Final Report 2012 IR-4 Biopesticide Project Biologically based alternatives for broadleaf weed control in turf and

ornamentals (2012) J. C. Neal and B. Shew North Carolina State University Raleigh NC 27695 Email address of corresponding author: joe_neal@ncsu.edu Abstract Field and container experiments were conducted to evaluate the efficacy of Phoma macrostoma, MBI-005 and Fe-HEDTA for preemergence and postemergence control of common annual and perennial weeds. Applied preemergence in field soil, MBI-005 controlled crabgrass, swinecress, and pigweed but not sida, carpetweed, common chickweed, or field madder. In container experiments, variable results were observed between years with good control of spotted spurge, dandelion, henbit, galinsoga, eclipta, rorippa, bittercress, annual bluegrass, henbit, pearlwort and yellow woodsorrel with MBI-005 in 2012 but poorer control of these species in 2013. Additional research is underway to investigate possible reasons for variable results between years. In the same tests, P. macrostoma controlled eclipta, bittercress, common groundsel, dandelion, and rorippa, but did not control spotted spurge. Neither MBI-005 nor P. macrostoma controlled common chickweed, vetch or pearlwort. In a golf course fairway, two applications of MBI-005 at 38L/ha provided preemergence control of smooth crabgrass through mid-summer. Longevity of control was less than pendimethalin; additional applications will likely be required for season-long control. Applied postemergence, P. macrostoma consistently controlled white clover and hairy bittercress; whereas many other species including common chickweed and field madder were symptomatic but not controlled. Seedling dandelions were controlled but established dandelions in turf were symptomatic, but not controlled. In the absence of white clover or chickweed, MBI-005 applied postemergence provided broadleaf weed control comparable to the industry standard herbicide, but in the presence of these tolerant weed species, overall weed control was poor. Postemergence weed control with MBI-005 or Fe-HEDTA was improved by sequential treatments, increased spray volume and increased concentration. Data suggest that the dose of MBI-005 or Fe-HEDTA per m2 is more important than concentration or carrier volume. For Fe-HEDTA a dose of > 48 oz/1000 ft2 was required for optimum weed control, yet additional research is needed to determine re-application intervals for winter annual and perennial weed species. P. macrostoma did not injure seedling tall fescue. In 2011, FeHEDTA injured seedling tall fescue turf, causing necrotic leaf tips which were black in color; however no significant injury was observed in the 2012 experiment. MBI-005 reduced turfgrass density when applied at seeding but when applied about 6 weeks after emergence, no reduction in turfgrass density was observed. Dormant woody ornamentals were not injured by these treatments; however, actively growing spirea was injured by Fe-HEDTA and MBI-005 but not by P. macrostoma. Heuchera, liriope, gardenia and ligustrum were injured by Fe-HEDTA but not by MBI-005 or P. macrostoma. These data show that biobased products can provide control of broadleaf weeds in turfgrass comparable to some industry standard herbicides yet weed control selectivity and variable responses under field conditions necessitate additional development research.

INTRODUCTION Turfgrass and landscape management and nursery crop production are multi-billion dollar industries in the United States. Traditional pest management programs in these sites rely heavily upon synthetic chemistry for insect, disease and weed management. However, significant concerns exist over exposure to lawn care and landscape maintenance chemicals applied in urban watersheds. Interest in alternative and organic pest management options for urban lawns and landscapes continues to grow, with retail sales of organic lawn and garden products estimated to be $460 million in 2008, a gain of 12% over 2007. Yet few bio-based alternatives for weed control are available. Non-selective weed control products such as vinegar and pelargonic acid are available to the market. However, aside from hand weeding, traditional synthetic herbicides are the only effective options for selective weed control in turfgrass, landscapes or nursery crops. Fe-HEDTA is currently labeled for control of certain broadleaf weeds in turf, but research on its efficacy is lacking. Recently, registration packages for two bio-based products for selective broadleaf weed control in turf have been submitted to US-EPA: Phoma macrostoma and MBI-005. Phoma macrostoma is a fungus isolated from Canada thistle (Cirsium arvense). Experiments conducted in Canada have demonstrated efficacy on common broadleaf weeds of turfgrass and landscape plantings (Bailey et al. 2009) with limited mobility and persistence in soil (Bailey and Derby 2005; Zhou et al. 2004). However, the temperature optima for this biocontrol agent may limit its effectiveness in warmer climates. Field experiments are needed to determine if this biocontrol agent will be effective under field conditions in the Southeastern U.S. Furthermore, Phoma macrostoma has been reported on a variety of woody plant hosts but is generally considered a weak, wound pathogen. Preliminary data from our lab suggest application of the inoculum around woody ornamental plants does not produce disease symptoms, nor does application to turf weeds adjacent to landscape plants. However, it is imperative to understand the potential impacts that P. macrostoma inoculum may have on landscape plant safety. The active ingredient in MBI-005 is thaxtomin, a non-systemic plant toxin produced by Streptomyces acidiscabies RL110 during fermentation. Thaxtomin phytotoxins have been characterized and shown to have herbicidal properties, with species selectivity varying with changes in phytotoxin molecular structure (King and Lawrence 2001). MBI-005 has been demonstrated to selectively control several broadleaf plants without injuring certain grass species. Preliminary research (unpublished data from registrant) suggested soil residual may provide preemergence broadleaf and grass weed control as well. These observations need to be confirmed under field conditions in the Southeastern U.S. before recommendations for use can be developed. Our 2011 research results support continued evaluation of these biologically based alternatives for turf, landscape and nursery crop sites. Core objectives of this research were to: 1. Evaluate and compare P. macrostoma, MBI-005 and Fe-HEDTA efficacy on common cool-season turf, landscape and nursery weeds in North Carolina, USA; and 2. Evaluate potential non-target effects of P. macrostoma on dicot crops grown for ornamental purposes

MATERIALS AND METHODS A series of experiments was initiated to investigate the performance of these bio-based weed management products in established turfgrass settings, in bare soil, and in containers. Specific experiments were slightly modified from the original proposal because of limited availability of products for testing, a significant delay in funding allocation (funding allocated in late August 2012), as well as results from on-going experiments. Common Methods All field and outdoor container experiments were conducted at the Horticultural Field Laboratory, Raleigh, NC. Field experiments were established in areas of existing weed infestations. In 2012-2013 preemergence field tests were over-seeded with several species to achieve more even distribution of species. Container experiments were seeded with locally collected weed seeds. Each experiment was conducted in a randomized complete block design with a minimum of 4 replications. P. macrostoma was applied as a dry granule in pre-weighed aliquots. Two formulations were compared. These formulations differed in the concentration of the herbicidally active fungal toxin, macrocidin. Doses were adjusted to apply equivalent macrocidin units or mus. P. macrostoma treatments were irrigated after application either by hand or with overhead irrigation. Due to constraints of space and product availability, not all doses were included in all experiments. Experiments included industry standard preemergence or postemergence herbicides for comparison. Previous research (Neal 1993) has demonstrated that treatments to wetted foliage will improve weed control with granular phenoxy herbicides; therefore, all plots treated with Preen Broadleaf Control (the granular industry standard) were wetted prior to treatment. Unless otherwise noted, individual plot sizes were 1 m2. MBI-005 and Fe-HEDTA spray applications were made with a CO2 pressurized sprayer equipped with flat fan nozzles and calibrated prior to each treatment. Weed control was evaluated using several metrics. In field tests, visual estimates of percent ground cover by species were conducted before and after treatment. Visual estimates of weed control were conducted on a 0 to 10 scale, where 0 = no control and 10 = 100% control, with incremental values based on a visual estimate of above ground biomass reduction compared to the non-treated plants (similar to that described by Frans et al. 1986). These visual ratings were converted to percent control for presentation. Where feasible, plant counts also were recorded. In container trials, visual control evaluations were conducted about 2, 4 and 6 weeks after treatment, and above-ground fresh weights were recorded at the termination of the experiment typically 4 to 6 weeks after treatment. Data from fresh weights were highly correlated with visual ratings; and were therefore not conducted for all species or experiments. Injury to crop plants was visually estimated using a 0 to 10 scale where 0 = no injury (equivalent to non-treated plants) and 10 = dead plants (100% injury); incremental values were based on a visual reduction in plant size and quality. Percent turfgrass cover was also visually estimated. Preemergence experiments Preemergence control of winter annual broadleaf weeds and safety to seedling tall fescue turf (fall 2012 spring 2013) Preemegence control of summer annual weeds in containers (Summer 2012 & 2013)

