The lipopolysaccharide biosynthesis core of the Mexican pathogenic strain Serratia entomophila is associated with toxicity to larvae of Phyllophaga blanchardi.

The Mexican bacteria Serratia entomophila strain Mor4.1 (Enterobacteriaceae) is pathogenic to coleopteran species of the Phyllophaga genus, which are considered important soil-dwelling pests. Mor4.1 causes anti-feeding activity and mortality to larvae after oral and injection bio-assay either, by bacteria or by cell free culture broth inoculation. The pathogenic determinants of Mor4.1 have not been elucidated. We hypothesize that Mor4.1 produces several toxins and other virulence factors, some acting at the level of the insect gut and others at the hemocoel. To identify and characterize virulence factors, a fosmid library of S. entomophila Mor4.1 was made in Escherichia coli. Five different insecticidal clones were isolated by injecting individual clones into Phyllophaga blanchardi larvae. The complete 40 kb DNA sequence and gene organization of clone G8 was determined. By comparative genomics, 21 genes were associated with virulence. By transposon (Tn5) insertion mutagenesis of G8 and further bio-assays we show that a dUTPase, a flavoprotein and a heptosyltransferase III, are key factors for G8 toxic activity. The heptosyltransferase III, is part of the lipopolysaccharide (LPS) biosynthesis core. We demonstrated that purified LPS from G8 and Mor4.1 are toxic to P. blanchardi larvae by injection bio-assay.

Identification of a putative Mexican strain of Serratia entomophila pathogenic against root-damaging larvae of Scarabaeidae (Coleoptera).

The larvae of scarab beetles, known as "white grubs" and belonging to the genera Phyllophaga and Anomala (Coleoptera: Scarabaeidae), are regarded as soil-dwelling pests in Mexico. During a survey conducted to find pathogenic bacteria with the potential to control scarab larvae, a native Serratia sp. (strain Mor4.1) was isolated from a dead third-instar Phyllophaga blanchardi larva collected from a cornfield in Tres Marías, Morelos, Mexico. Oral bioassays using healthy P. blanchardi larvae fed with the Mor4.1 isolate showed that this strain was able to cause an antifeeding effect and a significant loss of weight. Mortality was observed for P. blanchardi, P. trichodes, and P. obsoleta in a multidose experiment. The Mor4.1 isolate also caused 100% mortality 24 h after intracoelomic inoculation of the larvae of P. blanchardi, P. ravida, Anomala donovani and the lepidopteran insect Manduca sexta. Oral and injection bioassays were performed with concentrated culture broths of the Mor4.1 isolate to search for disease symptoms and mortality caused by extracellular proteins. The results have shown that Mor4.1 broths produce significant antifeeding effects and mortality. Mor4.1 broths treated with proteinase K lost the ability to cause disease symptoms and mortality, in both the oral and the injection bioassays, suggesting the involvement of toxic proteins in the disease. The Mor4.1 isolate was identified as a putative Serratia entomophila Mor4.1 strain based on numerical taxonomy and phylogenetic analyses done with the 16S rRNA gene sequence. The potential of S. entomophila Mor4.1 and its toxins to be used in an integrated pest management program is discussed.

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

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.