Evaluation of Pseudomonas fluorescens PICF7 as potencial biocontrol agent against Plasmopara halstedii in sunflower

Abstract

One of the most important biotic constraints for sunflower production is the soil-borne, obligate-parasitic oomycete Plasmopara halstedii Farl. Berl. & de Toni, which causes sunflower downy mildew (SDM). Control of SDM by means of biocontrol agents (BCAs) has been poorly investigated so far. Our previous studies have demonstrated that Pseudomonas fluorescens PICF7 is an effective BCA against Verticillium wilt of olive, a disease caused by the soil-borne pathogenic fungus Verticillium dahliae Kleb. Moreover, strain PICF7 was confirmed as endophyte in olive roots. Recently, colonization and persistence in Arabidopsis thaliana roots of strain PICF7 and one spontaneous rifampicin-resistant mutant derivative (PICF7Rf) were assessed, thus confirming the ability of this strain to colonize different plant species. Considering these antecedents, we aimed in this study to determine whether P. fluorescens PICF7: (1) colonize and persist in sunflower roots; (2) promote growth of sunflower at early stages; and (3) control SDM. Hybrid seeds genetically susceptible to SDM were dipped in bacterial suspensions of PICF7 (aprox. 1 x 109 cfu/mL) or PICF7Rf, (aprox. 1 x 109 cfu/mL) or in 10 mM MgSO4•7H2O (control treatment) at 23ºC, 150 rpm for 5h. Seeds of each treatment were germinated and subsequently immersed in a zoosporangial suspension of an isolate of P. halstedii collected in southern Spain in 2010 (2 x 104 zoosporangia/mL) prepared in water for 5-h at 23ºC, 150 rpm. Bioassays were then designed and conducted under controlled growth conditions. Ten pots per treatment were filled with perlite and five sunflower seeds were sown in each pot. Plants were grown at 23ºC, 70-100% relative humidity and a 12h photoperiod (100 µE•m-2•s-1) for 18-25 days. Colonization and persistence of PICF7 and PICF7Rf in roots were assessed at different time points along the bioassays. Root macerates were diluted and sown in King’s B (KB) agar (PICF7) or KB agar amended with rifampicin (PICF7Rf). To assess growth promotion of sunflower seedlings upon bacteria inoculation, the dry-weight of the above-ground part of plants was determined at the end of the experiments. Disease incidence (percentage of diseased plants per pot) and symptoms were scored at 10 days after bacterial inoculation and at the end of the experiments. Results showed that strains PICF7 and PICF7Rf are able to colonize sunflower roots from the very first moment after seed bacterization (107-108 cfu/g) and persist until the end (about three weeks) of the experiments (107cfu/g). On the other hand, no significant differences of dry-weight were found between control and bacteria-inoculated plants. Plants inoculated with the pathogen displayed similar final disease incidences whether they were bacterized (52%, PICF7; 68%, PICF7Rf) or not (64%). Although results showed that Pseudomonas fluorescens PICF7 and PICF7Rf are good colonizers of sunflower roots, neither increase of the above-ground biomass, nor control of SDM were observed under the tested experimental conditions. Root colonization ability by an enhanced green fluorescent protein (EGFP)-tagged derivative of strain PICF7 is currently being evaluated by confocal laser scanning microscopy (CLSM). The aim will be to determine whether the lack of SDM suppression could be explained by a failure of PICF7 to establish as an endophyte in sunflower root tissues. Biological control may offer an environmentally-friendly approach fitting criteria of modern sustainable agriculture and may be a complementary action to other disease control measures. This study is one of the first attempts to evaluate the potential biocontrol effectiveness of a beneficial, naturally-occurring bacteria originating from the same bioclimatic area as sunflower is cultivated.