom Line: Oral and injection bioassays using healthy Phyllophaga blanchardi larvae fed with the S. marcescens isolates showed different degrees of antifeeding effect and mortality.S. marcescens (Sm81) cell-free culture supernatant caused significant antifeeding effect and mortality to P. blanchardi larvae by oral bioassay and also mortality by injection bioassay.This insecticidal protein could have applications in agricultural biotechnology.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación en Dinámica Celular, Instituto de Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Col. Chamilpa, CP 62209, Cuernavaca, Morelos, Mexico. mlpc@uaem.mx.

ABSTRACT
Serratia marcescens is a Gram negative bacterium (Enterobacteriaceae) often associated with infection of insects. In order to find pathogenic bacteria with the potential to control scarab larvae, several bacterial strains were isolated from the hemocoel of diseased Phyllophaga spp (Coleoptera:Scarabaeidae) larvae collected from cornfields in Mexico. Five isolates were identified as Serratia marcescens by 16S rRNA gene sequencing and biochemical tests. Oral and injection bioassays using healthy Phyllophaga blanchardi larvae fed with the S. marcescens isolates showed different degrees of antifeeding effect and mortality. No insecticidal activity was observed for Spodoptera frugiperda larvae (Lepidoptera: Noctuidae) by oral inoculation. S. marcescens (Sm81) cell-free culture supernatant caused significant antifeeding effect and mortality to P. blanchardi larvae by oral bioassay and also mortality by injection bioassay. Heat treated culture broths lost the ability to cause disease symptoms, suggesting the involvement of proteins in the toxic activity. A protein of 50.2 kDa was purified from the cell-free broth and showed insecticidal activity by injection bioassay towards P. blanchardi. Analysis of the insecticidal protein by tandem- mass spectrometry (LC-MS/MS) showed similarity to a Serralysin-like protein from S. marcescens spp. This insecticidal protein could have applications in agricultural biotechnology.

No MeSH data available.


Related in: MedlinePlus

Electrophoretic analysis of S. marcescens 81 cell-free culture supernatants. Panel A. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%) analysis of cell-free culture supernatant of S. marcescens 81 strain. Cell-free culture supernatant was concentrated and adjusted to 30 µL per line. Panel B. SDS-PAGE analysis of the Sm81 50 kDa protein purified by HiTrap Q FF ion exchange column. Twenty µg of Sm81P50 was charged in the gel line. Panel C. Zymography showing the 50 kDa protease from the S. marcescens strains cell-free culture supernatants. Gelatin-SDS-PAGE stained with coomassie brilliant blue. The protein concentration was adjusted to 6 µg per line. For Sm65 the protein concentration was 0.6 µg per line.
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pathogens-04-00210-f006: Electrophoretic analysis of S. marcescens 81 cell-free culture supernatants. Panel A. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%) analysis of cell-free culture supernatant of S. marcescens 81 strain. Cell-free culture supernatant was concentrated and adjusted to 30 µL per line. Panel B. SDS-PAGE analysis of the Sm81 50 kDa protein purified by HiTrap Q FF ion exchange column. Twenty µg of Sm81P50 was charged in the gel line. Panel C. Zymography showing the 50 kDa protease from the S. marcescens strains cell-free culture supernatants. Gelatin-SDS-PAGE stained with coomassie brilliant blue. The protein concentration was adjusted to 6 µg per line. For Sm65 the protein concentration was 0.6 µg per line.

Mentions: In a next step, in order to study whether the S. marcescens isolates produced one or several protease(s) of similar or different molecular size, the S. marcescens cell-free culture supernatants were analyzed by in gel zymography as reported [11]. It was observed in the zymography (Figure 6) that the five S. marcescens isolates produced a single band with protease activity, corresponding to an extracellular protease of about 50 kDa (Figure 6, Panel C). No other protein with protease activity was detected by this system.


Pathogenicity of Isolates of Serratia Marcescens towards Larvae of the Scarab Phyllophaga Blanchardi (Coleoptera).

