Effect of Spray Tank Materials on Anti-Bacterial Activities of Sodium Hypochlorite Jean M. Bonasera and Steven V. Beer Section of Plant Pathology and Plant Microbe Interaction School of Integrative Plant Biology Cornell University, Ithaca, NY 14853 INTRODUCTION In 2017, we had obtained disparate results from the several grower trials with Surchlor sprayed on growing onions to reduce bacterial rot at harvest. We suspected that materials present in growers spray tanks, along with sodium hypochlorite, the active ingredient (A.I.) in Surchlor, may have affected the activity of the A.I. Based on analyses of growers spray records, the 2017 cooperating growers had included a broad range of other materials in their spray tanks together with Surchlor. (Grower-cooperators, collectively, had mixed 6 insecticides, 12 fungicides, 1 herbicide and 14 adjuvants or other materials with Surchlor.) We hypothesized that some of these may have affected the anti-bacterial activity of the A.I. Therefore, we designed assays to test the compatibility of the A.I. together with the materials that growers had combined with Surchlor in 2017. GENERAL TEST PROCEDURES First, we obtained fresh samples of the spray materials that growers had combined with sodium hypochlorite in 2017. Small samples (a few milliliters or grams of each) were donated by a cooperating onion grower and Cornell agricultural scientists. We combined preparations of each of the spray materials at their maximum labeled rates with a range of sodium hypochlorite concentrations (1 to 120 ppm) and tested the anti-bacterial activity of the mixture by adding a small amount of Pantoea ananatis, the center rot pathogen. Survival of P. ananatis, was assessed by placing 5 microliter (5 μl) drops of each mixture on the surface of microbiological medium in Petri plates. The plates were placed in an incubator for one or two days. If the mixture was toxic to the P. ananatis, due to the presence of active sodium hypochlorite, no growth was seen where the 5 μl drops had been placed. If bacterial colonies were visible, where the 5 μl drop had been placed, no anti-bacterial activity had been present in the mixture at the time of the addition of the indicator bacteria. We recorded the most dilute concentration of the A.I. (in ppm) with 100% kill activity and calculated the anti-bacterial activity of the test material plus sodium hypochlorite relative to the anti-bacterial activity of water plus sodium hypochlorite (Figure 1). The spray materials tested were divided into three groups: those that completely inactivated 120 ppm of sodium hypochlorite within 4 hours; those that reduced the effectiveness of sodium hypochlorite within 4 hours and those that did not change the effectiveness of sodium hypochlorite.
Based on the results of these tests, we have developed revised recommendations for mixing (or not) specific components together with Surchlor for spraying growing onions in New York State. Materials that STRONGLY interfered with the anti-bacterial activity of Surchlor Inspire Super Induce Lannate Luna Tranquility Manganese Roper (Dithane) Scala Viathon Materials that SOMEWHAT interfered with the anti-bacterial activity of Surchlor Agrimek Choice Weather Master Goal Lamcap Merivon Radiant Rovral Materials that DID NOT interfere with the anti-bacterial activity of Surchlor Movento Initiate Quadris Top Tilt Based on these results, we suggest that Surchlor be applied alone, or only together with the four materials we found that did not inactivate the anti-bacterial activity of Surchlor. We suggest that the materials found to interfere with the anti-bacterial activity of Surchlor might be applied separately on separate days. Our detailed results have been submitted to NYS DEC as suggestions for revision of the SLN label for use of Surchlor on growing onions. DETAILED TEST PROCEDURES and ILLUSTRATIONS Serial 1:2 dilutions of household bleach in autoclaved water were prepared in 1.2 ml tubes to yield 100 µl of A.I. concentrations from 360 ppm to 3 ppm, a 3X concentration. Test materials were diluted in autoclaved water to a 3X concentration of the rate as shown in Table 1. One hundred microliters (100 µl) of the 3X test concentration was added to two sets of serial dilution tubes. Table 1. Materials tested for effect of spray materials added to sodium hypochlorite
Test Material Amount added to 100 gallons Concentration converted to metric units AgriMek 3.5 oz 0.27 ml/liter Choice Weather Master 48 oz 3.75 ml/liter Diathane (liquid) = Roper (powder) 1.43 lbs 1.71 g/liter Goal 2 oz 0.156 ml/liter Induce 64 oz 5.0 ml/liter Initiate = Bravo = Echo 720 45 oz 3.5 ml/liter Inspire 15 oz 1.17 ml/liter Lannate 38 oz 3 ml/liter Luna Tranquility 19 oz 1.48 ml/liter Mancozeb 0.5 lbs 0.59 g/liter Manganese 18 oz 1.406 ml/liter Manzate 64 oz 5 ml/liter Merivon (Marivon?) 11 oz 0.86 ml/liter Movento 5 oz 0.39 ml/liter Quadris top 4.38 oz 0.342 ml/liter Radiant 10 oz 0.78 ml/liter Rovral 19 oz 1.48 ml/liter Scala 18 oz 1.406 ml/liter Tilt 7 oz 0.547 ml/ liter Viathon 38 oz 2.97 ml/liter Warrior = Lamcap 0.73 oz 0.057 ml/liter Then, ninety microliters of sterile water were added to each tube. The tubes were incubated at room temperature without shaking for 4 hours. Following the 4-hour incubation, 10 microliters of a 10 7 cfu/ml suspension of P. ananatis strain Oc5a were added to each tube (ca. 100,000 cells) and mixed by multiple pipetting the mixture up and down. Five microliter samples from each tube were transferred to LB agar plates and allowed to adsorb into the agar. Plates were examined for bacterial growth after 1 to 2 days of incubation at 28 C. The mixture with the lowest concentration of sodium hypochlorite that killed 100% of the cells of P. ananatis is noted (Figure 2). Survival of P. ananatis cells indicated failure of the mixture to perform as desired.
Antibacterial Activity Relative to Sodium Hypochlorite mixed with water Figure 1. Effect of spray materials on anti-bacterial activity of sodium hypochlorite. Antibacterial activity remaining after 4 hours in mixtures of sodium hypochlorite with various spray materials: Controls: Water: No effect on anti-bacterial activity. Ascorbic Acid: Caused complete loss of anti-bacterial activity
Figure 2. Bioassay results of the effect of several spray materials on the anti-bacterial activity of sodium hypochlorite. Manganese (Mn) strongly inactivated the anti-bacterial activity of sodium hypochlorite as did the negative control ascorbic acid (AA), while water (W) the positive control, had no effect. The yellow spots in the figure are bacterial growth, the green dots mark the spots where 5 microliters of mixtures were applied to the agar plates one to two days prior to photographing the plates. The concentrations of sodium hypochlorite ranged from 1 ppm (top row on the plates) to 120 ppm (bottom row on the plates).