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Pseudomonas aureofaciens Applied by Irrigation to Extend the Window of Dollar Spot Control of Various Fungicides

Glenn Hardebeck and Zac Reicher

Objective

The objective of this study was to determine if nightly applications via the BioJect system of Pseudomonas aureofaciens strain Tx-1 could extend the window of dollar spot control of single applications of fungicides in Penneagle creeping bentgrass maintained at fairway height.

Rationale

The use of biological disease control products is a popular idea due to the public's ongoing concern for safety and the environment. The integration of a "natural", biological disease control could double as a public relations tool as well as a disease control tool. Pseudomonas aureofaciens strain Tx-1 may be one such tool. Data from studies conducted during 1998 suggest that P. aureofaciens applied through the BioJect system can reduce dollar spot, but not to a level of commercially acceptable control when used without fungicide. This study is designed to evaluate the potential for P. aureofaciens applied via the BioJect through irrigation to extend the disease control of various commercial fungicides.

How It Was Done

The study was located at the Wm. H. Daniel Turfgrass Research and Diagnostic Center at Purdue University, West Lafayette IN. The soil type was Starks-Fincastle silt loam with pH of 7.0, 163 lbs P/A, and 534 lbs K/A. The area was previously in corn with this being the initial turf planting of Penneagle creeping bentgrass established during August of 1997. The bentgrass was mowed at 0.5 inches with clippings returned. The area received annual fertilization of 0.5 lbs N per 1000 sq. ft. applied in May, 1.0 lbs N per 1000 sq. ft. applied in Sep, and 1.5 lbs N per 1000 sq. ft. applied in Nov.

Treatments were a 2x8 factorial with 2 levels of P. aureofaciens application (none and nightly) and 8 fungicide treatments (Table 1). Experimental design was a split block with P. aureofaciens as main plots and fungicides as subplots (5.0 ft x 5.0 ft). Main plots were replicated 3 times and the experiment was completed four times during the summer of 1999 on separate, but adjacent areas. Each experiment began with fungicide application occurring on Jun 2, Jun 23, Jul 14 and Aug 4, heretofore referred to as trials 1, 2, 3 and 4, respectively. Dollar spot was minimized on experimental areas with applications of Daconil Weatherstik (4 oz per 1000 sq. ft.) applied not less than 7 days before the start of each trial.

P. aureofaciens application. A field bioreactor (BioJect system) was utilized to culture P. aureofaciens each day and then inject the culture into the irrigation system each evening after dusk during a two-minute/zone syringe cycle (0.05 inch of water). Plots that did not receive P. aureofaciens treatment received identical irrigation following the completion of the injected syringe cycle. Additional irrigation was done in the morning only as needed to prevent drought stress. Bacterial applications during 1999 were initiated Jun 2 and continued throughout the trials. To reduce bacterial application to the control plots injected and non-injected irrigation zones were supplied through separate piping and tarps were used to cover the non-injected plots for approximately 30 minutes while the injected irrigation system was running.

Fungicide treatments included an untreated check, BannerMaxx (0.5 oz per 1000 sq. ft.), Chipco 26GT (3.0 oz per 1000 sq. ft.), Cleary's 3336 (2.0 oz per 1000 sq. ft.), Fore (6.0 oz per 1000 sq. ft.), Daconil Weatherstik (3.0 oz per 1000 sq. ft.), Compass (0.15 oz per 1000 sq. ft.), and a tankmix of BannerMaxx (0.5 oz per 1000 sq. ft.) and Compass (0.15 oz per 1000 sq. ft.) (Table 1). Fungicide applications were made with a CO₂ backpack sprayer in 4 gal H₂O/1000 sq. ft.

Data samples were collected weekly for Tx-1 enumeration. A sample was collected directly from the bioreactor just prior to the injection cycle. Irrigation water was collected from the injected irrigation zones by placing an autoclaved catch pan on the turf surface immediately before the injection/irrigation cycle. Samples were stored at 35±2⁰F for 10 hours before enumeration. P. aureofaciens concentrations were determined through plate counts with three replicate plates per sample utilizing 1/10^th trypticase soy agar as the plating medium supplemented with rifampicin and cycloheximide each at 50 micrograms/ml. Dilution blanks consisted of 1/10^th Ringer solution.

