Non Technical Summary
The goal of this project is to enhance efficacy of biocontrol tactics for pesticide-free and integrated pest management (i.e., IPM) through research to understand factors which influence the establishment and persistence of introduced biocontrol antagonists applied as biofungicides to lawns/athletic fields. Results will support turfgrass disease IPM recommendations for sustainable home and school communities. This project is closely aligned with CPPM Program focus areas (Plant Protection Tactics and Tools & IPM for sustainable communities) to help the program meet its goals of improved IPM practices, reduced human health and environmental risks. Multiple greenhouse and field studies will be conducted in CT and SC to identify application strategies and cultural practices turfgrass managers can implement to enhance biofungicide efficacy. Population dynamics of introduced biocontrol antagonists applied under various cultural practices will be quantified using DNA based methods. Efficacy of biofungicides for brown patch and large patch disease control will be assessed through disease severity ratings and quantification of the Rhizoctonia fungal development also through DNA-based methods. Implementation of research results to stakeholders will be facilitated through workshops in CT and SC, and development of a best practices biocontrol bulletin. Impact will be assessed through prospective surveys of workshop participants. This project addresses a significant need to expand turfgrass disease IPM strategies for school athletic fields and lawns, particularly in states and municipalities where synthetic pesticides are prohibited.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
216	1620	1160	50%
216	1621	1160	50%

Knowledge Area
216 - Integrated Pest Management Systems;

Subject Of Investigation
1620 - Warm season perennial grasses; 1621 - Cool season perennial grasses;

Field Of Science
1160 - Pathology;

Goals / Objectives
Our project intends to provide new guidance on the implementation of biofungicides, also referred to as introduced biological antagonists (i.e., IBAs),as efficacious management tools for turfgrass diseases. Currently, published reports examining IBAs have showninconsistent results in controlling turfgrass diseases. Lack of efficacy has resulted in poor adoption of IBAs, even in pesticide-free areas where local or state legislation limts the use of synthetic pesticides for turfgrass maintenance. Our work intends to combine specific cultural practices with IBA applications as a means to enhance survivalof living bacteria contained in these products. We believe that enhancing survival of these bacteria will improve efficacy against common foliar diseases impacting both cool and warm season turfgrasses maintained in transition climates. Specific goals include:1. Determine if nutritional supplements applied to foliage increase survival of IBAs in the phyllosphere and how increased survival impacts production of antibiotics produced by the organisms contained in the products examined.2. Determine if IBAs have a host preference among cool and warm season grasses.3. Determine how application timing (i.e., diurnal timing) impacts IBA survival and disease caused by Rhizoctonia solani.