Preemergence control of common cool-season weeds in containers (Fall 2012) Residual longevity of weed control in containers with MBI-005 (summer 2013) Preemergence control of smooth crabgrass in golf turf (spring / summer 2013)

Preemergence control of winter annual broadleaf weeds and safety to seedling tall fescue turf The experiment was conducted on an area of newly seeded fescue (Festuca arundinacea) turf. In September 2012 the area was fertilized with a starter fertilizer at 0.5 lb N/1000 ft2, amended with composted leaves, cultivated, raked smooth, then seeded with a turf-type tall fescue variety blend at 4 pounds of seed per 1000 square feet. Treatments were applied after seeding and were arranged in a randomized complete block design with four replications. Turfgrass germination and establishment were evaluated as previously described; weed control was evaluated using the visual rating scale previously described and weed counts in 25 dm2 sub-plots. From the weed counts the number of weeds per m2 was estimated and weed frequency was estimated by recording presence or absence of each weed species in each of twenty-five 1-dm2 sub-plots evenly distributed in each plot; then expressed as a percentage of grids with weeds. Weed control was visually evaluated on a scale of 0 to 10 as previously described. In the 2012 experiment, two formulations of P. macrostoma were compared as well as a combination of P. macrostoma and MBI-005. Doses were adjusted to apply equivalent amounts of the herbicidally active fungal toxin, macrocidin, designated as macrocidin units or mus a measure of the macrocidin concentration. Preemergence control of summer annual weeds A container experiment was established to compare two formulations of P. macrostoma, MBI 005 alone or combined with P macrostoma, and labeled standards Corn Gluten, Pendulum 2G and Gallery for preemergence control of common summer-germinating weeds. On June 6, 2012, 3-liter pots were filled with hammer milled pine bark amended with Harrell's 18-4-8 slow release fertilizer added @ 8 #/yd3. Pots were hand watered to settle the substrate then treatments were applied. Treatments included two formulations of Phoma macrostoma, designated EP02-16 and EP02-13, each applied at three doses. Doses were adjusted to apply equivalent macrocidin doses of 3200, 6700 and 13000 mu/m2. Treatments also included MBI-005 at 19 and 32 L/ha, Corn Gluten (Preen Organic Garden Weeder) at the manufacturers recommended dose of 97 g/m2, and two common landscape herbicides Gallery (isoxaben) and Pendulum 2G (pendimethalin); as well as a non-treated control. Pots were irrigated with about 0.2 inches of water immediately after treatment then received 0.6 inches of overhead irrigation each day in three equal increments, morning, mid-day and late afternoon. Spotted spurge (Chamacyse maculata ), eclipta (Eclipta prostrata ), common groundsel (Senecio vulgaris), were surface seeded one day after treatment. Other weeds included in the experiment did not emerge in sufficient quantities to evaluate. The experiment was repeated on June 11, 2013 using the same methods except: because no differences were observed between P. macrostoma formulations, a single formulation of P. macrostoma was utilized; fertilizer was Osmocote 17-4-8; and weeds included in the 2013 experiment were large crabgrass (Digitaria sanguinalis), Japanese stiltgrass (Microstegium viminium), goosegrass (Eleusine indica), eclipta, spotted spurge, dandelion (Taraxacum officinale), flexuous bittercress (Cardamine flexuosa), and prostrate pigweed (Amaranthus blitum).

Preemergence Control of Cool-Season Weeds in Containers On September 12, 2012 1-liter pots were filled with a pine bark substrate amended with an industry standard slow release fertilizer, then hand watered to settle the potting substrate. Woodsorrel (Oxalis stricta), marsh yellowcress (Rorippa islandica), ivyleaf speedwell (Veronica hederifolia), hairy vetch (Vicia villosa), flexuous bittercress, hairy galinsoga (Galinsoga ciliata), pearlwort (Sagina procombens), annual bluegrass (Poa annua), dandelion, henbit (Lamium amplexicaule) and common chickweed (Stellaria media) were surface seeded. Treatments were arranged in a randomized complete block design with 5 replications (a single pot of each weed species per treatment per replicate). MBI-005, P. macrostoma, corn gluten and an industry standard preemergence herbicide were applied on September 13, 2012. Pots were overhead irrigated immediately after treatment and daily thereafter with about 0.6 inches of irrigation applied in 3 equal increments (early am, mid-day and midafternoon). Weed control was visually evaluated 4 and 6 WAT using the 0 to 10 scale previously described. Preemergence Control of Smooth Crabgrass in Golf Turf with MBI-005 An experiment was established to evaluate the efficacy of MBI-005 for control of smooth crabgrass in turf. The experiment was conducted on a golf course fairway in Oxford, NC with a history of heavy populations of smooth crabgrass (Digitaria ischaemum). The turfgrass was low-density of common bermudagrass mowed weekly at about 1.8 cm height. No other herbicides or pesticides were used on this fairway. The first treatments were applied on March 28, 2013, about 2 weeks before crabgrass emergence. Treatments included MBI-005 at 19, 38 and 57 L/ha applied individually or in combinations of 19 L/ha applied twice or 3 times at 4 week intervals, 38 L/ha followed by 19 L/ha or 38 L/ha 4 weeks after initial treatment. These treatments were compared an organic standard, corn gluten at 980 kg/ha, and a herbicide standard, pendimethalin 2% granule at 1.68 kg ai/ha, each applied twice at an 8 week interval. Percent weed control was visually evaluated monthly as previously described. Percent crabgrass cover was visually estimated 8 and 12 weeks after initial treatments. Turfgrass injury was evaluated 1 and 2 months after initial treatment on a % scale where 0 no injury and 100 = dead turf. The primary symptom observed was a delay in greening of the bermudagrass turf. By 2 months after treatment, turf injury evaluations were no longer possible because smooth crabgrass infestations had covered most of the bermudagrass. Postemergence Experiments Five experiments were conducted to evaluate postemergence control of broadleaf weeds. Those experiments included: Established dandelion control with MBI-005 and P. macrostoma combinations (Fall 2012 and Spring 2013); Postemergence control of established dicondra (Fall 2012 and evaluated spring 2013) Winter annual weed control in newly seeded turf (Fall 2012 and evaluated spring / summer 2013.); Postemergence control of cool-season weeds container experiment (Fall 2012 and Spring 2013); and Fe-HEDTA spray carrier volume / concentration influence on postemergence efficacy

Established Dandelion Control with MBI-005 and P. macrostoma combinations. Previous work suggested that a combination of P. macrostoma plus MBI-005 may enhance postemergence control of some weeds. To test this hypothesis, two experiments were established one in containers using seedling broadleaf weeds and the other on established dandelion in low maintenance turf. On October 1, 2012, 3-liter pots were filled with a pine bark substrate amended with slow release nursery fertilizer, hand watered to settle the substrate, then surface seeded with weeds of differing susceptibilities to the biocontrol agents (based on previous research). Weeds included were: common chickweed (tolerant), dandelion (susceptible), common grounsel (susceptible) and flexuous bittercress (intermediate susceptibility). Plants were initially outside where they received 0.5 inches of overhead irrigation each day applied in 3 equal increments. On November 21st, treatments were applied. MBI-005 was applied at 19, 38 and 57 L/ha. Phoma macrostoma was applied at two doses to achieve approximately 6700 and 13,000 macrocidin units per m2. Additionally the two lower doses of MBI-005 and the two doses of P. macrostoma were applied in a factorial arrangement. Pots were irrigated one day later then moved to an unheated, covered hoop house for winter protection. Weed control was visually evaluated for 6 weeks and above ground fresh weights were measured about 7 weeks after treatment. In the fall of 2012 an experiment was established on dormant bermudagrass turf with a uniform infestation of dandelion (Taraxacum officinale). Treatments were applied on November 21, 2012 and included P. macrostoma, formulation EP02-16-2012, at 31 and 63 g/m2 (6600 and 13,200 mu/m2) and MBI-005 at 19 and 38 L/ha, each applied alone or in combinations. Due to limited P. macrostoma availability, plot sizes were limited to 0.5 m2 and only one treatment was possible. Chlorosis induced by treatments was evaluated on a subjective scale of 0 to 3 where 0 = no chlorosis, 1 is slight chlorosis, 2 nearly all growing points are chlorotic or bleached, and 3 = nearly entire plant chlorotic or bleached. Weed control was evaluated about 1 month after treatment and again in February 2013 when new growth was observed on dandelions in the field. Pre- and post-treatment dandelion counts were also conducted. The field had inconsistent populations of other winter annual broadleaf weeds, control of which were also evaluated in an overall broadleaf weed control rating.

