Leafy Spurge (Euphorbia esula) Cell Cultures for Screening Deleterious Rhizobacteria

Abstract

Bioassays using cell cultures and callus tissues of leafy spurge were devised to evaluate the potential of rhizobacteria as biocontrol agents. Rhizobacteria isolated from roots of leafy spurge seedlings were screened in suspension-cultured leafy spurge cells. Cell viability was assessed using the Evan's blue bioassay 48 h after bacterial inoculation. Among the 30 isolates tested, LS102 and LS105 consistently caused intensive cell death determined by measuring the A630 of the inoculated cell cultures. Cell death was 2.5 to 3 times higher in cultures inoculated with LS105 and LS102, respectively, than in the control. Population levels of the two isolates within cell cultures and callus tissues of leafy spurge increased during the first 48 h. Leafy spurge callus tissues were inoculated with rhizobacteria either directly or by using the Host Pathogen Interaction System (HPIS). The latter exposes calli to bacteria without any physical contact. LS102 caused cellular leakage and eventually death of the callus tissue. Callus growth was reduced by about 30 to 70% when exposed to LS102 and LS105, respectively. Results suggest that these two isolates may affect leafy spurge at the cellular level by different mechanisms. A screening method based on cell cultures and callus tissues offers a good and rapid technique for detecting deleterious rhizobacteria with potential as biocontrol agents for leafy spurge.

EVALUACIÓN DEL EFECTO DEL CADMIO SOBRE LA ACTIVIDAD DE FENOLOXIDASAS DE TEJIDO CALLOSO DE Epithelantha micromeris INOCULADO CON RIZOBACTERIAS

“…Se obtuvo el peso fresco de las masas celulares y se evaluó la viabilidad celular de éstas, con el empleo del colorante azul de Evans, que no es permeable y produce baja toxicidad. El empleo de este colorante esta descrito por varios autores (Gaff y Okong'o-Ogola 1971, Turner y Novacky 1974, Souissi y Kremer 1994, 1998, Song et al 1999, Iriti et al 2006, Kollárová et al 2017) para determinar el número relativo de células muertas en cultivos de células en suspensión y de callo, mediante la cuenta de células muertas que se tiñen con el azul y el número de células vivas que no presentan esta coloración. La tinción se realizó de acuerdo con el método modificado por Toledo (2012) y consistió en colocar las masas celulares en tubos de ensaye, adicionando 1 mL de solución de azul de Evans al 0.5 % durante 10 min.…”

Constraints in implementing biological weed control: A review

“…Some concerns have been raised about the efficacy and rapidity of bacterial‐screening techniques. Souissi and Kremer (1994) have described a screening method based on cell cultures and callus tissue that offers a rapid, aseptic and inexpensive technique for detecting deleterious rhizobacteria. Differences between field tests and chamber tests create uncertainty in the validity of preliminary tests.…”

Influence of Deleterious Rhizobacteria on Leafy Spurge (Euphorbia esula) Roots

“…Two DRB strains originally collected from roots of leafy spurge in Montana (LS102) and South Dakota (LS174) were used in this study. Both strains were identified as P. fluorescens using the API 20NE test system with verification by fatty acid methyl ester analyses as described previously (Souissi and Kremer 1994). These strains also severely inhibited leafy spurge growth in greenhouse assays (Souissi et al 1997).…”

“…The biocontrol tactic based on use of DRB delivers selected nonparasitic bacteria that colonize plant roots and suppress plant growth (Kennedy et al 1991;Kremer et al 1990;Kremer and Kennedy 1996). Several strains of P. fluorescens have been isolated from leafy spurge roots and are phytotoxic and host-specific toward leafy spurge calli and seedlings in laboratory and greenhouse assays (Souissi and Kremer 1994). The objective of this study was to determine if selected strains of DRB collected from leafy spurge infestations in South Dakota and Montana could colonize and influence plant growth under field conditions.…”