Project Methods
Greenhouse studies will be conducted to evaluate the impact of niche-clearing and nutritional supplement applications on IBA population dynamics and antibiotic production. P. chlororaphis and B. subtilis responses will be examined separately in asynchronous companion studies. Treatments will include a non-treated control and an incomplete factorial of P. chlororaphis AFS009 applied as Zio at 6 oz/1000ft2 in combination with niche-clearing pre-treatment [no vs. yes (ZeroTol 2.0, peroxyacetic acid and hydrogen peroxide)] and nutritional supplements [blackstrap molasses alone (3.0 fl.oz./1,000 ft2); hydrolyzed soy protein alone (Amino Pro V at 1.0 fl.oz./1,000 ft2); or combination]. The companion study will include the same treatments, but substitute B. subtilis QST713 (Rhapsody at 10 fl.oz./1000ft2) in lieu of P. chlororaphis. Biofungicides tank-mixed with nutritional supplements will be applied 2-3 hours after niche-clearing pre-treatment. Treatments will be applied twice on a 7-d interval during each experimental run. Studies will be conducted in a randomized complete block design with four blocks. Each study will be completed twice. IBAs will be quantified from turf foliage at timepoint 0, weighed to ascertain total mass, and immediately frozen using liquid nitrogen. Sampling of tissue will proceed 1, 3, and 7 days after each treatment (DAT). To measure phyllosphere population dynamics ofP. chlororaphisAFS009 (Zio), we will employ a proprietary strain-specific qPCR TaqMan assay developed by AgBiome to quantify DNA of the target organism. Similarly,B. subtilisQST713 (Rhapsody) populations will be quantified using a strain-specific qPCR TaqMan protocol developed by Mendis et al. (2018). Secondary metabolite (i.e., antibiotics) production from both IBAs will be quantified through high performance liquid chromatography and mass spectroscopy. Verdure samples (i.e., 10 g) will be transferred to 250 mL polypropylene conical tube, combined with 30 mL acetonitrile and placed on an orbital shaker for 30 min (200 rotations min-1). A 10 mL aliquot will then be transferred to a 20 mL vial and centrifuged for 15 min at 3500 rpm before filtering 1 mL through a 0.45 µm nylon membrane syringe filter in a 2 mL HPLC chamber vial. Purified secondary metabolites will be quantified for bothB. subtilisQST713 (i.e., surfactin) andP. chlororaphisAFS009 (i.e., pyrrolnitrin and phenazines) using HPLC diode-array detection on an Agilent-1260 Infinity equipped with a C18column.A two-year field study will be conducted to evaluate the impact of niche-clearing and supplemental nutrient application on IBA populations and control of diseases caused by R. solani (i.e., brown patch on ryegrass and large patch on zoysia grass). Field sites established as perennial ryegrass or zoysia grass will be used. All plots will measure 1.8 x 1.8 m and buffer strips between plots will be used to minimize movement of applied material into adjacent plots. Both locations will be inoculated withRhizoctoniasolanito promote disease development within each plot. The pathogenR. solaniwill be grown in the laboratory on sterilized rye grain for 14 d and 10 cm3of infested grain will be used to inoculate 4 points within 0.6 m from each corner of all individual experimental plots. All plots will be niche-cleared with ZeroTol 2.0 prior to treatment. Nutrient supplements Amino Pro V and Blackstrap Molasses will be applied together at low labeled rates and in combination with Rhapsody (10 fl.oz./1,000 ft2) or Zio (6 oz/1,000 ft2). Treatments will be replicated four times in a randomized complete block design. IBA populations will be quantified from foliage harvested at timepoint 0 as described above, and treatments will be applied shortly after. A second application will be applied 7 DAT, then turf sampling and IBA quantification will continue for one week at 1, 3, and 7 DAT. At 14-days after initial treatment, all plots will be inoculated with R. solani. The treatment regimen will continue two more weeks and disease ratings will be visually assessed 14 days after inoculation. The same plot randomization will be utilized in both years to observe the cumulative impact of treatment implementation on biofungicide establishment. Treatment efficacy will be evaluated based on visual assessments of symptoms and Rhizoctonia quantification (qPCR) in thatch and foliage using a qPCR assay developed by Schulze and others (2016). The pathogen will be quantified within cores (2.5-cm diam × 5-cm deep) containing foliage and thatch. During each sampling event, three cores spaced at 5-cm intervals, 5 to 15 cm from inoculation points will be collected and pooled to determine the pathogen abundance before inoculation, and spread of the pathogen from inoculation points, after inoculation. Samples will be collected at timepoint 0 to establish pre-treatment pathogen population and again 7 days after the second application to assess treatment effects on resident pathogen populations prior to inoculation. A final sampling event will occur at the initial onset of symptoms at point-inoculations. Treatment effects on Rhizoctonia populations will be determined by comparing abundance of Rhizoctonia DNA in foliage and thatch during both the pre- and post- inoculation periods.A panel of hosts will be assayed in the greenhouse for persistence of P. chlororaphis and B. subtilis using qPCR as described above. For cool season turf in CT, we will survey Perennial Ryegrass, Tall Fescue, Kentucky Bluegrass, and Creeping Bentgrass. For warm season turf in SC, we will survey Perennial Ryegrass (as a control), Zoysia japonica, St. Augustine, Centipede, and Bermuda. Both locations will also survey Bean as a host established previously to be highly conducive of IBA establishment. Grass tillers cut at the chlorophyll line or leaf disks from bean leaves providing comparable surface area as turf samples will be collected from each experimental potting at timepoint 0, weighed to ascertain total mass of sample, and immediately frozen using liquid nitrogen. After timepoint 0 sample collection, all hosts will be treated with a biofungicide (Rhapsody-10 fl.oz./1,000 ft2 or Zio-6 oz./1,000 ft2). All plots will be niche-cleared with ZeroTol 2.0 prior to biofungicide treatment. Sampling of turf tissue will procced 1, 3, and 7 DAT.Field studies will be conducted to evaluate the impact of application time of day on IBA establishment and persistence in the phylloplane. This experiment will be conducted in CT and SC on their respective host turf species. High-rate applications of P. chlororaphis or B. subtilis will be applied every 7 d at 8am, 2pm, or 8pm. The experiment will be completed twice in the same field location on established turfgrass at respective locations to examine multi-year effects of treatments. Turf plants will be collected at the chlorophyll line at timepoint 0 and14 d after initial treatment for qPCR as described above. Treatment applications will continue every 7-d for 42 d to evaluate IBA application timing on brown patch and large patch symptom development. All plots will be niche-cleared with ZeroTol 2.0 prior to treatment and IBAs will be tank-mixed with nutritional supplements.For all experiments, DNA (IBA and/or pathogen), metabolite concentration, and visual disease assessments will be analyzed to identify significant treatment effects over time and locations using the MIXED procedure of SAS 9.4.