Colonization ofPythium oligandrumin the Tomato Rhizosphere for Biological Control of Bacterial Wilt Disease Analyzed by Real-Time PCR and Confocal Laser-Scanning Microscopy

Abstract

It recently has been reported that the non-plant-pathogenic oomycete Pythium oligandrum suppresses bacterial wilt caused by Ralstonia solanacearum in tomato. As one approach to determine disease-suppressive mechanisms of action, we analyzed the colonization of P. oligandrum in rhizospheres of tomato using real-time polymerase chain reaction (PCR) and confocal laser-scanning microscopy. The real-time PCR specifically quantified P. oligandrum in the tomato rhizosphere that is reliable over a range of 0.1 pg to 1 ng of P. oligandrum DNA from 25 mg dry weight of soil. Rhizosphere populations of P. oligandrum from tomato grown for 3 weeks in both unsterilized and sterilized field soils similarly increased with the initial application of at least 5 x 10(5) oospores per plant. Confocal microscopic observation also showed that hyphal development was frequent on the root surface and some hyphae penetrated into root epidermis. However, rhizosphere population dynamics after transplanting into sterilized soil showed that the P. oligandrum population decreased with time after transplanting, particularly at the root tips, indicating that this biocontrol fungus is rhizosphere competent but does not actively spread along roots. Protection over the long term from root-infecting pathogens does not seem to involve direct competition. However, sparse rhizosphere colonization of P. oligandrum reduced the bacterial wilt as well as more extensive colonization, which did not reduce the rhizosphere population of R. solanacearum. These results suggest that competition for infection sites and nutrients in rhizosphere is not the primary biocontrol mechanism of bacterial wilt by P. oligandrum.

Influence of Pythium oligandrum Biocontrol on Fungal and Oomycete Population Dynamics in the Rhizosphere

“…In particular, Pythium oligandrum is an effective biocontrol agent (2,14,49,64). This oomycete colonizes roots without damaging the host plant cells (24,45) and survives in the rhizosphere, where it exerts its biocontrol (57). P. oligandrum acts through both direct effects (mycoparasitism, antibiosis, and competition for nutrients and space) and indirect effects (stimulation of plant defense reactions and plant growth promotion) (49).…”

Biocontrol of Sugar Beet Seedling and Taproot Diseases Caused by Aphanomyces cochlioides by Pythium oligandrum Treatments before Transplanting

“…Collected rhizosphere soil was mixed well and part of the soil sample was used to determine soil dry weight after incubation at 70℃ for 48 hours. Total soil DNA was extracted from 0.2 to 0.3 g of soil (expressed as dry weight) using a Bio 101 FastD-NA SPIN kit as described by the manufacturer (Qbiogene, Inc., Irvine, CA) with some modifications 26 , and then used for the detection of PO DNA by PCR. Soil sample DNA concentrations were adjusted to correspond to the amount of DNA extracted from 25 mg of soil (expressed as dry weight) in 2 µl of solution.…”

Visualization of Ralstonia solanacearum cells during biocontrol of bacterial wilt disease in tomato with Pythium oligandrum

“…Recently, the biocontrol agent Pythium oligandrum (PO) was found to suppress bacterial wilt in tomato (Hase et al 2006;Takenaka et al 2008). PO has a number of properties that are advantageous for biocontrol agents, including the ability to parasitize a wide range of plant pathogens (Benhamou et al 1999;Lewis et al 1989), production of antibiotics including hydrolytic enzymes (Benhamou et al 1999;Lewis et al 1989), competition for rhizosphere colonization , and induction of plant resistance (Benhamou et al 1997;Takenaka et al 2003).…”