Postemergence Control of Dicondra (Dicondra repens). In previous field experiments, small patches of dicondra were observed to be symptomatic following Phoma macrostoma treatment. November 21, 2012, an experiment was established on a field of low maintenance turf with over 90% ground cover of dicondra to compare the efficacy of P. macrostoma, MBI-005 and Fe-HEDTA. Due to limited product availability only a single dose of each treatment was included, and plots size was 0.5 m2. Treatments were P. macrostoma 13,000 mu/m2, MBI-005 at 38.2 L/ha, MBI-005 combined with P. macrostoma, FeHEDTA (Fiesta) at 160 L/ha, and WeedBGon Max RTU (2,4-D+MCPP+Dicamba). Weed control was evaluated in the 1, 2, and 4 weeks after treatment, and again in March 2013. Percent weed control was visually evaluated and at the end of the experiment percent ground cover of dicondra in each plot was visually estimated.

Winter annual weed control in newly seeded turf (fall 2012; evaluated spring 2013) An established turfgrass site was treated with glyphosate to kill existing grass and weeds then on September 26, 2012 the site was cultivated, amended with leaf compost and lime, then rototilled again. The soil was raked smooth then seeded with The Rebels tall fescue blend on September 27, 2012. The same day the area was fertilized with Pennington 18-24-6 starter fertilizer at a rate of 0.5 lb N/1000 ft2. The site was irrigated after seeding. On November 5, 2012 the area was fertilized again using Scotts TurfBuilder for Southern Lawns at a rate of 0.5 lb N/1000 ft2. Treatments were applied on November 16th and included two doses of P. macrostoma applying approximately 6700 and 13000 mu/m2 of macrocidins, MBI-005 at 19 and 38 L/ha, FeHEDTA (Fiesta) at 80 and 160 L/ha compared to non-treated plots, a granular auxin herbicide (Preen Broadleaf Control at 98 kg/ha, and a ready to use (RTU) auxinic herbicide spray (Weed B Gon RTU). Visual estimates of weed control were conducted on a 0 to 10 scale, where 0 = no control and 10 = 100% control, with incremental values based on a visual estimate of above ground biomass reduction compared to the non-treated plants (similar to that described by Frans et al. 1986). These visual ratings were converted to percent control for presentation. A subjective rating of foliar chlorosis was conducted using a 0 to 3 scale where 0 = no chlorosis, 1 = barely noticeable yellowing, 2 and 3 are increasing severity and extent of chlorosis and bleaching.

Postemergence control of cool-season weeds in containers. On September 12, 2012, 1-liter pots were filled with a pine bark substrate then hand watered to settle the potting substrate. Oxalis stricta, Rorippa islandica, Veronica hederafolia, Vicia sativa, Cardamine flexuosa, Galinsoga ciliata, Sagina procombens, Poa annua, Taraxacum officinale, Lamium amplexicaule and Stellaria media were surface seeded. Treatments were arranged in a randomized complete block design with 5 replications (a single pot of each weed species per treatment per replicate). Plant size was used as a blocking factor for replications. Treatments listed in Table 2 were applied on October 16 and again on November 6th. Pots were overhead irrigated about 4 hours after treatment and daily thereafter with about 0.6 inches of irrigation applied in 3 equal increments (early am, mid-day and midafternoon). Weed control was visually evaluated 1, 6, 13, 21 and 24 days after treatment on a 0 to 10 scale where 0 = no control (equal to the non-treated) and 10 = 100% control. Intermediate values are visual estimates of percent reduction in above ground plant biomass abbreviated to a 0 to 10 scale.

Fe-HEDTA spray carrier volume / concentration influence on postemergence efficacy Fe-HEDTA (trade name: Fiesta) is currently marketed for broadleaf weed control in warmseason and cool-season turf. Application instructions on the label suggest a dilution of 5 oz product per gallon applied in approximately 2.5 to 10 gallons per 1000 sq ft (109 to 436 gallons per acre) spray volume. These guidelines provide a large range of dosing and cost per use. An experiment was established to test a range of doses and concentrations to determine the optimum combination of concentration and spray volume for control of common perennial turfgrass weeds. Two experiments were conducted to evaluate the influence of spray carrier volume, spray concentration and dose of Fe-HEDTA on weed control. Both tests were conducted in a randomized complete block design with 4 replications. Individual plots were 1 m2 in size. Treatments are outlined in Table 1. Fe-HEDTA product was diluted 1.95%, 3.9% and 7.8% by volume in water and each dilution applied in 50, 100, 200 and 400 GPA spray volume using a

CO2 pressurized sprayer equipped with 8008 flat fan nozzles calibrated to deliver 50 GPA. Higher spray volumes were achieved by multiple (2, 4, or 8) passes over the plot using the same calibrated sprayer.
Table 1. Concentrations and spray volumes tests; and resulting product dose. Dilution oz/Gal % v/v Spray volume 2 GPA oz/1000 ft Equivalent product dose 2 oz /1000 ft 2.87 5.73 11.46 22.92 5.73 11.46 22.92 45.84 11.46 22.92 45.84 91.68

2.5

1.95

3.9

10

7.8

50 100 200 400 50 100 200 400 50 100 200 400

147 294 588 1175 147 294 588 1175 147 294 588 1175

One experiment was conducted on an established stand of white clover (Trifolium repens) and dandelion (Taraxacum officinale) with little or no actual turfgrass cover. Treatments were applied on October 16, 2012 and again on November 6, 2012 when re-growth was observed on most plants. Pre-treatment data included a visual estimate of percent cover of white clover and dandelion counts. One day after treatment % foliage necrosis was visually evaluated. Subsequently, weed control was visually evaluated on a 0 to 10 scale where 0 = no control (equal to non-treated plots) and 10 = 100% mortality of the weed. Percent cover of clover and number of dandelion plants were also recorded. Weed control evaluations were recorded approximately weekly until weather was cold enough to halt weed growth. On December 17th clover evaluations also included a 1-m2 grid count in which a 1 m2 frame was divided into 100 dm2 grid. Each grid section containing living white clover was counted; the resulting counts express a % of the grids with living clover labeled % Frequency. On April 19, 2013, after plant growth had resumed % ground cover of clover was evaluated; dandelion control evaluations were not feasible at this time as the germination of dandelions after treatment confounded the results. The second site was a low maintenance bermudagrass turf mowed at 3 inches and infested with a mixture of winter annual broadleaf weeds including persian speedwell (Veronica persica), ivyleaf speedwell (Veronica hederifolia), and field madder (Sherardia arvense). Treatments were applied on March 29, 2013 and re-applied on April 26, 2013. Percent broadleaf weed cover was visually estimated before treatment and 20 days after the 2nd treatment. Percent weed control was visually evaluated about 3, 7, and 20 days after each treatment. Evaluations were discontinued when warmer weather resulted in natural senescence of the winter annual weeds.

Data from both experiments were subjected to analysis of variance and means separated using a least significant differences procedure. Data were also averaged by actual Fe-HEDTA dose, across all spray volume and concentrations variables, then plotted against percent control or percent ground cover. Regression equations were fit to these data.

