Last updated 6/9/99 |
Table of Contents
Von Sigler
The objective was to determine the environmental fate of P. aureofaciens in the turfgrass ecosystem through DNA amplification coupled with nucleic acid hybridization.
Biological organisms for the prevention of turfgrass diseases are now available and in widespread use. Biological control is achieved through antagonistic action of one organism toward another such as a pathogen or weed. At present, no genetically engineered organisms are available in the turfgrass industry, however, as advances in biotechnology increase the activity of wild type (natural occurring) organisms through genetic manipulation, engineered varieties will likely enter the market. Although not genetically engineered, the biological control organism P. aureofaciens may serve as a predictive model for the behavior of future organisms that may potentially harbor altered genetics and thus, require sensitive post-application monitoring.
Approximately 106 cfu ml-1 of P. aureofaciens was applied nightly to an established stand of creeping bentgrass through the existing irrigation system. The bacteria were applied in 0.1 inch of water. To determine the environmental fate of the bacteria, samplings of the turfgrass leaves, thatch, and soil (~1 inch depth) were made periodically. DNA was extracted from each matrix and subsequently PCR amplified using primers designed to amplify selected regions of the 16S rDNA of Pseudomonas spp., including P. aureofaciens. An oligonucleotide probe was developed by aligning the sequence of the P. aureofaciens 16S rDNA amplified by the primers mentioned above with the sequences of other closely related Pseudomonads. A variable region within the amplified sequence provided adequate dissimilarity between the target organism and related organisms to allow the design of a 21 base oligonucleotide probe specific to P. aureofaciens. The degree of probe specificity was assessed by testing the hybridization of the probe to several closely related Pseudomonads as well as genera of more distant relation. A dilution series of P. aureofaciens was inoculated into samples of turfgrass leaves, thatch, and soil followed by DNA extraction and probe hybridization in order to determine the limit of detection for the organism in the turfgrass environment. A qualitative fate assessment of P. aureofaciens was determined using dot-blot hybridizations to PCR product derived from leaves, thatch, and soil. In brief, 1 ul of each PCR product was applied to a neutral membrane, fixed with UV irradiation, and probed with the probe labeled with the digoxygenin protein.
Fig. 1 shows the results of adding dilutions of P. aureofaciens to leaves, thatch, and soil followed by probe hybridization. The results indicate that at least 103 cells per gram dry weight, 103 cells, and 102 cells are detectable in the leaves, thatch, and soil, respectively. Signal strengths suggest that more dilute samples are assumed to be within the limits of detection.
Testing of the oligonucleotide probe against other Pseudomonads indicates sufficient specificity to detect P. aureofaciens amongst other closely related bacteria (Fig. 2).
Dot blot hybridizations to leaf surface, thatch, and soil bacterial DNA suggest that the majority of the applied bacteria are collected on the leaf surface with a lesser amount entering the thatch layer (Fig. 3). No hybridization signal was detected in soil-derived PCR products suggesting that most of the P. aureofaciens is effectively filtered by the canopy and thatch layer.
From an environmental standpoint, the turfgrass ecosystem effectively arrests the movement of the bacteria after it is applied. This result also proves positive for the use of the organism as a biological control as the intended pathogen, dollar spot (Sclerotinia homoecarpa), is most damaging when active in the turfgrass canopy and upper thatch regions. Thus, the usefulness of P. aureofaciens as a biological control organism, assuming the organism is, in fact, active in the turfgrass environment, couples well with its ultimate environmental fate.
| Leaf
Thatch
Soil
|
| Figure 1. The lowest dilution (white arrows) of P. aureofaciens inoculated into turfgrass leaves (103 cells per gram dry weight), thatch (103 cells), and soil (102 cells) is detectable following hybridization with the P. aureofaciens probe. |
 |
Figure 2. Dot blot hybridization test of P. aureofaciens probe specificity. Blot assignments: 1. P. syringae pv. tomato, 2. P. cepacia, 3. P. aeruginosa, 4. P. putida AC10R, 5. P. desmolytica 1123, 6. P. fluorescens 55, 7. P. stutzeri, 8. P. testosteroni 31, 9. P. viridiflava pv5, 10. P. aureofaciens, 11. Comomonas testosteroni, 12. Alcoligenes sp., 13. Ralstonia sp., 14. R. eutrophus, 15. P. putida, 16. blank, 17. P. desmolytica 1123, 18. P. syringae pv 61, 19. P. fluorescens B2265, 20. blank. |
 1
2
3
4
5 |
Figure 3. Dot blot hybridizations indicate the presence of P. aureofaciens in the leaf canopy (lane 2), and the thatch layer (lane 4). Blot assignments: 1. Leaf surface DNA without applied P. aureofaciens (PA); 2. Leaf with PA; 3. Thatch without PA; 4. Thatch with PA; 5. Positive control. |
Table of Contents
Send corrections, suggestions, and comments to biehlj@purdue.edu
