® Pest Technology ©2011 Global Science Books Application of a Bacterial Pathogen, Ralstonia solanacearum, with a Wet-blade Mower for Biological Control of Tropical Soda Apple, Solanum viarum James T. DeValerio1 • R. Charudattan2* • J. Jeffrey Mullahey3 • Pamela D. Roberts4 1 2266 N Temple Ave., Starke, FL 32091-1612, USA 2 Plant Pathology Department, P.O. Box 110680, University of Florida-IFAS, Gainesville, FL 32611-0680, USA 3 5988 Hwy 09, Bldg 4900, West Florida REC-Milton, Milton, FL 32583-1713, USA 4 2686 Hwy 29 N, Southwest Florida REC-Immokalee, FL 34142-9515, USA Corresponding author : * [email protected] ABSTRACT Tropical soda apple (Solanum viarum Dunal; TSA) is an invasive noxious weed in Florida and several southeastern U.S. states. To develop a bioherbicide agent that could be integrated with mowing, a recommended management practice for TSA, we screened several isolates of bacterial pathogens of Solanaceous plants and established that a Ralstonia solanacearum (=Pseudomonas solanacearum) (RS) isolate (10 Q, Race 1), originally from tomato, was capable of killing TSA without affecting tomato. RS is a xylem-invading, wilt-causing pathogen that when applied to cut main stems of TSA prevented regrowth and killed TSA plants under greenhouse conditions. To determine the effectiveness of this model bacterial bioherbicide agent under field conditions, a novel application method using a wet- blade mower was tested. Whereas wet-blade systems are used to deliver chemical herbicides and plant growth regulators to target weeds while mowing, none has been tested to deliver a biocontrol agent. We used the Burch Wet BladeTM mower system (BWB) and conducted the study in a pasture with 18% TSA coverage. RS cells suspended in sterile tap water at 1.4 x 109 CFU units/ml were used as inoculum. Treatments included a BWB-applied control (culture medium without RS), RS applied at 23 L/ha with BWB, and RS applied at 23 L/ha with BWB plus RS over-sprayed at 560 L/ha with a backpack sprayer. Both RS treatments reduced TSA regrowth compared to the control (P = 0.0003). There was no difference between the wet-blade-applied RS treatment and the wet-blade + over-sprayed RS treatment. The wet-blade mower was an effective, practical means of application of the bacterial wilt pathogen to control TSA and it may have broader applicability to other types of wilt-causing pathogens. _____________________________________________________________________________________________________________ Keywords: application technology, bacterial wilt, bioherbicide, Burch Wet-Blade, host range, Lifetest analysis, noxious weed Abbreviations: BWB, Burch Wet-BladeTM; GPA, gallons per acre; IFAS, Institute of Food and Agricultural Sciences; RS, Ralstonia solanacearum; TSA, tropical soda apple INTRODUCTION are used to manage TSA in Florida (Sellers et al. 2009). A combination of methods and materials are needed to control Tropical soda apple (Solanum viarum Dunal; TSA), a mem- TSA because no single approach is satisfactory under all ber of the Solanaceae family, is an exotic invasive plant conditions. Accordingly, in an effort to find suitable biolo- from South America that is a designated noxious weed in gical control agents, the University of Florida-IFAS began a the United States (Anonymous 2009). First observed in program in 1994 which resulted in the release and establish- South Florida around 1987, it spread rapidly throughout the ment of a chrysomelid beetle, Gratiana boliviana, as a clas- state covering an estimated half million hectares by 1995 sical biocontrol agent (Mullahey et al. 1994; Medal et al. (Mullahey et al. 1993, 1998). TSA infests open pastures 1995, 2006). Several fungal and bacterial pathogens were and wooded areas in cattle ranches as well as natural areas also collected in Brazil and Florida and screened against (Langeland and Burks 2008). The plant is copiously covered TSA. While none of the fungi proved promising as a bio- with sharp spines that can range from 1 to 2 cm in length. A control candidate, a few isolates of the bacterial pathogen defense against herbivory, these spines discourage animals Ralstonia solanacearum (RS) were highly virulent on TSA from consuming the leaves and stems but not the fruit. (Charudattan and DeValerio 1995). Because of the needle-like spines, dense infestations of RS is a soilborne bacterial pathogen of worldwide dis- TSA hinder movement of cattle, workers, and field manage- tribution. It consists of a diverse group of related strains ment practices. A prolific producer of seeds with high rates representing more than one species (Prior and Fegan 2005). of germination (Mullahey et al. 1993), TSA is seed-dis- RS strains cause bacterial wilt that is characterized by chlo- persed by cattle, water, and wildlife, and transported in con- rosis, necrosis, systemic wilting, and rapid plant mortality taminated manure, soil, hay, grass seeds, and sod (Mullahey on a wide range of hosts. The host range includes tomato, et al. 1998). Consequently, TSA