Safety to Ornamental Plants Two experiments were initiated at the Horticultural Field Laboratory, Raleigh, NC to investigate the safety of bio-based herbicides on ornamental plants. Rooted cuttings Juniperus conferta Blue Pacific, Ligustrum japonica, Spirea japonica, and Gardenia radicans in 18-cell trays were obtained from a local nursery. Plants were arranged in a randomized complete block design with 4 replications. Treatments were applied on September 13, 2012 and again on October 16, 2012. Phoma macrostoma at 6700 and 13000 mu/m2 was applied using a hand-held shaker jar. MBI-005 at 38.2 l/ha and Fe-HEDTA at 160 L/ha were applied with a a CO2 pressurized sprayer calibrated to deliver 100 GPA. Pots were overheadirrigated daily with about 0.5 inches of irrigation applied in 3 equal increments (early AM, noon and late afternoon). Treatments were reapplied November 6th. Plants were placed in an unheated, plastic-covered shelter for winter protection. Phytotoxicity was visually evaluated 1, 2 and 4 weeks after treatment on a 0 to 10 scale where 0 = no damage (equal to the non-treated) and 10 = dead. Intermediate values are visual estimates of percent reduction in above ground plant biomass abbreviated to a 0 to 10 scale. Re-growth the following spring was evaluated. On August 6, 2012 the same treatments were applied to container grown Shasta daisy (Leucanthemum x superbum), variegated liriope (Lirope muscari Silver Sunproof), and heuchera (Heuchera x Palace Purple). Plant injury was visually evaluated weekly for 3 weeks at which time the evaluations were discontinued due to high plant mortality caused by an unrelated plant disease. Safety to Newly Seeded Tall Fescue Turf Two experiments were conducted at the Horticultural Field Laboratory, Raleigh NC on an area of newly seeded fescue turf to evaluate the safety of treatments applied at seeding and to seedling turf. Methods for these experiments are described above. Turfgrass stand density and % foliar necrosis / injury were recorded.

RESULTS Preemergence broadleaf weed control in newly seeded tall fescue turf. MBI-005 and two formulations of Phoma macrostoma (EP02-16 and EPO2-13) were compared. When applied at equivalent macrocidin doses, no differences between the two P. macrostoma formulations were observed. Abundance and size of broadleaf weeds were reduced by both P. macrostoma and MBI-005. Corn gluten reduced weed populations 6 WAT but increased vigor of surviving weeds resulting in no visual reduction in dandelion cover as reflected in the visual evaluations of % weed control (Table 2a). MBI-005 applied preemergence at seeding reduced dandelion and hop clover counts with weed populations decreasing with increased dose. Other

weeds in the test area, including parsley piert, henbit, chickweed, hop clover, vetch, Carolina geranium, and field madder, were present in variable populations but were evaluated in composite broadleaf weed evaluations. Chickweed, henbit, hop clover and field madder in P. macrostoma treated plots were symptomatic (new growth was chlorotic to white) but growth was not significantly reduced through 6 weeks after treatment (data not shown).

Table 2a. Preemergence efficacy of bio-based products on newly seeded tall fescue turf. Treated Fall
2012; evaluated 5-6 weeks after treatment Treatment Dose Dandelion Hop Other Clover BrdLf* ------------------ #/m2 ---------------88 a 135 a 156 a 4c 0c 4c 0c 16 bc 4c 4c 40 b 56 b 16 cd 48 bc 12 d 32 bcd 12 cd 32 bcd 36 bcd 88 bcd 64 de 76 cde 40 e 132 ab 108 bc 84 cde 116 abc Dandelion Other BrdLf* ------- % control ------0c 0d 100 a 100 a 100 a 100 a 83 b 97 a 100 a 0c 73 ab 70 ab 35 c 75 ab 70 ab 87 a 65 b 0d

Non-Treated P. macrostoma EP02-16 6600 mu/m2 (31 g/m2 ) 13000 mu/m2 (63 g/m2) 6600 mu/m2 (55 g/m2) 13000 mu/m2 (110 g/m2) 19.1 L/ha 38.2 L/ha EP02-16 + MBI-005 Corn Gluten 6600 mu/m2 + 19 L/ha 970 kg/ha

P.macrostoma EP02-13

MBI-005

*Other Brdlf = composite counts or ratings for broadleaf weeds, other than dandelion, present in the plots. Other broadleaf weeds included henbit, common chickweed, parsley piert, hairy vetch, hop clover and Carolina geranium. Populations of these weeds were variable on these evaluation dates.

In contrast, when plots were evaluated the following spring, hop clover was well controlled by P macrostoma and corn gluten but not by MBI-005 (Table 2b). Field madder was also reduced in plots treated with P. macrostoma but populations were inconsistently distributed in the field leading to high variability in the data. Plots treated with MBI-005 had reduced turf density compared to other treatments, perhaps leading to weed establishment in those plots the following spring. In contrast, P. macrostoma and corn gluten plots had nearly complete turfgrass cover before winter. Consequently, based on this data it is unclear if the observed control of hop clover and field madder in P macrostoma and corn gluten treatments can be attributed to direct treatment effects or to indirect effects associated with turfgrass density and competition. Turfgrass tolerance is discussed in more detail later in this manuscript.

Table 2b. Preemergence efficacy of bio-based products on newly seeded tall fescue turf. Treated Fall
2012; evaluated May 2013 Treatment Non-Treated P. macrostoma EP02-16 6600 mu/m2 (31 g/m2 ) 13000 mu/m2 (63 g/m2) 6600 mu/m2 (55 g/m2) 13000 mu/m2 (110 g/m2) 19.1 L/ha 38.2 L/ha 6600 mu/m2 + 19 L/ha 970 kg/ha Dose Broadleaf Hop Field Weeds Dandelion Clover Madder ---------------------- % control ---------------------0 c 0c 0d 0 c 83 a 80 a 80 a 98 a 73 a 86 a 88 a 27 cd 40 bc 74 a 68 ab 100 a 100 a 100 a 38 b 38 b 100 a 5 c 88 a 93 a 99 a 23 b 18 bc 89 a 80 a 50 abc 45 abc 97 a 0c 0c 45 abc 0

P.macrostoma EP02-13

MBI-005 EP02-16 + MBI-005 Corn Gluten

Means followed by same letter do not significantly differ (P=.05, Waller-Duncan k=100). Broadleaf weeds = overall broadleaf weed control including dandelion, hop clover, field madder and all other broadleaf weeds present in the field at varying populations. Note: field madder populations were variable in the field and these data should be confirmed by further testing.

Preemergence Control of Summer Annual Weeds in Containers 2012 and 2013. In 2012, MBI-005 controlled eclipta, spotted spurge, and common groundsel equal to the industry standard preemegence herbicide, Gallery. Spurge was controlled by both doses of MBI005 (Table 3a), but control declined by 8 WAT (data not shown). However, in 2013 spurge control was less than 50%. Similarly, in 2012 eclipta was controlled MBI-005 but in 2013 eclipta control was less than 25%. Common groundsel was controlled by both doses of MBI-005 in 2012 and by the higher dose in 2013. No differences were observed between the two P macrostoma formulations therefore data for the two formulations used in 2012 are averaged for presentation in Table 3. Phoma macrostoma controlled eclipta and common groundsel but not spotted spurge. Percent control of both susceptible species was improved with increased dose. As was observed with the MBI-005 treatments, eclipta and groundsel control with P. macrostoma was better in 2012 than in 2013. Combining low doses of the two agents did not enhance control of these weeds. The list of species tested was expanded in 2013. Summer annual grasses, large crabgrass, goose grass and Japanese stiltgrass, were not controlled by P macrostoma (Table 3b). MBI-005 did not control large crabgrass or goosegrass but did provide 74% control of Japanese stiltgrass. These data are in contrast to other results from 2011 and 2013 field experiments in which MBI-005 controlled large crabgrass (data not shown) and smooth crabgrass (See Table 5). The industry standard herbicide for summer annual grass control, Pendulum 2G, controlled goosegrass and

Japanese stiltgass, but surprisingly provided only 57% control of large crabgrass. The organic industry standard, corn gluten, did not control any of the weed species tested. Prostrate pigweed was not controlled by MBI-005, P. macrostoma or corn gluten, but was well controlled by the two labeled herbicides. Flexuous bittercress control with P. macrostoma was improved by increased dose, but even at the highest dose tested control did not exceed 68%. Flexuous bittercress control with MBI-005 was less than 50% in 2013. Dandelion was controlled by P. macrostoma with control increasing with increasing dose. MBI-005 at 38L/ha provided 73% dandelion control. It is unclear why preemergence weed control was poorer in 2013 than in 2012. The tests were established on the same site, with a similar watering regime. Weather patterns were not remarkably different between years. Small experiments were established to compare efficacy of different lots of products with no differences observed. The substrates used in each year were similar but in 2013 the milled pine bark did appear to be coarser textured and had a significantly lower pH. Experiments are planned to compare efficacy in different substrates and to compare the influence of substrate pH on product efficacy.