Pineda-Castellanos ML, Rodríguez-Segura Z, Villalobos FJ, Hernández L, Lina L, Nuñez-Valdez ME - Pathogens (2015)

Electrophoretic analysis of S. marcescens 81 cell-free culture supernatants. Panel A. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%) analysis of cell-free culture supernatant of S. marcescens 81 strain. Cell-free culture supernatant was concentrated and adjusted to 30 µL per line. Panel B. SDS-PAGE analysis of the Sm81 50 kDa protein purified by HiTrap Q FF ion exchange column. Twenty µg of Sm81P50 was charged in the gel line. Panel C. Zymography showing the 50 kDa protease from the S. marcescens strains cell-free culture supernatants. Gelatin-SDS-PAGE stained with coomassie brilliant blue. The protein concentration was adjusted to 6 µg per line. For Sm65 the protein concentration was 0.6 µg per line.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493471&req=5

pathogens-04-00210-f006: Electrophoretic analysis of S. marcescens 81 cell-free culture supernatants. Panel A. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%) analysis of cell-free culture supernatant of S. marcescens 81 strain. Cell-free culture supernatant was concentrated and adjusted to 30 µL per line. Panel B. SDS-PAGE analysis of the Sm81 50 kDa protein purified by HiTrap Q FF ion exchange column. Twenty µg of Sm81P50 was charged in the gel line. Panel C. Zymography showing the 50 kDa protease from the S. marcescens strains cell-free culture supernatants. Gelatin-SDS-PAGE stained with coomassie brilliant blue. The protein concentration was adjusted to 6 µg per line. For Sm65 the protein concentration was 0.6 µg per line.
Mentions: In a next step, in order to study whether the S. marcescens isolates produced one or several protease(s) of similar or different molecular size, the S. marcescens cell-free culture supernatants were analyzed by in gel zymography as reported [11]. It was observed in the zymography (Figure 6) that the five S. marcescens isolates produced a single band with protease activity, corresponding to an extracellular protease of about 50 kDa (Figure 6, Panel C). No other protein with protease activity was detected by this system.

Bottom Line: Oral and injection bioassays using healthy Phyllophaga blanchardi larvae fed with the S. marcescens isolates showed different degrees of antifeeding effect and mortality.S. marcescens (Sm81) cell-free culture supernatant caused significant antifeeding effect and mortality to P. blanchardi larvae by oral bioassay and also mortality by injection bioassay.This insecticidal protein could have applications in agricultural biotechnology.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación en Dinámica Celular, Instituto de Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Col. Chamilpa, CP 62209, Cuernavaca, Morelos, Mexico. mlpc@uaem.mx.

ABSTRACT
Serratia marcescens is a Gram negative bacterium (Enterobacteriaceae) often associated with infection of insects. In order to find pathogenic bacteria with the potential to control scarab larvae, several bacterial strains were isolated from the hemocoel of diseased Phyllophaga spp (Coleoptera:Scarabaeidae) larvae collected from cornfields in Mexico. Five isolates were identified as Serratia marcescens by 16S rRNA gene sequencing and biochemical tests. Oral and injection bioassays using healthy Phyllophaga blanchardi larvae fed with the S. marcescens isolates showed different degrees of antifeeding effect and mortality. No insecticidal activity was observed for Spodoptera frugiperda larvae (Lepidoptera: Noctuidae) by oral inoculation. S. marcescens (Sm81) cell-free culture supernatant caused significant antifeeding effect and mortality to P. blanchardi larvae by oral bioassay and also mortality by injection bioassay. Heat treated culture broths lost the ability to cause disease symptoms, suggesting the involvement of proteins in the toxic activity. A protein of 50.2 kDa was purified from the cell-free broth and showed insecticidal activity by injection bioassay towards P. blanchardi. Analysis of the insecticidal protein by tandem- mass spectrometry (LC-MS/MS) showed similarity to a Serralysin-like protein from S. marcescens spp. This insecticidal protein could have applications in agricultural biotechnology.

No MeSH data available.


Related in: MedlinePlus