has great propensity to potato, and banana as well as several important plants in the spread and is now established in several southeastern U.S. Solanaceae, Musaceae, Zingiberaceae, and other monocot states (Cuda et al. 2002; Bryson and Byrd 2007). Helped by and dicot families, depending on the race of the pathogen the mild winters in Florida, TSA grows year-round and strain. In preliminary trials, some of the RS isolates we perennates in some parts of the state, which further in- screened were highly virulent on TSA but not tomato or creases the chances of its spread. several other hosts, which suggested the possibility to find a Chemical herbicides, mowing, and biological control TSA-restricted isolate to evaluate as a biocontrol agent. Received: 7 September, 2009. Accepted: 1 December, 2010. Research Note Pest Technology 5 (Special Issue 1), 102-107 ©2011 Global Science Books As a vascular wilt pathogen that normally requires natu- reaction characterized by the absence of disease symptoms. ral openings or wounds to enter plants, it is necessary to deliver RS into the plant in order to obtain satisfactory lev- 2. Cut-and-swab inoculation els of weed control. Since mowing is one of the recom- mended practices for controlling TSA (Mislevy et al. 1999; In the second greenhouse experiment, isolates 446 and 506, both Sellers et al. 2009), we tested the Burch Wet-BladeTM from tomato, which were the most virulent of the isolates screened, mower (BWB) system for field application of RS. The wet- were tested on large, mature TSA plants. The 187-day-old, 0.6-m- blade mower system, of which there are a few different tall plants were inoculated by clipping the main stem 3 cm above types, enables application of an herbicide while mowing. the soil surface and swabbing the cut surface with 1-day-old bac- Designed for liquid pesticide applications, these systems terial cells suspended in sterile tap water. The inoculum was have been used primarily with chemical herbicides and applied at two cell densities of 0.74 and 1.74 A at 600 nm, which plant growth regulators for weed control, weed suppression, equaled approximately 1 × 109 and 3 × 109 cells per ml, respec- or to reduce the mowing frequency (Henson et al. 2003; tively. Control plants were inoculated by swabbing with sterile tap Hixson et al. 2007). A feature of the wet-blade systems that water. There were five replicates per treatment and the plants were was important to our work was the capability for instanta- observed for resprouts from the cut stems for 12 weeks after ino- neous introduction of the bacterial suspension as the TSA culation. The number resprouts per stem and the average height of stems are cut. This was expected to maximize bacterial resprouts were analyzed by analysis of variance and Duncan’s entry into the cut stem due the suction created by xylem mean separation. cavitation that pulls the liquid inward (Wahlers et al. 1997). Other advantages of wet-blade systems include the ability 3. Host-range study to apply low volumes (L/ha) and rates (active ingredient/ha) of herbicides, site-directed application, low or no drift, wor- In the third experiment, the host ranges of five RS isolates, W2, ker safety, low or no nontarget effects, single-step mowing W4, 10, 10Q, and H2, were compared on 30 plant species in the and spraying, and cost savings. In studies done by Mullahey Solanaceae, including five cultivars of tomato, two cultivars of and Williams (2001) and Sellers and Mullahey (2008), the pepper, two accessions of eggplant, and 27 other Solanum spp. BWB system was used with different levels of success to Twenty to 30-cm-tall greenhouse-grown seedlings were screened control TSA, southern wax myrtle, and melaleuca. With by one of two methods: 1) injecting a suspension of fresh bacterial TSA, 70% to 95% control could be achieved with several cells or 2) stabbing plant stems with a sterile wooden toothpick chemical herbicides (Mullahey and Williams (2001). dipped in fresh cells. The bacterial cells for inoculation were pre- The objectives of this study were two-fold, namely to pared from 24 h cultures on Difco nutrient agar (Difco Labora- determine the feasibility of using RS as a bioherbicide agent tories, Detroit, MI). For stem injection, the cells were rinsed off for TSA and to test the feasibility of the BWB system as an the Petri plates with sterile water, centrifuged, and suspended in effective, practical means of application of this pathogen in sterile 0.1 M PBS at approximately 1 × 108 cells per ml. For stem- the field. In this paper, we present the results from different stab inoculation, the cells were picked directly off the plates with methods of inoculation of TSA with RS in a greenhouse and the toothpick. Controls consisted of injecting with a phosphate in the field, selection of a strain for field trials based on its buffer solution or stabbing them with a culture medium-coated host range and relative virulence to TSA and some Solana- wooded pick without the bacterium.