Preemergence Control of Cool Season Weeds in Containers MBI-005 at 38 L/ha provided preemergence control equivalent to isoxaben for: henbit, dandelion, galinsoga, yellow woodsorrel, northern yellow cress, and pearlwort (Table 4). Additionally this treatment controlled bittercress about 80% and controlled annual bluegrass equivalent to pendimethalin. Neither MBI-005 nor P. macrostoma controlled hairy vetch nor common chickweed (data not shown). Corn gluten provided less than 25% control of all species. Doses of the two P. macrostoma formulations were adjusted to apply equivalent amounts of macrocidin (the active fungal toxin). Control of susceptible species increased with increasing dose of P. macrostoma. No differences were observed between the two formulations of Phoma macrostoma. Dandelion, bittercress and rorippa were controlled by P. macrostoma equivalent to industry standard herbicides. Dandelion was well controlled at low rates but other species required higher doses. At the highest dose evaluated, P. macrostoma controlled henbit and oxalis were controlled 50 to 78% but control of pearlwort, chickweed, vetch and annual bluegrass averaged less than 50%.

Table 3a. Preemergence control of broadleaf weeds in containers with Phoma macrostoma, MBI-005 and corn gluten compared to industry
standard herbicides -- % control from visual evaluations 4 to 5 weeks after treatment -- weeds included in both 2012 and 2013 tests..

Treatment

Dose

Eclipta 2012 2013

Spotted Spurge 2012 2013

2012

Groundsel 2013

------------------------------------------------- % control -----------------------------------Non-Treated P. macrostoma* P. macrostoma P. macrostoma MBI-005 MBI-005 EP02-16 + MBI005 Corn Gluten Gallery Pendlulum 2G 3300 mu/m2 6600 mu/m2 13000 mu/m2 19.1 L/ha 38.2 L/ha 6600 mu/m2 + 19 L/ha 97 kg/ha 1.12 kg ai/ha 3.3 kg ai/A 0e 57 c 88 a 98 a 83 ab 98 a 98 a 25 d 100 a 42 cd 0d 15 cd 28 c 98 a 17 cd 23 c 77 b 12 cd 99 a 13 cd 0e 5e 0e 8e 85 a 98 a 57 b 8c 100 a 100 a 0c 2c 17 c 0c 17 c 47 b 17 c 3c 97 a 78 a 0d 30 cd 77 ab 100 a 100 a 100 a 100 a 17 d 100 a 52 bc 0 d* 40 cd 60 a-d 74 abc 55 bcd 92 ab 82 ab 34 cd 100 a 28 d

*P. macrostoma formulations used: EP02-16 in 2012 and EP111312 in 2013.

Means followed by same letter do not significantly differ (P=.05, Waller-Duncan k=100). Because weed control was visually evaluated relative to the non-treated plants, data for the non-treated were omitted from the analysis

*variable results between replicates in 2013 and experiment needs to be repeated on this species.

Table 3b. Preemergence control of broadleaf weeds in containers with Phoma macrostoma, MBI-005 and corn gluten compared to industry
standard herbicides -- % control from visual evaluations 5 weeks after treatment. Additional species included in summer 2013.

Treatment Non-Treated P. macrostoma EP111312

Dose

Large Crabgrass 0c
2

Goosegrass 0d 5 cd 13 bcd 3d 8 bcd 30 b 28 bc 20 bcd 15 bcd 95 a

Japanese Stiltgrass 0e 20 de 3e 3e 38 cd 74 ab 55 bc 30 cd 49 bc 85 a

Flexuous Bittercress 0f 17 ef 20 def 68 bc 42 cde 40 cde 50 cd 0f 100 a 83 ab

Dandelion 0e 28 cd 85 ab 98 a 27 cd 73 b 80 ab 5 de 100 a 37 c

Prostrate Pigweed 0b 3b 7b 0b 3b 8b 8b 0b 80 a 88 a

3300 mu/m (16 g/m2) 6600 mu/m2 (31 g/m2 ) 13000 mu/m2 (63 g/m2) 19.1 L/ha 38.2 L/ha

12 bc 5c 0c 8c 13 bc

MBI-005

EP02-16 + MBI-005 Corn Gluten Gallery Pendlulum 2G

6600 mu/m + 19 L/ha 97 kg/ha 1.12 kg ai/ha 3.3 kg ai/A

12 bc 0c 23 b 57 a

Means followed by same letter do not significantly differ (P=.05, Waller-Duncan k=100). Because weed control was visually evaluated relative to the nontreated plants, data for the non-treated were omitted from the analysis

Table 4. Preemergence control of cool-season broadleaf weeds in containers with two formulations of Phoma macrostoma, MBI-005 and corn
gluten compared to industry standard herbicides -- % control from visual evaluations 8 weeks after treatment Marsh Dande- Yellow- Pearl- Bitter- Galin- ChickAnnual Treatment Dose Henbit Oxalis lion cress wort cress soga weed Vetch Bluegrass ---------------------------------------------------- % control -----------------------------------------------------------Non-Treated P. macrostoma EP02-16 3300 mu/m (16 g/m2) 6600 mu/m2 (31 g/m2) 13000 mu/m2 (63 g/m2) 3300 mu/m2 (28 g/m2) 6600 mu/m2 (55 g/m2) 13000 mu/m2 (110 g/m2) 19.1 L/ha 38.2 L/ha EP02-16 + MBI-005 Corn Gluten Gallery Pendlulum 2G 6600 mu/m + 19 L/ha 97 kg/ha 1.12 kg ai/ha 3.3 kg ai/A
2 2

0e 5e 28 d 55 c 13 de 10 de 60 c 70 bc 93 a 85 ab 13 de 100 a 100 a

0e 6e 8e 52 d 12 e 14 e 68 cd 80 bc 100 a 96 ab 8e 100 a 100 a

0d 90 ab 100 a 100 a 90 ab 100 a 100 a 74 b 96 a 100 a 20 c 100 a 74 b

0e 88 bc 100 a 100 a 60 d 100 a 100 a 82 c 100 a 100 a 8e 100 a 98 ab

0d 10 d 10 d 32 c 20 cd 4d 54 b 98 a 100 a 100 a 14 cd 100 a 100 a

0e 42 d 54 cd 96 a 16 e 48 d 84 ab 14 e 68 bc 76 b 2e 100 a 84 ab

0f 6 ef 16 def 24 def 30 de 10 ef 28 de 30 de 85 ab 60 bc 40 cd 100 a 68 b

0d 10 cd 14 c 4 cd 26 b 8 cd 10 cd 6 cd 26 b 28 b 2d 100 a 94 a

0f 0f 2f 22 bc 0f 4 ef 24 bc 26 b 16 bcd 14 cde 10 def 92 a 18 bcd

0f 38 c 18 de 4 ef 12 def 16 de 12 def 96 a 100 a 100 a 22 d 74 b 94 a

P. macrostoma EP02-13

MBI-005

Means followed by same letter do not significantly differ (P=.05, Waller-Duncan k=100). Because weed control was visually evaluated relative to the nontreated plants, data for the non-treated were omitted from the analysis Abbreviations are standardized EPPO codes for weed names (http://eppt.eppo.org/).

Preemergence control of smooth crabgrass in turf with MBI-005 Corn gluten did not control crabgrass in this experiment (Table 5). Crabgrass control increased with increasing dose of MBI-005, but single applications, even at the highest dose of 57 L/ha, did not provide control equivalent to the industry standard herbicide (Table 5). Multiple applications provided longer residual control. Two applications of MBI-005 at 38 L/ha controlled crabgrass as well as the industry standard herbicide through mid-summer (Figure 1). By late summer crabgrass emergence and growth was greater in the MBI-005 treated plots compared to pendimethalin treated plots. These data suggest that multiple applications of MBI005 > 38 L/ha have the potential for commercially acceptable crabgrass control. MBI-005 treatments resulted in delayed green-up of bermudagrass turf but at no point was this injury greater than that observed in the industry standard herbicide-treated plots (Table 5). The number and frequency of applications required for full season control as well as the impact on turfgrass quality need to be evaluated through additional research.