Dollar spot disease severity was recorded two times per week following the application of fungicide treatments at the beginning of each trial. Disease severity was measured as the number of dollar spots per plot up to 300. Area under the disease progress curve (AUDPC) was calculated as ?ⁿ [(x₂-x₁)y₁ + (x₂-x₁)(y₂-y₁)/2] using eight ratings in all trials. To determine the number of days after fungicide application to reach thresholds of 25 and 50 spots per plot, interpolation of the disease severity data was done per plot and then analyzed.

A Box-Cox evaluation was applied to the disease severity and AUDPC data (Y) prior to other analyses. The linear regression of the log of the standard deviation of Y on the log of the mean of Y was significant on the majority of dates, suggesting a logarithmic transformation would provide the most homogeneous error variance. Disease severity and AUDPC data were transformed to log₁₀(Y+1) for further analysis. One was added to Y to prevent the occurrence of log₁₀(0). Threshold data were not found to be appropriate for transformation. Analysis of variance was performed on the transformed disease severity data with PROC ANOVA of SAS (SAS Institute, Inc., Cary, NC) and threshold data with PROC GLM of SAS due to several plots that did not reach the thresholds.

Results

P. aureofaciens production. Bacterial counts of culture from the bioreactor ranged from low 10⁷cfu/ml to low 10⁹ cfu/ml while bacterial counts of the irrigation applied to the experimental area were generally low 10⁶ to mid 10⁷ cfu/ml and the counts were fairly uniform across the area on most sampling dates. Lower counts on Jul 5 and Aug 2 seemed to correspond to older Tx-l inoculum use for incubation.

Dollar Spot Disease severity. As expected, disease severity varied among the fungicide treatments with Chipco 26GT, Cleary's 3336 and BannerMaxx alone and tank mixed with Compass performing the best throughout all four trials. P. aureofaciens reduced dollar spot 5, 23 and 26 days after fungicide application during the Trial 1 (Table 2). Numerical reductions in disease severity were present on 9, 12, 16 and 19 days after fungicide application during the same trial. There was no significant reduction in disease severity due to the application of P. aureofaciens in Trials 2-4 (Tables 3-5). There were no significant interactions between P. aureofaciens and fungicide application in any of the trials of this study. This suggests that the effects of P. aureofaciens are not enhanced nor worsened by different fungicides or vice versa.

Area under the disease progress curve (AUDPC). AUDPC data was consistent with the disease severity data. The application of P. aureofaciens via the BioJect reduced AUDPC values during Trial 1 (Table 6). There was no significant reduction of AUDPC due to the application of P. aureofaciens in Trials 2-4. There were no significant interactions between P. aureofaciens and fungicide application in any of the trials of this study.

Days to reach 25 and 50 spots per plot (Table 7 and 8). The application of P. aureofaciens via the BioJect increased the time to reach the 50 spot threshold by over two days in Trial 1. Although P. aureofaciens numerically increased the number of days to reach both 25 and 50 spots per plot in Trials 3 and 4, elevated dollar spot disease pressure during trials 2, 3 and 4 reduced P. aureofaciens effect. As mentioned with the disease severity and AUDPC data, there were no significant interactions between P. aureofaciens and fungicide application.

Summary

The BioJect unit operated very well during the course of this study with no applications being missed due to the bioreactor. P. aureofaciens applied via the BioJect system reduced dollar spot severity and is capable of extending control of dollar spot by commercial fungicides. Control was extended in Trial 1 by over two days due to the P. aureofaciens applied through the BioJect. However, this trial occurred during relatively low disease pressure in June and P. aureofaciens had no effect on disease control during the high disease pressure of July and August. Therefore, we can conclude that P. aureofaciens applied via the BioJect system may provide only minimal control of dollar spot under relatively low disease pressure.

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