Figure 1. Percent control of smooth crabgrass in a bermudagrass fairway with two applications each of MBI-005 at 19 and 38 L/ha compared to pendimethalin at 1.5 lb ai/A. The lines represent the LSD at p= 0.05.
100 90 80 70

Percent Control

60 50 40 30 20 10 0 May June July August

MBI 19 L/ha MBI 38 fb 19 MBI 38 L/ha Pendimethalin

Table 5. Preemergence control of smooth crabgrass and turfgrass injury from MBI-005, Corn Gluten and Pendimethalin applied to a low maintenance bermudagrass (Cynodon dactylon) golf fairway.
Dose at each application date Treatment Non-treated MBI-005 MBI-005 MBI-005 MBI-005 MBI-005 MBI-005 MBI-005 Corn Gluten Pendimethalin Smooth Crabgrass % Control 6/28 7/19 0e 0f 0e 35 d 63 c 43 d 70 bc 85 ab 70 bc 0e 97 a 17 ef 33 de 48 cd 45 cd 63 bc 80 ab 58 c 0f 97 a Smooth Crabgrass % Cover 6/28 7/19 80 b 98 a 80 b 50 c 37 cd 47 c 22 de 14 ef 25 de 100 a 1f 80 ab 73 bc 55 cd 52 cd 39 de 16 ef 50 cd 100 a 1f Bermudagrass turf % Injury 5/23 6/28 0c 0c 3 bc 13 b 25 a 0c 3 bc 0c 3 bc 0c 13 b 0c 8 bc 25 a 13 abc 18 ab 18 ab 13 abc 0c 23 a

3/28
0 19 L/ha 38 L/ha 57 L/ha 19 L/ha 38 L/ha 38 L/ha 19 L/ha 980 kg/ha 1.68 kg ai/ha

4/26 0 0 0 0 19 L/ha 19 L/ha 38 L/ha 19 L/ha 0 0

5/23 0 0 0 0 0 0 0 19 L/ha 980 kg/ha 1.68 kg ai/ha

Postemergence Weed Control Postemergence control of weeds in containers. The only treatments to control seedling or mature hairy vetch were the auxin herbicides which provided nearly complete mortality of vetch plants. None of the bio-based treatments were effective; therefore vetch was not included in the 2013 experiment. Annual bluegrass growth was reduced ~ 50% and 25% by MBI-005 in 2012 and 2013, respectively; and ~ 23% by auxinic herbicides in 2013. In 2012 there was some moderate leaf necrosis from Fe-HEDTA in 2012 but not in 2013. Phoma macrostoma did not affect annual bluegrass. Weed species responded differentially to the treatments. When treatments are averaged across species and compared to the industry standard auxinic herbicide it is clear that bio-based treatments can control some species as well as auxinic herbicides, but bio-based products have a more limited spectrum of activity. When compared to Weed B Gon herbicide, Phoma macrostoma treatments did not provide equivalent control of any species tested. However, MBI005 at 38 L/ha controlled 6 of 11 species; FeHEDTA at either rate controlled 5 of 11 species tested as well as or better than Weed B Gon (Table 6). It is interesting to note that the granular auxinic herbicide, Preen Broadleaf, provided equivalent control of only 1 of 11 species tested.

Table 6. Number of weed species (out of the 11 broadleaf weed species tested) for which each treatment provided control equal to or better than that observed with the industry standard herbicide, Weed B Gon. Number of species Treatment Dose controlled P. macrostoma 3300 mu/m2 0 6600 mu/m2 0 13000 mu/m2 0 MBI-005 19.1 L/ha 38.2 L/ha 6600 mu/m2 + 19 L/ha 80 L/ha 160 L/ha 98 kg/ha ~40 ml/m2 2 6 3 5 5 1 na

EP02-16 + MBI-005 Fe-HEDTA Preen Broadleaf WeedBGon

Henbit was controlled 85% to 98% by 38 L/ha of MBI -005 but other treatments were ineffective. Henbit control with MBI-005 was significantly better than that provided by Weed B Gon. Fe-HEDTA caused up to 56% tissue necrosis but plants grew back. Phoma treatments caused chlorosis of the new growth but increased stem elongation and did not reduce overall above-ground growth of henbit. Pearlwort was suppressed by MBI-005 @ 38 L/ha and by Weed B Gon, with each herbicide providing about 65% control 6 weeks after the 2nd treatments. Little or no control was observed with other treatments. Fe-HEDTA provided nearly complete control of dandelion at both doses in both years. Dandlion control with MBI-005 and Phoma macrostoma was improved with increased dose and application. Following the 2nd application dandelion control improved to 54% and 64% suppression by the high doses of Phoma and MBI-005, respectively at 29 days after the 2nd application (See Figure below). Weed control to this rating date was comparable to that provided by the auxin herbicide, Weed B Gon. However, following that rating, dandelions regrew rapidly in both Phoma and MBI-005 treatments which Weed B Gon plants continued to decline. Figure 2. Dandelion control in containers with P. macrostoma and MBI-005 compared to an industry standard auxinic herbicide, WeedBGon.
100 90 80 70

Reapplied

% Control

60 50 40 30 20 10 0 0 10 20 30 40 50 60 Days after First Treatment Phoma MBI-005 WeedBGon

Annual bluegrass was suppressed 58% by MBI-005 at 32L/ha but not significantly controlled by other treatments. Bittercress was suppressed about 65% by MBI-005 at 32L/ha. Phoma treated plants were bleached and chlorotic but were not controlled. FE-HEDTA caused tissue necrosis but plants grew back rapidly from the treatments. Ivyleaf speedwell was controlled over 90% by Fe-HEDTA treatments and up to 87% by MBI005 at 32 L/ha. Phoma treated plants were bleached and chlorotic but were not controlled.

Oxalis was controlled over 90% by Fe-HEDTA and by MBI-005 but was not controlled by Phoma. Marsh yellowcress was controlled over 90% by Fe-HEDTA and over 80% by MBI-005 applied at 32 L/ha. Phoma-treated plants were bleached and chlorotic but overall control did not exceed 55%. Combining MBI-005 at 19L/ha with the middle-dose of Phoma did improve control of marsh yellowcress, bittercress, dandelion, and oxalis compared to either product applied alone.

Postemergence control of seedling broadleaf weeds in newly seeded tall fescue turf. When applied early postemergence to broadleaf weeds in a newly seeded tall fescue in the fall of 2011, P. macrostoma induced chlorosis and bleaching in susceptible species but did not reduce overall weed cover one month after treatment (Table 7a). However, when applied in the fall of 2012, P. macrostoma provided significant control of dandelion and hop clover. Similarly, FeHEDTA and MBI-005 did not reduce overall broadleaf weed cover one month after treatment in 2011 but did reduce weed cover in 2012. When the following spring, the high dose of P. macrostoma provided weed control and reduced broadleaf weed cover equivalent to the industry standard auxinic herbicide (Table 7a&c). In 2011-2012, neither MBI-005 nor Fe-HEDTA controlled broadleaf weeds as well as the industry standard herbicide, Weed B Gon. However, in spring of 2013, Fe-HEDTA treatments provided control equivalent to Weed B Gon. MBI-005 controlled weeds in the fall but significant regrowth was observed the following spring. Using the industry standard herbicide for comparison - The high dose of P. macrostoma provided equivalent control of hop clover, dandelion and overall winter annual broadleaf weeds but not vetch; The high dose of MBI-005 controlled hop clover and dandelion but was generally less effective on winter annual broadleaf weeds. FeHEDTA controlled hop clover, dandelion and overall winter annual broadleaf weeds but not vetch. These data suggest that fall applications of these natural product treatments can provide control of most seedling broadleaf weeds equivalent to that observed with an industry standard herbicide. This experiment utilized a single treatment in the fall. Data from other experiments have shown that multiple applications are necessary for optimum efficacy. Multiple applications could not be made in this experiment due to limited product availability. However, the experiment is being repeated in the fall of 2013 to evaluate the efficacy of multiple applications.

Table 7a. Postemergence control of seedling broadleaf weeds* in newly seeded tall fescue turfgrass; treated November 2011, evaluated 4 weeks after treatment and the following spring.
Treatment Non-Treated Phoma macrostoma Phoma macrostoma Phoma macrostoma MBI-005 MBI-005 Fe-HEDTA Fe-HEDTA WeedBGon -- RTU Preen Brdlf Control 42 g/m 84 g/m 168 g/m
2 2 2

Dose

~ 4 WAT % Cover 10 bc 18.8 a 12.5 b 11.3 bc 11.3 bc 7.5 bc 7.5 bc 7.5 bc 6.3 c 10 bc

April 2012 % Cover 69 ab 55 b 30 c 10 d 75 a 75 a 65 ab 60 ab 14 cd 28 c

April 2012 % Control 0 d 28 c 64 b 89 a 8 cd 5 cd 20 cd 25 c 86 ab 71 ab

19.1 38.2 79.5 159 ~28 ml/m

2

98

*Evaluations are of overall broadleaf weed cover. Broadleaf weeds present were a mixture of species including henbit, common chickweed, dandelion, and vetch but the vegetation was dominated by hop clover. Means followed by same letter do not significantly differ (P=.05, Waller-Duncan k=100).

Table 7b. Postemergence control of seedling broadleaf weeds in newly seeded tall fescue turfgrass; treated November 16, 2012, evaluated ~4 weeks after treatment
Treatment Dose DandeHop Vetch Chick lion clover weed ------------------- % control -------------------0 e 0 e 0 d 0 d 58 bc 23 d 28 c 5 de 60 bc 45 bcd 20 c 10 cde 45 cd 58 bc 20 c 78 a 67 bc 73 ab 18 c 78 a 75 ab 45 bcd 10 c 33 bc 100 a 38 cd 90 a 28 bcd 100 a 58 bc 100 a 48 b 53 bc 90 a 90 a 85 a 20 d 70 ab 53 b 3 e

Non-treated P. macrostoma EP02-16-2012 P. macrostoma EP02-16-2012 MBI-005 MBI-005 P. macrostoma + MBI Fe-HEDTA Fe-HEDTA WeedBGon RTU Preen Broadleaf

na 31 g/m2 63 g/m2 19 L/ha 38 L/ha 31 g + 19L 79 L/ha 159 L/ha 28 ml/m2 98 kg/ha

Means followed by same letter do not significantly differ (P=.05, Waller-Duncan k=100).

Table 7b. Postemergence control of seedling broadleaf weeds in newly seeded tall fescue turfgrass; treated November 16, 2012, May 9, 2013.
Treatment Dose DandeHop Vetch Winter Annual lion clover Broadleaves % Cover ------------------- % Control ----------------0 c 0 c 0 c 0 e 33 a 28 b 67 ab 33 b 63 ab 65 ab 85 a 93 a 88 a 30 b 43 b 93 ab 58 60 75 70 50 99 83 ab ab ab ab ab a ab 0 c 30 bc 0 30 70 25 30 100 23 c bc ab bc bc a bc 43 bcd 68 abc 38 17 71 70 77 93 50 cd d abc abc abc a bcd 21 ab 6 b 20 32 12 8 8 3 20 ab a ab b b b ab

Non-treated P. macrostoma EP02-16-2012 P. macrostoma EP02-16-2012 MBI-005 MBI-005 P. macrostoma + MBI Fe-HEDTA Fe-HEDTA WeedBGon RTU Preen Broadleaf

31 g/m2 63 g/m2 19 L/ha 38 L/ha 31 g + 19L 79 L/ha 159 L/ha 28 ml/m2 98 kg/ha

Means followed by same letter do not significantly differ (P=.05, Waller-Duncan k=100). W.A. Brdlf. = overall winter annual broadleaf weed control including all winter annual broadleaf weeds, including hope clover, vetch, henbit, chickweed, field madder and other species with non-uniform distributions in the site.

Postemergence control of established dicondra. Fe-HEDTA produced significant phytotoxicity with up to 98% control one week after treatment (Figure 3). Other treatments produced little or no control of dicondra before spring. By March 2013, P. macrostoma provided essentially complete control of dicondra, equivalent to that observed in the industry standard herbicide treatment plots. Plots treated with P. macrostoma had <10% cover of dicondra compared to >95% in the non-treated and MBI-005 treated plots (Figure 4). Fe-HEDTA-treated dicondra had recovered from the initial control and covered 82% of the plot area. Due to limited product availability only one dose of P. macrostoma was evaluated. In the future, experiments should be conducted to determine the dose required for dicondra control.

Figure 3. Percent control of dicondra with P. macrostoma, MBI-005 and Fe-HEDTA applied in the fall of 2012; evaluated 1 week after treatment and again the following spring, March 2013.
100 90 80 70 % Control 60 50 40 30 20 10 0 Non-Treated P.macro MBI-005 38L/ha P macro. + MBI-005 Fe-HEDTA 160L/ha WeedBGon Max

1 WAT
March

Figure 4. Effects of fall-applied P. macrostoma, MBI-005 and Fe-HEDTA on percent cover of dicondra the following spring evaluated March 2013
100

80

% Cover

60

40

20

0 Non-Treated P.macro MBI-005 38L/ha P macro. + MBI-005 Fe-HEDTA 160L/ha WeedBGon Max

Fe-HEDTA concentration and spray volume comparisons for control of broadleaf weeds. Fe-HEDTA produces rapid necrosis of white clover and dandelion foliage. Foliage turns black within 48 hours of treatment but re-growth or foliage which did not receive adequate spray coverage is visible within one week of treatment. Weed control increases with increasing concentration and spray volume (Figure 5). However, when dandelion and clover control data are plotted against actual Fe-HEDTA dose (across all concentrations and spray volumes), the results fit a logistic model (R2 = 0.93 and 0.83, respectively) suggesting that the actual dose applied has a greater influence on resulting levels of weed control than do the concentration or spray volume selected (Figure 6). Treatments were reapplied 4 weeks after initial treatment and evaluations continued with similar results (data not shown). Fe-HEDTA controlled only existing vegetation. Weeds not completely controlled resumed growth when weather was conducive. Plant distribution measures (grid counts) in April 2013 revealed that, despite nearly complete control in earlier evaluations, white clover was still evenly distributed in nearly all plots (data not shown).
Figure 5. Dandelion (TAROF) and white clover (TRFRE) control 3 weeks after initial treatment with 3 concentrations of Fe-HEDTA applied at 4 spray volumes. Note: Regrowth from most treatments was evident by 3 WAT.

Figure 6. Dandelion (TAROF) and white clover (TRFRE) dose response curves. Data for all spray volume and concentrations were plotted by actual dose of Fe-HEDTA applied then regression curves fit to the data.

At a separate site the experiment was repeated on winter annual broadleaf weeds in the spring of 2013. Results were similar to those reported for clover and dandelion. Increasing the dose of Fe-HEDTA applied improved broadleaf weed control independent of the spray concentration or spray volume at which the target dose was applied (see Figure 7).

Figure 7. Winter annual broadleaf weed control in dormant bermudagrass turf, 3 weeks after 2nd application treatment with 3 concentrations of Fe-HEDTA applied at 4 spray volumes.
100 90 80 70 60 50 40 30 20 10 0

Winter annual broadleaf weed control with FeHEDTA

% Control

y = 21.395ln(x) + 9.6411 R = 0.8496

10

20

30

40 50 60 oz Product/1000ft2

70

80

90

100

Safety to Newly Seeded Tall Fescue Turf When applied at the time of seeding, tall fescue seedling emergence and establishment were not reduced by Phoma macrostoma treatments and corn gluten increased turfgrass density. Corn gluten treated plots were noticeably denser and darker in color, likely as a result of the organic nitrogen released from the product. In contrast, MBI-005 reduced turfgrass stand density (see Table 8). Similar results were observed in 2012-treated turf. Table 8. Effect of bio-based products applied preemergence, at seeding of tall fescue turf on turfgrass
cover. Seeded September 27, 2012; treated September 27 and November 11, 2012 Treatment Dose 6 WAS 12 WAS

--------- % turf cover ---------Non-Treated P. macrostoma EP02-16 P. macrostoma EP02-16 P.macrostoma EP02-13 P.macrostoma EP02-13 MBI-005 MBI-005 EP02-16 + MBI-005 Corn Gluten 6600 mu/m2 (31 g/m2 ) 13000 mu/m (63 g/m ) 13000 mu/m (55 g/m ) 13000 mu/m (110 g/m ) 19 L/ha 38 L/ha 31 g/m + 19 L/ha 970 kg/ha
2 2 2 2 2 2 2

53 b 48 b 57 b 53 b 50 b 30 c 15 d 22 cd 73 a

89 a 86 a 76 ab 90 a 86 a 53 cd 50 d 61 cd 94 a

*WAS = weeks after seeding

When applied postemergence to seedling tall fescue neither P. macrostoma nor MBI-005 caused significant injury to the turf. Fe-HEDTA caused 20 to 35% foliar necrosis, but new growth was unaffected and the % damaged foliage decreased over time as new growth emerged and damaged foliage was removed by mowing (Table 9). The blacked foliage caused by these treatments (Figure 8) would likely be objectionable to the end-user and treatments should probably be limited to times of year when the turfgrass is actively growing so damaged foliage would be rapidly removed in mowing operations and replaced by new growth.
Table 9. Percent phytotoxicity of bio-based products applied postemergence to seedling tall fescue turf, Seeded September 27, 2012; treated November 11, 2012 Treatment Dose 1 WAT 3 WAT 5 WAT ------------------- % -----------------0c 0d 0c Non-Treated P. macrostoma EP02-16 P. macrostoma EP02-16 MBI-005 MBI-005 EP02-16 + MBI-005 Fe-HEDTA Fe-HEDTA Weed-B-Gone RTU Preen Broadleaf Control 6600 mu/m2 (31 g/m2 ) 13000 mu/m (63 g/m ) 19 L/ha 38 L/ha 31 g/m + 19 L/ha 80 L/ha 160 L/ha ~42 ml/m 98 kg/ha
2 2 2 2

0c 0c 0c 0c 0c 20 b 35 a 0c 0c

0d 3d 0d 3d 0d 22 b 35 a 3d 10 c

1c 1c 0c 3c 0d 8 ab 12 a 4 bc 1c

*WAT = weeks after treatment

Figure 8. Tall fescue seedling injury from Fe-HEDTA applied at 160 L/ha (left) vs the non-treated plant (right). Photos taken 2 weeks after treatment. New grow emerging after treatment was not affected.

Safety to Ornamental Plants P. macrostoma did not cause injury to any of the four woody plants tested. Of the four woody ornamentals tested, spirea was the most sensitive to injury from both MBI-005 and Fe-HEDTA with about 12% of the foliage necrotic or damaged following the 2nd application (See Table 6). Only new growth present at the time of application was affected. Older foliage was not affected, nor was new growth affected. Similarly, new growth on gardenia was affected by MBI-005 and Fe-HEDTA. MBI-005 caused discoloration of about 4% of the foliage; Fe-HEDTA caused necrosis of about 6% of the foliage. Ligustrum japonicum new growth was injured by FeHEDTA but was not injured by MBI-005 or Phoma macrostoma. Blue pacific juniper was not injured by any treatment.
Table 6. Phytotoxicity of bio-based weed control products from over the top applications to container grown woody ornamentals. Treatment Dose Spirea Ligustrum Gardenia Juniper ----------------- % foliar necrosis -----------------Non-Treated P. macrostoma EP02-16 P. macrostoma EP02-16 MBI-005 MBI-005 Fiesta (Fe-HEDTA) Fiesta (Fe-HEDTA) 6600 mu/m2 (31 g/m2 ) 13000 mu/m2 (63 g/m2) 19 L/ha 38 L/ha 80 L/ha 160 L/ha 1.5 c 0c 0c 5.0 abc 11.3 abc 4.0 bc 11.5 a 0b 0b 0b 0b 0b 7.4 a 8.0 a 0c 0c 0c 1.3 bc 3.8 ab 2.5 bc 6.3 a 0 0 0 0 0 0 0

Photos: left to right phytotoxicity from over the top applications of FeHEDTA to spirea, ligustrum and gardenia new growth.

Safety to herbaceous ornamental plants, Heuchera Palace Purple and Liriope spikata variegata, was also evaluated in 2012. Slight foliar injury was observed on both species from Fe-HEDTA. No injury from P. macrostoma or MBI-005 treatments was observed. Combined with previous

years data, we can conclude that when applied over the top of actively growing broadleaf ornamentals, foliar injury is likely. However, these data also suggest that treatments would likely not be injurious when applied as a directed spray (avoiding contact with the foliage) or over the top of plants that do not have a flush of tender foliage present at the time of treatment. These data further suggest that properly applied turfgrass applications would have little potential for off-target damage to nearby ornamental plantings.

OVERALL SUMMARY Results from these experiments demonstrate there is still much yet to learn about the performance of these bio-based weed control alternatives. Seedling broadleaf weeds were well controlled by Fe-HEDTA treatments of greater than 48 oz/10002 yet control was contact in nature and re-application intervals need to be refined. For P. macrostoma and MBI-005, significant variability in efficacy was observed between species. P. macrostoma provided adequate postemergence control of common groundsel, seedling dandelion and white clover, but limited postemergence control of common chickweed, henbit and field madder. MBI-005 provided preemergence and postemergence control of several species but had little efficacy on chickweed or clover. Our data so far suggest that preemergence applications of both bio-based products may provide better and more consistent control of broadleaf weeds compared to postemergence applications. In particular, MBI-005 provided commercially acceptable control of crabgrass in golf turf, through mid-summer. Treatment regimes that may extend seasonal control need to be investigated. Additionally, substantial differences in efficacy between 2012 and 2013 preemegence efficacy studies suggest that soil / substrate physical or chemical characteristics may significantly influence preemergence efficacy of both MBI-005 and P. macrostoma. The underlying reasons for these variable responses to preemergence applications need to be investigated. Weed management in turfgrass systems requires effective control of several key weed species. Standard turfgrass herbicides are applied in combinations to achieve broader spectrum weed control. A similar strategy with bio-based products may be helpful. For example: MBI-005 controled henbit but lacked efficacy on white clover; whereas both P. macrostoma and FeHEDTA were very effective postemergence on white clover but lacked efficacy on other species. Strategies that combine these or other products, along with bio-based cultural management systems will be required to achieve commercially successful levels of weed control in turfgrass with natural products. Safety to dormant and semi-dormant ornamental species suggests potential additional uses for the biologically-based weed management products in nursery or landscape settings.

Literature Cited: Bailey, K.L. and J. Derby. 2005. Environmental fate of Phoma macrostoma, a fungus for broadleaf weed control in turfgrass. Proceedings of the 13th EWRS Symposium, Bari, Italy, 19-23 June 2005 (unpaginated) Bailey, K. L., S. Falk, and S. Lombardo. 2009. Status of Phoma macrostoma, a bioherbicide for broadleaf weed control in turfgrass. Proc. IXth Intern. Bioherbicide Group Workshop. pp 42-52. http://www4.ncsu.edu/~jcneal/IBG%20Workshop/IBG%202009%20Proceedings%20revise d%2020Apri09.pdf (accessed Jan 19 2010) Frans, R. E, R. Talbert, D. Marx, and H. Crowley. 1986. Experimental design and techniques for measuring and analyzing plant responses to weed control practices. pp 29-46 In: Camper, D. ed. Research Methods in Weed Science, 3rd edition. Southern Weed Sci. Soc. Champaign, IL. King, R. R. and C. H. Lawrence. 2001. Herbicidal properties of the thaxtomin group of phytotoxins. J. Agric. Food Chem. 49 (5):22982301 Neal, J. C. 1993. Granular herbicides for postemergent control of broadleaf weeds in turf without 2,4-D. Proc. Intern. Turfgrass Conf. 7:238-245. Zhou LeCong; Bailey, K. L.; Derby, J. 2004. Plant colonization and environmental fate of the biocontrol fungus Phoma macrostoma. Biological Control; 30(3):634-644

Publications from this research Neal, J. C., R. Schiavone, and C. Harlow. 2013. Seedling broadleaf weed control with MBI005. Proc. Northeastern Weed Sci. Soc. 53:98. Neal, J. C., B. Shew, and R. Schiavone. 2013. Temperature and Dose influence Phoma macrostoma efficacy on seedling broadleaf weeds. Proc. International Bioherbicide Group Workshop. (in press)